Systems and methods to perform assays for detecting or quantifying analytes within samples

0001(Priority claim) This application claims the benefit of US Provisional Application No. 60 / 659,874 (filed March 10, 2005), the content of which is incorporated herein by reference.

0002(Field of invention) The present invention generally relates to an automated analyzer for performing multiple nucleic acid assays at the same time, and more specifically, a system for performing multiple nucleic acid amplification assays, including both real-time and endpoint amplification assays. And how. The present invention also relates to an apparatus and method for continuously processing the contents of a plurality of reaction vessels, following the real-time amplification procedure. The present invention also relates to a method of reducing the presence of amplification inhibitors in the reaction vessel before carrying out the nucleic acid amplification reaction.

0003Nucleic acid assays enable highly specific and sensitive detection of nucleic acid specimens from a variety of sources, including clinical, industrial, environmental and food sources. Using these assays, the presence of biological antigens (such as prions), cellular abnormalities, disease states, and pathogens associated with the disease, including parasites, fungi, bacteria, and viruses present in the host organism or sample. You can judge the number. The nucleic acid assay may be qualitative or quantitative, and the quantitative assay provides the practitioner with useful information for determining the extent of infection or disease, or for determining the condition in the long term. Quantitative assays can also be used, for example, to assess the effectiveness of therapeutic treatment programs, or to determine the extent of infection or contamination with a particular organism or virus.

0004All nucleic acid assay formats involve a number of processing steps leading to the identification, detection, or quantification of one or more nucleic acid of interest in a sample. If desired, the nucleic acid arrangement of a specifically targeted nucleic acid assay may be specific to a identifiable group of organisms (the term "organism" as used herein includes viruses). This group is common to all members of the group and is defined by at least one common nucleic acid arrangement specific to the group. (A organism "group" is usually a phylogenetic classification of an organism, such as a strain, species, or genus of the organism.) Usually, the uniqueness of the nucleic acid arrangement or multiple arrangements of interest is analyzed. It should be limited to only certain types of samples (eg, human samples vs. industrial or environmental samples). Nucleic acid-based methods and means for detecting individual or group organisms are described by Kohne, "Method for Detection," in Patent Document 1. It is disclosed in "Identification and Quantitation of Non-Viral Organisms" and "Nucleic Acid Probes for Detection and / or Quantitation of Non-Viral Organisms" of Patent Document 2 by Hogan et al.

0005Once the organism to be assayed is determined, the first step is to select or design a probe that exhibits the specificity of the nucleic acid arrangement belonging to the organism that defines the group. Nucleic acid assays can be designed to detect either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including ribosomal RNA (rRNA), transfer RNA (tRNA), or messenger RNA (mRNA). For prokaryotes and eukaryotes, rRNA or encoded DNA (rDNA) is usually the preferred detection target. Ribosomal RNA arrangement is a non-amplified nucleic acid because of its relative abundance in the cell and because rRNA contains a range of arrangement changes that can be used to design probes that are distinguishable even among closely related organisms. It is a particularly preferred subject for the assay. Ribosome nucleic acid-free viruses, and cellular alterations, are often best detected by targeting DNA, RNA, or messenger RNA (mRNA) configurations. As an example, McDonough et al., US Pat. No. 6,649,749, "Detection of Human" See "Immunodeficiency Virus Type 1" and "Methods to Detect Prostate Cancer in a Sample" in US Patent Application Bulletin No. US 2005-0282170 A1 by Fradet et al. Such viruses include plus-strand RNA viruses (such as hepatitis C virus) whose RNA genome is mRNA, minus-strand RNA viruses (such as influenza virus), retroviruses (such as human immunodeficiency virus), and single-stranded DNA viruses (parvovirus). Etc.), and may include double-stranded DNA viruses (such as adenovirus) that require a lysis step to amplify or detect the double-stranded target region to a sufficient single-strand. When the focus of nucleic acid assays is on the detection of genetic abnormalities, probes are usually designed to detect identifiable genetic code changes. An example of this is the Philadelphia chromosomal abnormality associated with chronic myelogenous leukemia. As an example, Stephenson et al., U.S. Pat. No. 4,681,840, "Deoxynucleic Acid Molecules" See Useful as Probes for Detecting Oncogenes Incorporated Into Chromosomal DNA.

0006When performing a nucleic acid assay, sample preparation is required to release and stabilize the nucleic acid of interest that may be present in the material. Sample preparation also serves to eliminate nuclease activity and to exclude or inactivate potential inhibitors of nucleic acid amplification (discussed below) or detection of the nucleic acid of interest. Ryder et al., US Pat. No. 5,639,599, "Amplification of" discloses a method for preparing nucleic acids for amplification, including the use of complexing agents capable of complexing with ferric ions released by lysed erythrocytes. See Nucleic Acids From Mononuclear Cells Using Iron Complexing and Other Agents. This sample preparation method varies and depends to some extent on the nature of the sample being processed (blood, urine, stool, pus, or sputum, etc.). When the nucleic acid of interest is extracted from a group of leukocytes present in a diluted or undiluted whole blood sample, the fractional lysis method is usually followed. "Preparation" of European Patent Application No. 0 547 267 A2 by Ryder et al. See of Nucleic Acid From Blood. Fractional lysis methods are well known in the art and limit or eliminate the presence or activity of heme and other erythrocyte products that can specifically isolate nucleic acids from leukocytes and at the same time interfere with the amplification or detection of nucleic acids. Designed to do. Other dissolution methods include, for example, "Methods of Extracting Nucleic Acids and PCR Amplification Without Using a Proteolytic Enzyme" in US Pat. No. 5,231,015 by Cummins et al., And "Methods for Extracting Nucleic Acids" in US Pat. No. 5,837,452 by Clark et al. From a Wide Range of Organisms by Nonlytic Permeabilization, and Cunningham et al., US Patent Application Bulletin No. US 2002-0055116 A1, "Compositions, It is disclosed in "Methods and Kits for Determining the Presence of Cryptosporidium Organisms in a Test Sample".

0007To purify the sample and remove nucleases and other substances that can interfere with amplification or detection, the nucleic acid of interest is simply by the target capture means using a "capture probe" that constrains the nucleic acid of interest. It can be separated and is directly or indirectly bonded or becomes bonded to a solid substrate such as a magnetic or silica particle. Ranki et al., US Pat. No. 4,486,539, "Detection of Microbial Nucleic Acids by a One-Step Sandwich Hybridization Test," Stabinsky, US Pat. No. 4,751,177, "Methods and Kits for Performing Nucleic Acid Hybridization Assays," by Boom et al. , US Pat. No. 5,234,809, "Process for Isolating Nucleic Acid," Englehardt et al., US Pat. No. 5,288,609, "Capture Sandwich." Hybridization Method and Composition, Collins, US Pat. No. 5,780,224, "Target and Background Capture Methods and MFP for Affinity Assays," and Weisburg et al., US Pat. No. 6,534,273, "Two-Step Hybridization and Capture of a Polynucleotide." Please refer to. If the solid support is a magnetic particle, a magnet is used in the vicinity of the reaction vessel to pull the magnetic particle to the side of the vessel and grab it, thereby isolating any bound nucleic acid in the reaction vessel. Other methods for isolating the bound nucleic acid in the reaction vessel include centrifugation and immobilization of the capture probe in the reaction vessel. As an example, "Polynucleotide Capture Assay Employing" in US Pat. No. 5,200,314 by Boom et al., Supra, and Urdea. in Vitro See "Amplification". Once the bound nucleic acid has been isolated as described above, the unbound nucleic acid, by aspirating the fluid contents of the reaction vessel and optionally performing one or more washing steps with a washing solution, And other cellular and other cellular materials and sample materials can be separated.

0008In many cases, it is preferable to amplify the target sequence. Nucleic acid amplification involves the use of a nucleic acid polymerase to enzymatically synthesize a nucleic acid amplification product (copy) containing a template arrangement complementary or homologous to the nucleic acid arrangement to be amplified. The amplification product may be an extension product or a transcription product produced by a transcription-based amplification method. Examples of nucleic acid amplification methods performed in the art include polymerase chain reaction (PCR), chain substitution amplification (SDA), loop-mediated isothermal amplification (LAMP), ligase chain reaction (LCR), immunoamplification, and transcriptional intervention. There are various transcription-based amplification methods including amplification (TMA), nucleic acid placement-based amplification (NASBA), and self-placement replication (3SR). As an example, Mullis, U.S. Pat. No. 4,683,195, "Process for Amplifying, Detecting, "and / or Cloning Nucleic Acid Sequences", Walker's US Pat. No. 5,455,166 "Strand Displacement Amplification", Notomi et al. "Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction", Cashman, US Pat. No. 5,849,478, "Blocked-Polymerase Polynucleotide Immunoassay Method and Kit," Kacian et al., US Pat. No. 5,399,491, "Nucleic Acid." Sequence Amplification See Methods, Malek et al., US Pat. No. 5,130,238, Enhanced Nucleic Acid Amplification Process, and Lizardi et al., 6: 1197 (1988), BioTechnology. Nucleic acid amplification is particularly beneficial when the amount of target sequence present in the material is very small. Sensitivity of the assay because less target sequence is required at the start of the assay to reliably detect the nucleic acid arrangement of interest by amplifying the target sequence and detecting the synthesized amplification product. Can be greatly improved.

0009Detection of the target nucleic acid requires the use of a nucleotide base configuration that binds to the target sequence contained in the target nucleic acid, or the use of a probe having an amplification product containing the target sequence or its complement. Probes useful for distinguishing sources of nucleic acids are selected or designed so that they do not detectably bind to nucleic acids of non-target organisms that may be present in the sample under selected assay conditions. The probe may contain non-nucleotide components, but the target binding portion of the probe contains DNA, RNA, and / or analogs thereof to achieve hybridization to the target sequence or its complement. As an example, Becker et al., US Patent Application No. US 2003-0036058 A1, "Modified Oligonucleotides for Determining the Presence." "of a Nucleic Acid Analyte in a Sample" (disclosure of the use of a 2'-O-methyl modified probe), and "Peptide Nucleic" in US Pat. No. 5,539,082 by Nielsen et al. See Acids (disclosure of the use of probes with a 2-aminoethylglycine backbone that binds the nucleobase subunit to a central secondary amine using a carboxymethyl linker). For detection purposes, the probe may include a detectable label, such as a radioactive label, fluorescent dye, biotin, enzyme, or chemiluminescent compound, which may be labeled before, during, or after hybridization to the probe. Either can be provided to the target sequence or complement. Examples include Higuchi's US Pat. No. 5,994,056, "Homogenous Methods for Nucleic Amplifications and Detection," and Urdea et al., US Pat. No. 5,635,352, "Solution Phase Nucleic Acid." See Sandwich Assays Having Reduced Background Noise.

0010Nucleic acid assays may be based on homogeneous or non-homogeneous formats. One form of heterogeneous assay preferentially binds the probe, which is the complex of interest, to a solid support such as glass, mineral, or polymeric material and removes any unbound probe prior to detection. In the alternative method, it is the unbound probe that is associated with the solid support, during which the probe that combines with the target sequence remains free in solution and can be separated for detection. Homogeneity assays are usually performed without a solid phase separation step and make extensive use of the chemical differences between the probe free in solution and the probe forming part of the subject, which is a probe complex. An example of a homogenization assay is described in Arnold et al., US Pat. No. 5,639,604, "Homogenous Protection. There is a hybridization protection assay (HPA) disclosed in Assay. Detection in HPA allows specific detection of acridinium ester-labeled probes hybridized to the target sequence or its complement based on specific hydrolysis. Examples include Arnold et al., US Pat. No. 4,950,613, "Protected Chemiluminescent Labels," Campbell et al., US Pat. No. 4,946,958, "Chemilunescent Acridium Labeling Compounds," and Arnold et al., US Pat. No. 5,185,439, "Acridinium Ester." Labeling and Purification of Nucleotide Probes, and Arnold et al., US Pat. No. 5,585,481 Linking Reagents for Nucleotide. See Probes. This detection format includes a hybridization step and a selection step. In the hybridization step, excess acridinium ester-labeled probe is added to the reaction vessel, allowing annealing to the target sequence or its complement. Following the hybridization step, the label associated with the non-hybridized probe is made non-chemiluminescent by adding an alkaline reagent in the selection step. The alkaline reagent specifically hydrolyzes only the acridinium ester label associated with the non-hybridized probe, leaving the acridinium ester of the probe of interest intact intact and detectable. The chemiluminescence from the acridinium ester of the hybridized probe can be measured using an illuminometer and signaled at the associated optical device or RLU.

0011Other homogenization assays include those disclosed below. US Pat. No. 5,804,375 "Reaction Mixtures for Detection of Target Nucleic Acids" by Gelfand et al., US Pat. No. 5,958,700 "Detection of Nucleic Acids by Fluorescence Quenching" by Nadeau et al., US Pat. No. 5,925,517 by Tyagi et al. "Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays and Kits, US Pat. No. 5,928,862, "Competitive Homogenous Assay" by Morrison, and US Pat. No. 6,849,412, "Molecular Torches," by Becker et al. These patents describe single or bimolecular probes, which may be used to determine the amount of nucleic acid of interest in real time in amplification procedures, respectively. In this case, changes in the signal associated with the formation of the probe, which is the target complex, are detected during amplification and are used to calculate an estimate of the nucleic acid of interest present in the sample. An algorithm for calculating the number of nucleic acid of interest originally present in a sample, based on signal information collected during the amplification procedure, is described by Wittwer et al., US Pat. No. 6,232,079, "PCR Method for Nucleic Acid Quantification." Utilizing Second or Third Order Rate Constants, US Pat. No. 6,691,041 "Methods for the Efficiency-Corrected Real-Time Quantification of Nucleic Acids" by Sagner et al., US Pat. No. 6,911,327 "Methods for Quantitative Analysis of a Nucleic Acid Amplification Reaction" by McMillan et al. , And those disclosed by US Provisional Application No. 60 / 693,455, "Method and Algorithm for Quantifying Polynucleotides," which enjoys joint ownership by Chismar et al.

0012After the nucleic acid assay has been initiated, and to avoid possible contamination with germs in subsequent amplification reactions, the reaction mixture can be treated with inactivating reagents that destroy the nucleic acids and associated amplification products in the reaction vessel. Such reagents include oxidizing agents, reducing agents, and reactive chemicals that modify the original chemical structure of the nucleic acid. These reagents act by inactivating the nucleic acid in an amplification reaction in which the nucleic acid is RNA or DNA. Examples of such chemicals include sodium hypochlorite solution (bleach), potassium permanganate solution, formic acid, hydrazine, dimethyl sulfide, and analogs. For more details on the deactivation protocol, see "Method and Kit" in US Pat. No. 5,612,200 by Dattagupta et al. For Destroying the Ability of Nucleic Acid to Be Amplified, and by Nelson et al., US Patent Application Bulletin No. US 2005-0202491 A1, "Reagents, Described in Methods and Kits for Use in Deactivating Nucleic Acids.

0013Given the many complex steps involved in nucleic acid amplification assays and the different processing and equipment required for different amplification assays, an automated system capable of treating the contents of multiple reaction vessels with different amplification assay protocols. Is required, and in particular, it is most needed to perform both real-time and endpoint amplification assays on the same platform and / or in an internal housing. The real-time amplification assay periodically determines the amount of amplification product of interest when the amplification reaction occurs, which facilitates the provision of quantitative information about the nucleic acid of interest present in the sample. Endpoint amplification, on the other hand, is more useful for determining the amount of amplification product of interest after an amplification reaction has occurred, thereby providing qualitative information about the nucleic acid of interest. To reduce the time required to process large samples by improving flow, the contents of multiple reaction vessels to manually or automatically load a new batch of reaction vessels for processing without disrupting the system. There is also a need for a system that can continuously process things according to real-time amplification protocols. Therefore, there is a need for reagents and methods that reduce the amount of amplification inhibitors in the reaction vessel that can affect qualitative or quantitative judgment.<patcit num="1"><text>U.S. Pat. No. 4,851,330</text></patcit><patcit num="2"><text>U.S. Pat. No. 5,541,308</text></patcit>

<p num="0014"> The above needs are addressed by an automated analyzer configured and operated according to aspects of the invention. In general, an automated analyzer integrates and coordinates the behavior of various automated stations or modules associated with performing one or more assays on multiple reaction mixtures contained in a reaction vessel. The analyzer is preferably a built-in stand-alone unit. Assay sample materials and reaction vessels, as well as various solutions, reagents, and other materials used to perform the assay are preferably stored in the analyzer as well as the waste generated during the assay. The analyzer is an integrated nucleic acid testing system that fully automates all assay steps from sample processing to amplification and multi-format detection. In a preferred embodiment, the instrument can perform both real-time and endpoint amplification assays. Once the routine setup is complete, the operator may choose to perform the endpoint amplification assay, the real-time amplification assay, or both. For real-time amplification assays, the analyzers of the invention, in contrast to batch mode, continuously process objects in multiple reaction vessels, thereby greatly increasing the rate at which results can be calculated and reported. .. During operation, the analyzer is provided with a surface treatment agent used to cover the inner surface of the reaction vessel before or during the isolation and purification steps of removing sample material and / or reagents from the reaction vessel. You may use it.</p><p num="0015"> The analyzer includes a computer control device that performs analyzer control and assay scheduling software to coordinate the operation of the analyzer's stations and the movement of each reaction vessel throughout the analyzer.</p><p num="0016"> The reaction vessels can be loaded into the loading preparation chamber, which sequentially presents each vessel to the collection position for recovery by a transfer mechanism that automatically transfers the reaction vessels between analyzer stations.</p><p num="0017"> The sample container is transported to the first ring assembly and the disposable pipette tip is transported to the second ring assembly. A container of target capture reagent, including a suspension of solid support material, is configured and arranged to selectively agitate the container or present the container so that the probe of an automated robot pipette system can reach it. Transported to a rotatable assembly. The reaction mixture containing the fluid sample material and the subject capture test is prepared by a pipette system in each reaction vessel.</p><p num="0018"> The analyzer further includes a vessel stirrer for mixing the contents of the vessel to be installed. The stirrer may be fluid communication with the fluid vessel and may include a dispenser for dropping one or more fluids into the vessel. One or more incubators carry multiple containers into the temperature control chamber, allowing individual containers to be automatically installed indoors or removed outdoors. The magnetic separation station automatically performs magnetic separation and cleaning procedures on the contents of the container installed in the station.</p><p num="0019"> In a preferred procedure, assay results may be confirmed by the amount of light emitted from the vessel at the end of the appropriate preparation step. Therefore, the analyzer includes an illuminometer (a type of signal detector) for detecting and / or quantifying the amount of light emitted by the contents of the reaction vessel. An inactivation preparation room may be provided to inactivate the contents of the reaction vessel to be installed at the end of the assay.</p><p num="0020"> Reaction vessels are individually transferred between stations by a transfer mechanism, and by operating the stations in parallel, different assay procedures can be performed simultaneously in different reaction vessels, resulting in an efficient and high throughput analyzer. To facilitate the operation of. In addition, the present invention places various stations associated with nucleic acid assays on a single platform included, thereby achieving efficient space utilization.</p><p num="0021"> Other objects, features, and features of the invention, including the functions and interrelationships of operating methods and structural elements, will be further clarified by reviewing the following description and the accompanying claims with reference to the accompanying drawings. However, all of these form part of this disclosure, and similar reference numbers specify the corresponding parts in the various figures.<u style="single">The present invention provides, for example,:</u><u style="single">(Item 1)</u><u style="single"> A method for treating the contents of the reaction vessel in order to quantitatively determine the amount of the target nucleic acid present in each of the plurality of reaction vessels.</u><u style="single"> a) An automatic step in which a plurality of reaction vessels are continuously provided to a heater, and the presence of a target sequence or a complement thereof contained in the target nucleic acid is determined in each reaction vessel under the temperature conditions of the heater. Steps under which sufficient reagents are provided to amplify and detect, and</u><u style="single"> b) Automatically measure the amount of signal associated with the presence of the amplification product containing the target sequence and its complement in each of the reaction vessels provided to the heater according to step a). Steps and</u><u style="single"> c) Based on the above measurement results obtained in step b), an automatic step of quantifying the amount of the target nucleic acid present in each reaction vessel, and an automatic step.</u><u style="single"> Including,</u><u style="single"> At least a portion of the reaction vessel is continuously provided to the heater according to step a) as the contents of at least a portion of the reaction vessel are processed according to steps b) to c).</u><u style="single"> Method.</u><u style="single">(Item 2)</u><u style="single"> The method of item 1, wherein the reaction vessel is provided to the heater using an automated reaction vessel transfer system.</u><u style="single">(Item 3)</u><u style="single"> The reaction vessel having the contents processed according to steps a) to c) is when the reaction vessel having the contents to be processed according to steps a) to c) is continuously provided to the heater. The method according to item 2, wherein the automatic reaction vessel transfer system is used to continuously remove the heat from the heater.</u><u style="single">(Item 4)</u><u style="single"> The method according to any one of items 2 to 3, wherein the automatic reaction vessel transfer system includes a first rotatable transfer mechanism for providing the reaction vessel to the heater.</u><u style="single">(Item 5)</u><u style="single"> The method of item 4, wherein the automated reaction vessel transfer system comprises a second rotatable transfer mechanism for removing the reaction vessel from the heater.</u><u style="single">(Item 6)</u><u style="single"> The method according to any one of items 1 to 5, wherein the heater includes a reaction vessel carrier that can rotate around a rotating shaft.</u><u style="single">(Item 7)</u><u style="single"> The heater is a closed incubator, wherein the closed incubator has one or more doors for storing or removing the reaction vessel from any of items 1 to 6. The method described in item 1.</u><u style="single">(Item 8)</u><u style="single"> 7. The method of item 7, wherein the incubator comprises a rotatable reaction vessel carrier contained within a fixed housing for accommodating and moving the reaction vessel within the incubator.</u><u style="single">(Item 9)</u><u style="single"> The method according to any one of items 1 to 8, wherein the amplification and detection reagent is provided to the reaction vessel after the reaction vessel is provided to the heater.</u><u style="single">(Item 10)</u><u style="single"> The amplification reagent is a nucleic acid polymerase capable of extending 3 ́-ends of the primer or promoter primer, nucleoside triphosphate, and the primer or promoter primer hybridized to the target nucleic acid under the temperature conditions of the heater. The method according to any one of items 1 to 9, including.</u><u style="single">(Item 11)</u><u style="single"> The method according to any one of items 1 to 10, wherein the above temperature conditions are isothermal.</u><u style="single">(Item 12)</u><u style="single"> The detection reagent provides a self-hybridizing, detectable probe capable of binding to the target sequence or complement thereof under the temperature conditions of the heater, thereby emitting the signal. The method according to any one of items 1 to 11, including.</u><u style="single">(Item 13)</u><u style="single"> The method of item 12, wherein the probe is a molecular beacon or molecular torch and comprises an interacting label pair.</u><u style="single">(Item 14)</u><u style="single"> The method of item 13, wherein the label pair comprises a fluorescent probe and a quencher.</u><u style="single">(Item 15)</u><u style="single"> The method according to any one of items 1 to 13, further comprising the step of simultaneously measuring the signal from each of the plurality of reaction vessels.</u><u style="single">(Item 16)</u><u style="single"> 15. The method of item 15, wherein the signal is measured by a plurality of isolated fluorescence signal detectors associated with the heater so that it can operate.</u><u style="single">(Item 17)</u><u style="single"> The method of any one of items 1 to 16, further comprising the step of simultaneously measuring different signals in each of the plurality of reaction vessels, wherein each of the different signals is associated with a different amplification product. How to be.</u><u style="single">(Item 18)</u><u style="single"> 17. The method of item 17, wherein the different signals are measured by a plurality of isolated fluorescence signal detectors that are operably associated with and relative to the heater.</u><u style="single">(Item 19)</u><u style="single"> Prior to step a), the reagents and conditions sufficient to remove the amplification inhibitor from the reaction vessel further include an automated step of exposing the sample material present in each of the reaction vessels. , The method according to any one of items 1 to 18.</u><u style="single">(Item 20)</u><u style="single"> The above exposure steps</u><u style="single"> A step of providing a solid support material to each reaction vessel, wherein the solid support material can bind the target nucleic acid, and a step of providing the solid support material.</u><u style="single"> The step of isolating the solid support material in each reaction vessel,</u><u style="single"> The step of removing at least a part of the sample substance from each reaction vessel,</u><u style="single"> The step of cleaning the solid support material with a cleaning liquid and</u><u style="single"> including,</u><u style="single"> The method described in item 19.</u><u style="single">(Item 21)</u><u style="single"> 20. The method of item 20, wherein the solid support material is magnetically reactive particles and the isolation step is performed by a magnet placed adjacent to the reaction vessel.</u><u style="single">(Item 22)</u><u style="single"> The method of any one of items 19-21, further comprising the step of transferring the reaction vessel to the heater using an automated reaction vessel transfer system after the exposure step.</u><u style="single">(Item 23)</u><u style="single"> 22. The method of item 22, wherein the automated reaction vessel transfer system comprises a rotatable transfer mechanism.</u><u style="single">(Item 24)</u><u style="single"> The method according to any one of items 1 to 23, wherein each reaction vessel is one of a plurality of integrally formed reaction vessels.</u><u style="single">(Item 25)</u><u style="single"> The method according to any one of items 1 to 24, wherein the steps of this method are all performed within the housing of the built-in analyzer unit.</u><u style="single">(Item 26)</u><u style="single"> A system for processing the contents of the reaction vessel in order to quantitatively determine the amount of the target nucleic acid present in each of the plurality of reaction vessels.</u><u style="single"> (A) A temperature-controlled incubator</u><u style="single"> (1) A housing that defines an incubation chamber therein and for allowing movement of the reaction vessel to or from the incubation chamber through a reaction vessel reach opening, one or more of the above. A housing with a reaction vessel reach opening and</u><u style="single"> (2) A heat source that communicates with the incubation chamber</u><u style="single"> (3) A reaction vessel carrier including a plurality of reaction vessel stations arranged in the incubation chamber, each of the reaction vessel stations being configured and arranged to transport at least one reaction vessel, and said reaction vessel. The carrier is configured and arranged to present any of the plurality of reaction vessel stations at the reaction vessel transmission position with respect to the reaction vessel reach opening.</u><u style="single"> With an incubator and</u><u style="single"> (B) With a reaction vessel transfer system comprising at least one transfer mechanism configured and arranged to transfer the reaction vessel through the reaction vessel reach opening to or from the reaction vessel carrier. ,</u><u style="single"> (C) The signal measuring device configured and arranged to measure the amount of signal emitted by the contents of the reaction vessel placed in close proximity to the signal measuring device. The measuring device is continuous so that when the reaction vessel carrier moves the reaction vessel in the incubation chamber, the reaction vessel transported by the reaction vessel carrier can operate in close proximity to the signal measuring device. Placed against the reaction vessel carrier so that it can be moved</u><u style="single"> The reaction vessel carrier moves the reaction vessel in the incubation chamber so that the reaction vessel transported thereby is placed continuously and periodically so that it can operate in close proximity to the signal measuring device. Controlled, the signal measuring device is controlled to produce a periodic measurement result of the amount of signal emitted by the contents of the reaction vessel placed in close proximity to the signal measuring device. Ru,</u><u style="single"> Signal measuring device and</u><u style="single"> (D) A microprocessor adapted to quantify the amount of the nucleic acid of interest in the reaction vessel based on the periodic measurement results produced by the signal measuring device.</u><u style="single"> System with.</u><u style="single">(Item 27)</u><u style="single"> 26. The system of item 26, further comprising a signal measuring device transfer mechanism, configured and arranged to move the signal measuring device relative to the reaction vessel carrier.</u><u style="single"> Each reaction vessel station of the reaction vessel carrier is configured and arranged to carry more than one reaction vessel, and the reaction vessel carrier is continuous with the reaction vessel station at a signal measurement position relative to the signal measurement apparatus. The signal measuring device transfer mechanism moved the signal measuring device with respect to the reaction vessel transporting device, and was transported to the reaction vessel station moved to the signal measuring position. The signal measuring device is controlled to be continuously placed so that it can operate in close proximity to each of the reaction vessels.</u><u style="single"> system.</u><u style="single">(Item 28)</u><u style="single"> The housing includes a signal measurement opening, and the signal measurement device measures the amount of signal emitted by the contents of the reaction vessel in the incubation chamber through the signal measurement opening outside the incubation chamber. The system according to any one of items 26 to 27, which is placed in a position for measurement.</u><u style="single">(Item 29)</u><u style="single"> The system according to any one of items 26 to 28, comprising one or more signal measuring devices.</u><u style="single"> Each signal measuring device is configured and arranged to measure different signals emitted by the contents of the reaction vessel.</u><u style="single"> system.</u><u style="single">(Item 30)</u><u style="single"> 29. The system of item 29, further comprising a signal measuring device transfer mechanism configured and arranged to move the signal measuring device with respect to the reaction vessel carrier.</u><u style="single"> Each reaction vessel station of the reaction vessel carrier is configured and arranged to carry more than one reaction vessel, and the reaction vessel carrier is continuous with the reaction vessel station at a signal measurement position relative to the signal measurement apparatus. Configured and arranged as presented in, the signal measuring device transfer mechanism moves each of the signal measuring devices with respect to the reaction vessel carrier and moves to the signal measuring position corresponding to the signal measuring device. The signal measuring device is configured and arranged to be continuously placed so that it can operate in close proximity to each of the reaction vessels transported to the reaction vessel station.</u><u style="single"> system.</u><u style="single">(Item 31)</u><u style="single"> Each signal measuring device comprises a fluorometer configured and arranged to measure fluorescence emission from the contents of the reaction vessel, and each fluorescence meter measures fluorescence emission with different wavelengths, items 29-30. The system described in any one of the items up to.</u><u style="single">(Item 32)</u><u style="single"> Each fluorometer</u><u style="single"> Excitation components configured and arranged to direct excitation energy to the reaction vessel, wherein the excitation component of each fluorometer directs excitation energy of different wavelengths to the reaction vessel.</u><u style="single"> A detection component configured and arranged to detect the amount of emitted energy from the contents of the reaction vessel, wherein the detection component of each fluorometer detects emitted energy of different wavelengths. Elements and</u><u style="single">To prepare</u><u style="single"> The system described in item 31.</u><u style="single">(Item 33)</u><u style="single"> The reaction vessel carrier includes a substantially circular turntable mounted in the incubation chamber so that it can rotate around the rotation axis, and the reaction vessel station is placed around the periphery of the turntable. The system according to any one of items 26 to 32.</u><u style="single">(Item 34)</u><u style="single"> 33. The system of item 33, further comprising a signal measuring device transfer mechanism configured and arranged to move the signal measuring device with respect to the reaction vessel carrier.</u><u style="single"> Each reaction vessel station of the reaction vessel carrier is configured and arranged to transport more than one reaction vessel arranged radially with respect to the rotation axis of the turntable. , The reaction vessel station is configured and arranged to continuously present at the signal measurement position with respect to the signal measurement device, and the signal measurement device transfer mechanism attaches the signal measurement device to the rotation axis of the turntable. The signal measuring device is continuously placed so that the reaction vessel can be moved in the substantially radial direction and operated in close proximity to each of the reaction vessels transported to the reaction vessel station moved to the signal measuring position. Configured and placed,</u><u style="single"> system.</u><u style="single">(Item 35)</u><u style="single"> 34. The system of item 34, wherein each reaction vessel station comprises a radially oriented end open slot formed within the turntable.</u><u style="single">(Item 36)</u><u style="single"> The system according to any one of items 26 to 28, wherein the signal measuring device comprises a fluorometer configured and arranged to measure fluorescence emission from the contents of the reaction vessel.</u><u style="single">(Item 37)</u><u style="single"> The above fluorometer</u><u style="single"> Excitation components configured and arranged to direct excitation energy to the reaction vessel.</u><u style="single"> Detection components configured and arranged to detect the amount of energy released from the contents of the reaction vessel.</u><u style="single">To prepare</u><u style="single"> The system described in item 36.</u><u style="single">(Item 38)</u><u style="single"> The excitation component includes an excitation light housing and an excitation lens housing, the detection component includes a detection lens housing, the excitation light housing and the detection lens housing are at relative positions separated from each other, and the excitation lens housing is described above. The system according to any one of items 32 or 37, which extends between the excitation light housing and the detection lens housing.</u><u style="single">(Item 39)</u><u style="single"> The above fluorometer</u><u style="single"> With a light emitting part adapted to emit excitation light,</u><u style="single"> The optical element that defines the first optical path, the second optical path, and the third optical path,</u><u style="single"> Beam splitter and</u><u style="single"> Equipped with a light detector</u><u style="single"> The light emitting section is arranged so as to be operable with respect to the first optical path, and the optical path is configured and arranged to send at least a part of the excitation light emitted by the light emitting section.</u><u style="single"> The beam splitter is placed so as to be operable with respect to the first optical path and the second optical path, and receives the excitation light sent by the first optical path to obtain a desired excitation spectrum component. It is configured and arranged so that at least a part of the received excitation light having the light is directed to the second optical path.</u><u style="single"> The second optical path is placed relative to the reaction vessel so that the second optical path can operate in close proximity to the fluorometer so that the light having the desired excitation spectral component is directed at the reaction vessel. The second optical path receives at least a part of arbitrary fluorescent light emitted by the contents of the reaction vessel, and directs the received portion toward the beam splitter.</u><u style="single"> The beam splitter is placed so as to be operable with respect to the third optical path, and receives the fluorescent light sent by the second optical path, and has a desired excitation spectral component of the received light. At least partly configured and arranged to direct the third optical path above,</u><u style="single"> The photodetector is arranged to operate with respect to the third optical path, which is configured to send at least a portion of the light having the desired emission spectral component towards the photodetector. Be placed,</u><u style="single"> The system according to any one of items 31 to 38.</u><u style="single">(Item 40)</u><u style="single"> 39. The system of item 39, wherein the optical element of the first optical path is further configured and arranged to substantially eliminate any undesired spectral components of light transmitted from the light emitting section.</u><u style="single">(Item 41)</u><u style="single"> In the optical element of the first optical path, the light received from the first optical path by the beam splitter is substantially within a predetermined excitation angle range with respect to the optical axis of the first optical path. The system according to any one of items 39 to 40, further configured and arranged to adjust the light sent from the light emitting section.</u><u style="single">(Item 42)</u><u style="single"> The optical element of the first optical path is</u><u style="single"> In order to receive at least a part of the excitation light emitted by the light emitting unit and send the excitation light received from the light receiving end to the opposite end of the optical pipe, the light emitting unit can be operated. The optical pipe having the light receiving end and the light pipe, which is placed,</u><u style="single"> A mirror arranged and oriented to divert the light emitted by the opposite end of the optical pipe.</u><u style="single"> One or more lenses arranged to receive light redirected by the mirror and configured and arranged to collimate the received light within a substantially defined excitation angle range.</u><u style="single"> Excitation baffles that are placed to receive light collimated by one or more of the above lenses and are configured and arranged to block virtually all light that is not within the defined excitation angle range.</u><u style="single"> An excitation filter that is placed to receive light passing through the baffle and is configured and arranged to substantially remove the unwanted spectral component of the excitation light sent from the light emitting section.</u><u style="single"> To prepare</u><u style="single"> The system according to any one of items 39 to 41.</u><u style="single">(Item 43)</u><u style="single"> Items 39 to 42, wherein the optical element of the third optical path is configured and arranged to eliminate at least a portion of any unwanted spectral component of light sent towards the photodetector. The system described in any one paragraph.</u><u style="single">(Item 44)</u><u style="single"> In the optical element of the third optical path, the light received from the third optical path by the light detection unit is substantially within a predetermined detection angle range with respect to the optical axis of the third optical path. The system according to any one of items 39 to 43, which is further configured and arranged to adjust the light sent from the beam splitter.</u><u style="single">(Item 45)</u><u style="single"> The optical element of the third optical path is</u><u style="single"> An emission baffle that is placed to receive light from the beam splitter and is configured and arranged to block virtually all light that is not within the specified detection angle range.</u><u style="single"> An emission filter arranged to receive light passing through the emission baffle and configured and arranged to substantially remove the unwanted spectral component of the light sent towards the photodetector.</u><u style="single"> With at least one lens configured and arranged to focus the light passing through the emission filter towards the photodetector.</u><u style="single"> To prepare</u><u style="single"> The system according to any one of items 39 to 44.</u><u style="single">(Item 46)</u><u style="single"> The system according to any one of items 39 to 45, wherein the light emitting unit and the photodetecting unit are connected to a circuit board.</u><u style="single">(Item 47)</u><u style="single"> The system according to any one of items 39 to 46, wherein the beam splitter comprises a dichroic beam splitter.</u><u style="single">(Item 48)</u><u style="single"> The first, second, and third optical paths include the first, second, and third optical axes, respectively, and (a) the optical axis is substantially perpendicular to the second optical axis. Yes, (b) the second and third optical axes are substantially coaxial, and (c) the dichroic beam splitter is at an angle of about 45 degrees with respect to the first, second, and third optical axes. The system according to item 47, which is oriented.</u><u style="single">(Item 49)</u><u style="single"> Any of items 26-48 further comprising a signal detector configured and arranged to detect the signal emitted by the contents of the reaction vessel placed in close proximity to the signal detector. The system described in paragraph 1</u><u style="single"> The signal detector is placed outside the incubation chamber and</u><u style="single"> The reaction vessel transfer system is further configured and arranged to continuously transfer the reaction vessel to the signal detector following incubation of the reaction vessel in the incubator.</u><u style="single">(Item 50)</u><u style="single"> 49. The system of item 49, wherein the signal detector comprises an illuminometer configured and arranged to detect chemiluminescent emissions from the contents of the reaction vessel.</u><u style="single">(Item 51)</u><u style="single"> Each of the reaction vessel carriers further comprises a magnetic partition located adjacent to one of the reaction vessel stations, each of which exposes the reaction vessel transported to the adjacent reaction vessel station to a magnetic field. The system according to any one of items 26 to 50, comprising one or more magnetic elements for the purpose.</u><u style="single">(Item 52)</u><u style="single"> Process the contents of multiple reaction vessels to determine the amount of nucleic acid of interest present in one or more reaction vessels, or to determine the presence or absence of nucleic acid of interest in one or more of the reaction vessels. System for</u><u style="single"> (A) At least one temperature-controlled incubator comprising the above-mentioned incubation chamber for accommodating the reaction vessel and exposing the reaction vessel to the controlled temperature conditions in the incubation chamber.</u><u style="single"> (B) Nucleic acid quantification system</u><u style="single"> (1) Periodically measuring the amount of signal emitted by the contents of each reaction vessel,</u><u style="single"> (2) Quantifying the amount of target nucleic acid present in each reaction vessel based on the periodic measurement results, and</u><u style="single">And a nucleic acid quantification system configured and arranged to determine the amount of the nucleic acid of interest in the reaction vessel.</u><u style="single"> (C) Nucleic acid detection system</u><u style="single"> (1) To detect the signal emitted by the contents of each reaction vessel,</u><u style="single"> (2) Confirming the presence or absence of the target nucleic acid in the reaction vessel based on whether or not a signal exceeding the threshold level is detected.</u><u style="single">A nucleic acid detection system configured and arranged to confirm the presence of the nucleic acid of interest in the reaction vessel.</u><u style="single"> System with.</u><u style="single">(Item 53)</u><u style="single"> Item 52, further comprising an automated transfer system configured and arranged to automatically transfer the reaction vessel to or from the nucleic acid quantification system and automatically transfer the reaction vessel to or from the nucleic acid detection system. Described system.</u><u style="single">(Item 54)</u><u style="single"> 53. The system of item 53, wherein the transfer system comprises a transfer mechanism that is rotatable around a rotating shaft.</u><u style="single">(Item 55)</u><u style="single"> The nucleic acid quantification system is configured and arranged to measure the amount of signal emitted by the contents of a reaction vessel placed in the incubation chamber and placed in close proximity to the signal measuring device. Equipped with a signal measuring device</u><u style="single"> The nucleic acid detection system is such that it detects the signal emitted by the contents of a reaction vessel that is placed in a detection chamber different from the incubation chamber and placed in close proximity to the signal detector. Configured and arranged, equipped with a signal detector,</u><u style="single"> The system according to any one of items 52 to 54.</u><u style="single">(Item 56)</u><u style="single"> 55. The system of item 55, further comprising a signal measuring device transfer mechanism configured and arranged to operably move the signal measuring device in close proximity to a reaction vessel placed in the incubation chamber. ..</u><u style="single">(Item 57)</u><u style="single"> 56. The system of item 56, wherein the signal measuring device comprises a fluorometer configured and arranged to measure fluorescence emission from the contents of the reaction vessel.</u><u style="single">(Item 58)</u><u style="single"> The system according to any one of items 55 to 57, wherein the signal detector comprises an illuminometer configured and arranged to detect chemiluminescent emissions from the contents of the reaction vessel.</u><u style="single">(Item 59)</u><u style="single"> The system according to any one of items 55 to 58, wherein the nucleic acid quantification system and the nucleic acid detection system are contained in a housing.</u><u style="single">(Item 60)</u><u style="single"> The system according to any one of items 55 to 59, wherein the signal measuring device and the signal detecting device are located on a processing desk in the housing.</u><u style="single">(Item 61)</u><u style="single"> The system of item 60, wherein the housing defines a built-in stand-alone analyzer unit.</u><u style="single">(Item 62)</u><u style="single"> 61. The system of item 61, wherein the analyzer unit is mobile.</u><u style="single">(Item 63)</u><u style="single"> The temperature-controlled incubator includes a reaction vessel carrier that is located in the incubation chamber and includes a plurality of reaction vessel stations, each of which is configured and arranged to carry at least one reaction vessel. Being done</u><u style="single"> The nucleic acid quantification system comprises a signal measuring device configured and arranged to measure the amount of signal emitted by the contents of the reaction vessel placed in close proximity to the signal measuring device. The signal measuring device is provided so that when the reaction vessel carrier moves the reaction vessel in the incubation chamber, the reaction vessel transported to the reaction vessel carrier can operate in close proximity to the signal measuring device. Placed against the reaction vessel carrier so that it is continuously moved to</u><u style="single"> The reaction vessel carrier is controlled to move the reaction vessel in the incubation chamber and place the reaction vessel transported by the reaction vessel continuously and periodically so as to be able to operate in close proximity to the signal measuring device. The signal measuring device is controlled to produce a periodic measurement result of the amount of signal emitted by the contents of the reaction vessel placed in close proximity to the signal measuring device.</u><u style="single"> The system described in item 52.</u><u style="single">(Item 64)</u><u style="single"> Item 63, the nucleic acid quantification system further comprises a microprocessor adapted to quantify the amount of the nucleic acid of interest in the reaction vessel based on the results of periodic measurements made by the signal measuring device. The system described in.</u><u style="single">(Item 65)</u><u style="single"> The above nucleic acid quantification system</u><u style="single"> Further comprising a signal measuring device transfer mechanism configured and arranged to move the signal measuring device with respect to the reaction vessel carrier.</u><u style="single"> Each reaction vessel station of the reaction vessel carrier is configured and arranged to carry more than one reaction vessel, and the reaction vessel carrier is continuous with the reaction vessel station at a signal measurement position relative to the signal measurement apparatus. The signal measuring device transfer mechanism moves the signal measuring device with respect to the reaction vessel transporting device, and is transported to the reaction vessel station moved to the signal measuring position. The signal measuring device is controlled to be continuously placed so that it can operate in close proximity to each of the reaction vessels.</u><u style="single"> The system described in item 63 or 64.</u><u style="single">(Item 66)</u><u style="single"> 63 or 64, wherein the nucleic acid quantification system comprises more than one signal measuring device, each signal measuring device being configured and arranged to measure different signals emitted by the contents of the reaction vessel. System.</u><u style="single">(Item 67)</u><u style="single"> The above nucleic acid quantification system</u><u style="single"> Further comprising a signal measuring device transfer mechanism configured and arranged to move the signal measuring device with respect to the reaction vessel carrier.</u><u style="single"> Each reaction vessel station of the reaction vessel carrier is configured and arranged to carry more than one reaction vessel, and the reaction vessel carrier is continuous with the reaction vessel station at a signal measurement position relative to the signal measurement apparatus. The signal measuring device transfer mechanism moves each of the signal measuring devices with respect to the reaction vessel carrier to the signal measuring position corresponding to the signal measuring device. The signal measuring device is configured and arranged to be continuously placed so that it can operate in close proximity to each of the reaction vessels transported to the moved reaction vessel station.</u><u style="single"> The system according to item 66.</u><u style="single">(Item 68)</u><u style="single"> The reaction vessel carrier includes a substantially circular turntable mounted in the incubation chamber so that it can rotate around a rotation axis, and the reaction vessel station is located around the periphery of the turntable. The system according to any one of items 63 to 67, which is placed.</u><u style="single">(Item 69)</u><u style="single"> The above nucleic acid quantification system</u><u style="single"> Further comprising a signal measuring device transfer mechanism configured and arranged to move the signal measuring device with respect to the reaction vessel carrier.</u><u style="single"> Each reaction vessel station of the reaction vessel carrier is configured and arranged to transport more than one reaction vessel arranged radially with respect to the rotation axis of the turntable. , The reaction vessel station is configured and arranged to continuously present at the signal measurement position with respect to the signal measurement device, and the signal measurement device transfer mechanism attaches the signal measurement device to the rotation axis of the turntable. The signal measuring device is continuously placed so that the reaction vessel can be moved in the substantially radial direction and operated in close proximity to each of the reaction vessels transported to the reaction vessel station moved to the signal measuring position. Configured and placed,</u><u style="single"> The system described in item 68.</u><u style="single">(Item 70)</u><u style="single"> The system according to any one of items 63 to 69, wherein the signal measuring device comprises a fluorometer configured and arranged to measure fluorescence emission from the contents of the reaction vessel.</u><u style="single">(Item 71)</u><u style="single"> The above fluorometer</u><u style="single"> With a light emitting part adapted to emit excitation light,</u><u style="single"> The optical element that defines the first optical path, the second optical path, and the third optical path,</u><u style="single"> Beam splitter and</u><u style="single"> Light detector and</u><u style="single"> With</u><u style="single"> The light emitting section is arranged so as to be operable with respect to the first optical path, and the optical path is configured and arranged to send at least a part of the excitation light emitted by the light emitting section.</u><u style="single"> The beam splitter is placed so as to be operable with respect to the first optical path and the second optical path, and receives the excitation light sent by the first optical path to obtain a desired excitation spectrum component. It is configured and arranged so that at least a part of the received excitation light having the light is directed to the second optical path.</u><u style="single"> The second optical path is placed relative to the reaction vessel so that the second optical path can operate in close proximity to the fluorometer so that the light having the desired excitation spectral component is directed at the reaction vessel. The second optical path receives at least a part of arbitrary fluorescent light emitted by the contents of the reaction vessel, and directs the received portion toward the beam splitter.</u><u style="single"> The beam splitter is placed so as to be operable with respect to the third optical path, and receives the fluorescent light sent by the second optical path, and has a desired emission spectrum component of the received light. At least partly configured and arranged to direct the third optical path above,</u><u style="single"> The photodetector is arranged to operate with respect to the third optical path, which is configured to send at least a portion of the light having the desired emission spectral component towards the photodetector. Be placed,</u><u style="single"> The system described in item 70.</u><u style="single">(Item 72)</u><u style="single"> The system of item 71, wherein the optical element of the first optical path is further configured and arranged to substantially eliminate any unwanted spectral components of light sent from the light emitting section.</u><u style="single">(Item 73)</u><u style="single"> In the optical element of the first optical path, the light received from the first optical path by the beam splitter is substantially within a predetermined excitation angle range with respect to the optical axis of the first optical path. 72. The system of item 72, further configured and arranged to regulate the light sent from the light emitting section.</u><u style="single">(Item 74)</u><u style="single"> The optical element of the first optical path is</u><u style="single"> To be able to operate with respect to the light emitting unit so as to receive at least a part of the excitation light emitted by the light emitting unit and send the excitation light received from the light receiving end to the opposite end of the optical pipe. With the optical pipe having the light receiving end placed,</u><u style="single"> A mirror arranged and oriented to divert the light emitted by the opposite end of the optical pipe.</u><u style="single"> One or more lenses arranged to receive light redirected by the mirror and configured and arranged to collimate the received light within a substantially defined excitation angle range.</u><u style="single"> Excitation baffles that are placed to receive light collimated by one or more of the above lenses and are configured and arranged to block virtually all light that is not within the defined excitation angle range.</u><u style="single"> An excitation filter that is placed to receive light passing through the baffle and is configured and arranged to substantially remove the unwanted spectral component of the excitation light sent from the light emitting section.</u><u style="single"> To prepare</u><u style="single"> The system described in item 73.</u><u style="single">(Item 75)</u><u style="single"> Items 71 to 74, wherein the optical element of the third optical path is configured and arranged to eliminate at least a portion of any unwanted spectral component of light sent towards the photodetector. The system described in any one paragraph.</u><u style="single">(Item 76)</u><u style="single"> In the optical element of the third optical path, the light received from the third optical path by the light detection unit is substantially within a predetermined detection angle range with respect to the optical axis of the third optical path. 75. The system of item 75, which is further configured and arranged to adjust the light sent from the beam splitter.</u><u style="single">(Item 77)</u><u style="single"> The optical element of the third optical path is</u><u style="single"> An emission baffle that is placed to receive light from the beam splitter and is configured and arranged to block virtually all light that is not within the specified detection angle range.</u><u style="single"> An emission filter arranged to receive light passing through the emission baffle and configured and arranged to substantially remove the unwanted spectral component of the light sent towards the photodetector.</u><u style="single"> With at least one lens configured and arranged to focus the light passing through the emission filter towards the photodetector.</u><u style="single"> To prepare</u><u style="single"> The system described in item 76.</u><u style="single">(Item 78)</u><u style="single"> The system according to any one of items 71 to 77, wherein the light emitting unit and the photodetecting unit are connected to a circuit board.</u><u style="single">(Item 79)</u><u style="single"> The system according to any one of items 71 to 78, wherein the beam splitter comprises a dichroic beam splitter.</u><u style="single">(Item 80)</u><u style="single"> The first, second, and third optical paths include the first, second, and third optical axes, respectively. (A) The first optical axis is abbreviated with respect to the second optical axis. It is vertical, (b) the second and third optical axes are substantially coaxial, and (c) the dichroic beam splitter is about 45 degrees with respect to the first, second, and third optical axes. The system according to item 79, which is oriented at an angle.</u><u style="single">(Item 81)</u><u style="single"> The above fluorometer</u><u style="single"> Excitation components configured and arranged to direct excitation energy towards the reaction vessel,</u><u style="single"> Detection components configured and arranged to detect the amount of energy released from the contents of the reaction vessel.</u><u style="single"> To prepare</u><u style="single"> The system according to any one of items 70 to 80.</u><u style="single">(Item 82)</u><u style="single"> The above nucleic acid detection system</u><u style="single"> A signal detector that is configured and arranged to detect signals emitted by the contents of a reaction vessel placed in close proximity to the signal detector and placed outside the incubation chamber. The signal detector</u><u style="single"> Prepare</u><u style="single"> The system according to any one of items 52 to 54 or items 63 to 81.</u><u style="single">(Item 83)</u><u style="single"> 82. The system of item 82, wherein the signal detector comprises an illuminometer configured and arranged to detect chemiluminescent emissions from the contents of the reaction vessel.</u><u style="single">(Item 84)</u><u style="single"> To transfer the reaction vessel to or from the reaction vessel carrier through the reaction vessel reach opening provided in the incubator, and following incubation of the reaction vessel in the incubator, said. Further comprising a reaction vessel transfer system, including at least one transfer mechanism configured and arranged to continuously transfer the reaction vessel to the signal detector.</u><u style="single"> The system according to any one of items 70 to 83.</u><u style="single">(Item 85)</u><u style="single"> Each of the reaction vessel carriers further comprises a magnetic partition located adjacent to one of the reaction vessel stations, each of which exposes the reaction vessel transported to the adjacent reaction vessel station to a magnetic field. The system according to any one of items 63 to 84, comprising one or more magnetic elements for the purpose.</u><u style="single">(Item 86)</u><u style="single"> The above signal detection device</u><u style="single"> A structure in which a plurality of reaction vessels arranged adjacent to each other define a transfer path to be moved by the apparatus.</u><u style="single"> The photosensitizer, which is arranged along the transfer path and configured and arranged to detect light emitted from the contents of the reaction vessel placed so as to be operable with respect to the photosensitizer.</u><u style="single"> Placed adjacent to the transfer path, (1) a first position that allows the adjacently placed reaction vessel to move along the transfer path, and (2) placed on the transfer path. Isolation of a reaction vessel configured and arranged to swivel between a second position operably engaged with one of the reaction vessels placed operably with respect to the photosensitizer. The apparatus, wherein the reaction vessel isolator detects light from a source other than the operably placed reaction vessel engaged by the reaction vessel isolate by the photosensitizer. Configured and arranged to substantially prevent, the reaction vessel isolator is configured and arranged to swivel between the first and second positions, and the vessel tank isolate is configured. When in the second position, the reaction vessel isolator comprises a structure that at least partially surrounds the reaction vessel engaged by the reaction vessel isolator.</u><u style="single"> To prepare</u><u style="single"> The system according to any one of items 82-85.</u><u style="single">(Item 87)</u><u style="single"> The signal detector is an adjacent reaction for a period of time sufficient for the photosensitizer to detect the amount of light emitted from the contents of the reaction vessel placed so that it can operate. A transfer mechanism configured and arranged to move the plurality of reaction vessels arranged adjacent to each other along the transfer path in such a manner that each of the containers is placed so as to be operable with respect to the photosensitive device. The system according to item 86, further comprising.</u><u style="single">(Item 88)</u><u style="single"> The signal detection device further comprises an opening panel having an opening formed therein, which is arranged adjacent to the transfer path.</u><u style="single"> The above-mentioned photosensitive device is</u><u style="single"> A photomultiplier tube that detects light emitted from an object placed in front of a light intake opening at one end of the photomultiplier tube, and light detected by the photomultiplier tube. A photomultiplier tube adapted to generate an electronic signal indicating that the opening is placed to receive light emitted from the contents of the reaction vessel placed on the transfer path in front of the opening. A photomultiplier tube placed on the side of the opening panel opposite the transfer path having the light uptake opening.</u><u style="single"> A shutter assembly of the shutter assembly that is mounted on the opening panel and between an open position that allows light to pass through the opening and a closed position that prevents light from passing through the opening. With a shutter assembly configured and arranged to selectively take light into the photoelectron multiplier through the aperture formed in the aperture panel by movement.</u><u style="single"> With</u><u style="single"> The above shutter assembly</u><u style="single"> The shutters are configured and arranged for rotational movement between the open and closed positions corresponding to the open and closed positions of the shutter assembly, respectively, and when the shutter is in the closed position, the opening is opened. A shutter that blocks and does not block the opening when the shutter is in the open position,</u><u style="single"> A motor operably connected to the shutter to provide power rotation of the shutter between the open and closed positions.</u><u style="single"> To prepare</u><u style="single"> The system described in item 86 or 87.</u><u style="single">(Item 89)</u><u style="single"> The system according to any one of items 52 to 88, further comprising a separation device configured and arranged to isolate a solid support material for binding the nucleic acid of interest present in the reaction vessel.</u><u style="single">(Item 90)</u><u style="single"> 89. The system of item 89, wherein the separator comprises a fluid suction mechanism configured and arranged to suck fluid material from the reaction vessel.</u><u style="single">(Item 91)</u><u style="single"> The above separation device</u><u style="single"> A fluid dispensing mechanism configured and arranged to provide a cleaning solution to the reaction vessel after removing the fluid sample from the reaction vessel.</u><u style="single"> A mixing device configured and arranged to agitate the reaction vessel to resuspend the solid support material after the cleaning solution has been provided by the fluid dispensing mechanism.</u><u style="single"> Further prepare,</u><u style="single"> The system described in item 90.</u><u style="single">(Item 92)</u><u style="single"> The system according to any one of items 89 to 9, wherein the separation device comprises a magnetic element for placing a fluid substance in a magnetic field.</u><u style="single">(Item 93)</u><u style="single"> The system according to any one of items 52 to 92, wherein each reaction vessel is one of a plurality of integrally formed reaction vessels.</u><u style="single">(Item 94)</u><u style="single"> A system for measuring signals emitted by the contents of each of a plurality of reaction vessels.</u><u style="single"> A movable reaction vessel carrier including a plurality of reaction vessel stations, each of which transports two or more reaction vessels.</u><u style="single"> The signal measuring device configured and arranged to measure the amount of signal emitted by the contents of the reaction vessel placed in close proximity to the signal measuring device, the reaction vessel carrier. The signal measuring device is moved so that the reaction container transported to the reaction container carrier can be continuously moved so as to be able to operate in close proximity to the signal measuring device when the reaction vessel is moved. A signal measuring device and a signal measuring device placed on the reaction vessel carrier,</u><u style="single"> A signal measuring device transfer mechanism configured and arranged to move the signal measuring device with respect to the reaction vessel carrier, and a signal measuring device transfer mechanism.</u><u style="single"> With</u><u style="single"> The reaction vessel transport device continuously presents each reaction vessel station to the signal measurement position, and one of the reaction vessels transported to the reaction vessel station at the signal measurement position is close to the signal measurement device. Controlled to be operational, the signal measuring device transfer mechanism moved the signal measuring device with respect to the reaction vessel carrier and was transported to the reaction vessel station moved to the signal measurement position. The signal measuring device is controlled to be continuously placed so that it can operate in close proximity to each of the reaction vessels.</u><u style="single"> system.</u><u style="single">(Item 95)</u><u style="single"> The system according to item 94, wherein the signal measuring device is placed under the reaction vessel carrier.</u><u style="single">(Item 96)</u><u style="single"> The system according to any one of items 94 to 95, comprising one or more signal measuring devices.</u><u style="single"> Each signal measuring device is configured and arranged to measure different signals emitted by the contents of the reaction vessel.</u><u style="single"> The reaction vessel transport device is configured and arranged so as to continuously present the reaction vessel station at the signal measurement position with respect to the signal measurement device, and the signal measurement device transfer mechanism makes each of the signal measurement devices react. The signal measurement so as to be able to operate in close proximity to each of the reaction vessels transported to the reaction vessel station moved to the signal measurement position corresponding to the signal measurement device by moving with respect to the container carrier. Configured and arranged to place the device continuously,</u><u style="single"> system.</u><u style="single">(Item 97)</u><u style="single"> The system of item 96, comprising four signal measuring devices.</u><u style="single">(Item 98)</u><u style="single"> The system according to any one of items 94 to 97, wherein the signal measuring device comprises a fluorometer configured and arranged to measure fluorescence emission from the contents of the reaction vessel.</u><u style="single">(Item 99)</u><u style="single"> The above fluorometer</u><u style="single"> Excitation components configured and arranged to direct excitation energy to the reaction vessel.</u><u style="single"> Detection components configured and arranged to detect the amount of energy released from the contents of the reaction vessel.</u><u style="single"> To prepare</u><u style="single"> The system described in item 98.</u><u style="single">(Item 100)</u><u style="single"> The system according to any one of items 94 to 99.</u><u style="single"> (a) The reaction vessel carrier includes a substantially circular turntable attached so as to be rotatable around the rotation axis together with the reaction vessel station placed around the periphery of the turntable. The reaction vessel station comprises two or more reaction vessels arranged radially with respect to the rotation axis of the turntable.</u><u style="single"> (b) The rotation of the turntable continuously presents each of the reaction vessel stations to the signal measurement position, and at the signal measurement position, one of the reaction vessels arranged radially carried to the reaction vessel station , Can operate in close proximity to the above signal measuring device,</u><u style="single"> (c) The signal measuring device transfer mechanism is arranged radially to the reaction vessel station moved to the signal measuring position by moving the signal measuring device in a substantially radial direction with respect to the turntable. The signal measuring devices are configured and arranged to be continuously placed so that they can operate adjacent to each of the reaction vessels.</u><u style="single"> system.</u><u style="single">(Item 101)</u><u style="single"> The signal measuring device transfer mechanism is</u><u style="single"> A fixed plate oriented substantially perpendicular to the axis of rotation of the turntable, and</u><u style="single"> A translation assembly that is operably associated with the signal measuring device and includes a track attached to the fixing plate in a substantially radial direction with respect to the rotation axis of the turntable, wherein the signal measuring device is translated. With a translation assembly that is attached to the assembly and can be moved along the track above,</u><u style="single"> To prepare</u><u style="single"> The system described in item 100.</u><u style="single">(Item 102)</u><u style="single"> 101. The system of item 101, further comprising a mounting bracket for mounting the signal measuring device on the translation assembly.</u><u style="single"> The translation assembly further includes a bearing element to which the mounting bracket is attached, the bearing element being configured and arranged to translate along the track.</u><u style="single"> system.</u><u style="single">(Item 103)</u><u style="single"> Any one of items 101 to 102, further comprising a sensor located at a predetermined position along the track and configured and arranged to detect the presence of the signal measuring device at the predetermined position along the track. The system described in the section.</u><u style="single">(Item 104)</u><u style="single"> The signal measuring device transfer mechanism is</u><u style="single"> A cam disc rotatably mounted adjacent to the fixing plate, the tracks of the parallel moving assembly are arranged substantially radially with respect to the rotation axis of the cam disc, the cam disc including a cam slot. The cam slot is arranged on the cam disk so that the radial distance between the cam slot and the rotation axis of the cam disk varies from one end of the cam slot to the opposite end of the cam slot. Also, with a cam disc,</u><u style="single"> In order to bring about the power rotation of the cam disc, a motor connected to the cam disc so as to operate, and</u><u style="single"> An engaging pin that is associated with the signal measuring device and is engaged with the cam slot such that the power rotation of the cam disk traverses the engaging pin along the cam slot, thereby. With an engaging pin that changes the radial position of the signal measuring device according to a change in the radial distance between the rotating shaft of the cam disk and the portion of the cam slot in which the engaging pin is engaged. ,</u><u style="single"> Further prepare,</u><u style="single"> The system according to any one of items 101 to 103.</u><u style="single">(Item 105)</u><u style="single"> The cam slot includes a positioning point along the slot, which is defined by each end of the slot and one or more points along the slot where the curvature of the slot changes, and the engaging pin. 104. The signal measuring device is placed so that it can operate in close proximity to one of the reaction vessels carried to the reaction vessel station when is engaged in the slot at the positioning point. system.</u><u style="single">(Item 106)</u><u style="single"> The system according to any one of items 100 to 105, comprising one or more signal measuring devices.</u><u style="single"> Each signal measuring device is configured and arranged to measure different signals emitted by the contents of the reaction vessel.</u><u style="single"> The reaction vessel transport device is configured and arranged so as to continuously present the reaction vessel station at the signal measurement position with respect to the signal measurement device, and the signal measurement device transfer mechanism makes each of the signal measurement devices react. The signal so that it can operate in close proximity to each of the reaction vessels transported to the reaction vessel station that has been moved relative to the container carrier and moved to the signal measurement position corresponding to the signal measuring device. Configured and arranged to place the measuring device continuously,</u><u style="single"> system.</u><u style="single">(Item 107)</u><u style="single"> Each signal measuring device comprises a fluorometer configured and arranged to measure fluorescence emission from the contents of the reaction vessel, and each fluorescence meter measures fluorescence emission having different wavelengths, item 106. Described system.</u><u style="single">(Item 108)</u><u style="single"> Each fluorometer</u><u style="single"> Excitation components configured and arranged to direct excitation energy to the reaction vessel, wherein the excitation component of each fluorometer directs excitation energy of different wavelengths to the reaction vessel.</u><u style="single"> A detection component configured and arranged to detect the amount of emitted energy from the contents of the reaction vessel, wherein the detection component of each fluorometer detects emitted energy of different wavelengths. Elements and</u><u style="single"> To prepare</u><u style="single"> The system according to item 107.</u><u style="single">(Item 109)</u><u style="single"> The system according to any one of items 106 to 108.</u><u style="single"> (a) The reaction vessel carrier includes a substantially circular turntable attached so as to be rotatable around the rotation axis together with the reaction vessel station placed around the periphery of the turntable. The reaction vessel station comprises two or more reaction vessels arranged radially with respect to the rotation axis of the turntable.</u><u style="single"> (b) The rotation of the turntable continuously presents each of the reaction vessel stations to the signal measurement position, and at the signal measurement position, one of the reaction vessels arranged radially transported to the reaction vessel station. However, it can operate in close proximity to the signal measuring device corresponding to the signal measuring position.</u><u style="single"> (c) The signal measuring device transfer mechanism moves the signal measuring device in a substantially radial direction with respect to the rotation axis of the turntable, and is transported to the reaction vessel station moved to the signal measuring position. The signal measuring devices are configured and arranged to be continuously placed so that they can operate adjacent to each of the radially arranged reaction vessels.</u><u style="single"> system.</u><u style="single">(Item 110)</u><u style="single"> The signal measuring device transfer mechanism is</u><u style="single"> A fixed plate oriented substantially perpendicular to the axis of rotation of the turntable, and</u><u style="single"> A translation assembly operably associated with each signal measuring device, each translation assembly includes a track mounted on the fixed plate approximately radially with respect to the axis of rotation of the turntable and each signal. With the translation assembly, the measuring device is attached to the associated translation assembly and is movable along the track.</u><u style="single"> To prepare</u><u style="single"> The system according to item 109.</u><u style="single">(Item 111)</u><u style="single"> 110. The system of item 110, further comprising a mounting bracket for mounting each signal measuring device to the translation assembly.</u><u style="single"> Each translation assembly further includes a bearing element to which the mounting bracket is attached, the bearing element being configured and arranged to translate along the track of the translation assembly.</u><u style="single"> system.</u><u style="single">(Item 112)</u><u style="single"> Any one of items 110 to 111, further comprising a sensor located at a predetermined position along each track and further configured and arranged to detect the presence of the signal measuring device at the predetermined position along the track. The system described in the section.</u><u style="single">(Item 113)</u><u style="single"> The signal measuring device transfer mechanism is</u><u style="single"> A cam disc rotatably mounted adjacent to the fixing plate, with each track of the parallel moving assembly arranged substantially radially with respect to the axis of rotation of the cam disc, the cam disc having each parallel moving assembly. Each cam slot varies in radial distance between the cam slot and the rotation axis of the cam disk from one end of the cam slot to the opposite end of the cam slot. With the cam disc placed on the above cam disc,</u><u style="single"> In order to bring about the power rotation of the cam disc, a motor connected to the cam disc so as to operate, and</u><u style="single"> Engagement pins that are associated to operate with each signal measuring device and are associated with the associated cam slot such that the power rotation of the cam disk traverses the engagement pin along the cam slot. Combined, thereby the associated signal measuring device according to a change in the radial distance between the rotating shaft of the cam disk and the portion of the associated cam slot in which the engaging pin is engaged. With an engaging pin that changes the position of the</u><u style="single"> Further prepare,</u><u style="single"> The system according to any one of items 110 to 112.</u><u style="single">(Item 114)</u><u style="single"> Each cam slot includes a positioning point along the slot, which is defined by each end of the slot and by one or more points along the slot where the curvature of the slot changes. When the mating pin is engaged in the slot at the positioning point, the signal measuring device is placed so that it can operate in close proximity to one of the reaction vessels carried to the reaction vessel station, item 113. Described system.</u><u style="single">(Item 115)</u><u style="single"> The system according to any one of items 113 to 114, comprising four signal measuring devices, four translation assemblies, and four cam slots.</u><u style="single">(Item 116)</u><u style="single"> To quantitatively determine the amount of the first target nucleic acid present in the first part of the reaction vessel, and qualitatively determine the presence or absence of the second target nucleic acid present in the second part of the reaction vessel. A method for processing the contents of a plurality of said reaction vessels.</u><u style="single"> a) An automated step of continuously providing a plurality of reaction vessels to a first heater, each reaction vessel having a first or second contained in the corresponding first or second nucleic acid of interest. Sufficient amplification and enzyme reagents are provided to amplify the target sequence of the above first heater under the temperature conditions of the first heater.</u><u style="single"> b) The reaction vessel of the first part of the reaction vessel provided with sufficient detection reagent to detect the first amplification product containing the first target sequence or its complement is used as a signal measuring device. Continuously and so as to be able to operate in close proximity to the signal measuring device configured and arranged to measure the amount of signal emitted by the contents of the reaction vessel placed in close proximity. Associated with the presence of the first amplification product in each of the first parts of the reaction vessel that has been moved to operate in close proximity to the signal measuring device and the automatic steps that are moved periodically. With automatic steps to measure the amount of signal</u><u style="single"> c) Based on the measurement results obtained in step b), a microprocessor is used to quantify the amount of the first target nucleic acid present in each reaction vessel of the first portion of the reaction vessel. With automatic steps to do</u><u style="single"> d) The reaction vessel of the second part of the reaction vessel provided with sufficient detection reagents to detect the second amplification product containing the second target sequence or its complement is used in the signal detector. Continuously to operate in close proximity to the signal detector configured and arranged to detect the presence of signals emitted by the contents of a reaction vessel placed in close proximity. The signal associated with the automatic step to move and the presence of the second amplification product in each reaction vessel of the second portion of the reaction vessel moved to operate in close proximity to the signal detector. Automatic steps to detect and</u><u style="single"> f) Based on the measurement results obtained in step d), an automatic step using a microprocessor to determine the presence or absence of the second target nucleic acid in each reaction vessel of the second portion of the reaction vessel. When,</u><u style="single"> Including, methods.</u><u style="single">(Item 117)</u><u style="single"> The method of item 116, wherein the reaction vessel is provided to the first heater using an automated reaction vessel transfer system.</u><u style="single">(Item 118)</u><u style="single"> The method of any one of items 116-117, wherein the transfer system comprises a first rotatable transfer mechanism for providing the reaction vessel to the first heater.</u><u style="single">(Item 119)</u><u style="single"> The method of item 118, wherein the transfer system comprises a second rotatable transfer mechanism for removing the reaction vessel from the first heater.</u><u style="single">(Item 120)</u><u style="single"> In step d), the transfer system moves the reaction vessel of the second portion of the reaction vessel from the first heater so that it can operate in close proximity to the signal detector, items 117-119. The method described in any one of the items up to.</u><u style="single">(Item 121)</u><u style="single"> The method of item 120, further comprising an automated step of moving the reaction vessel of the second portion of the reaction vessel from the first heater to the second heater.</u><u style="single"> The detection reagent comprises the second portion of the reaction vessel under temperature conditions under which the probe present in the detection reagent can be detectable to hybridize with the second target sequence or complement thereof. Provided to the reaction vessel,</u><u style="single"> Method.</u><u style="single">(Item 122)</u><u style="single"> 12. The method of item 121, wherein the second heater is a closed incubator, the incubator having one or more doors for accommodating and removing the reaction vessel from the incubator.</u><u style="single">(Item 123)</u><u style="single"> 122. The method of item 122, wherein the incubator comprises a rotatable reaction vessel carrier for accommodating and moving the reaction vessel within the incubator.</u><u style="single">(Item 124)</u><u style="single"> Any of items 121 to 123, further comprising an automatic step of moving the reaction vessel of the second portion of the reaction vessel from the second heater so that it can operate in close proximity to the signal detector. The method described in paragraph 1.</u><u style="single">(Item 125)</u><u style="single"> The method according to any one of items 121 to 124, wherein the signal detector is a fluorometer and the probe is labeled with a chemiluminescent label.</u><u style="single">(Item 126)</u><u style="single"> The first heater is a closed incubator, which has one or more doors for accommodating and removing the reaction vessel from the incubator, any one of items 116 to 125. The method described in the section.</u><u style="single">(Item 127)</u><u style="single"> 126. The method of item 126, wherein the incubator comprises a rotatable reaction vessel carrier for accommodating and moving the reaction vessel within the incubator.</u><u style="single">(Item 128)</u><u style="single"> The method according to any one of items 116 to 127, wherein the amplification and enzyme reagents are provided in the reaction vessel after the reaction vessel is provided in the first heater.</u><u style="single">(Item 129)</u><u style="single"> The detection reagent is provided to the reaction vessel of the first portion of the reaction vessel after the reaction vessel of the first portion of the reaction vessel is provided to the first heater, and is provided to the reaction vessel of the first portion of the reaction vessel. 128. The method of item 128, wherein the reaction vessel of the portion is moved in close proximity to the signal measuring device in step b) before being removed from the first heater.</u><u style="single">(Item 130)</u><u style="single"> The reaction vessel of the second portion of the reaction vessel is removed from the first heater before being moved in close proximity to the signal detector in step d) so that the detection reagent is removed. 129. The method of item 129, which is provided in the reaction vessel of the second portion of the reaction vessel after being removed from the first heater.</u><u style="single">(Item 131)</u><u style="single"> The amplification and enzyme reagents provided in the reaction vessel of the first portion of the reaction vessel in step a) are the above under the temperature conditions of a primer or promoter primer, a nucleoside triphosphate, and the first heater. The above-mentioned primer which contains a nucleic acid polymerase capable of extending 3 ́-ends of the above-mentioned primer or promoter primer hybridized to the first target nucleic acid and is provided to the reaction vessel of the above-mentioned second part of the reaction vessel in step a). Amplification and enzyme reagents include primers or promoter primers, nucleoside triphosphates, and 3'ends of the primers or promoter primers hybridized to the second nucleic acid of interest under the temperature conditions of the first heater. The method of any one of items 116-130, comprising a nucleic acid polymerase that can be extended.</u><u style="single">(Item 132)</u><u style="single"> The method of item 131, wherein the temperature conditions are isothermal.</u><u style="single">(Item 133)</u><u style="single"> The detection reagent for detecting the first amplification product is self-hybridizing so as to be detectable so as to be detectable with the first target sequence or its complement under the temperature conditions of the first heater. The method of any one of items 116-132, comprising a probe.</u><u style="single">(Item 134)</u><u style="single"> 133. The method of item 133, wherein the probe is a molecular beacon or molecular torch and comprises an interacting label pair.</u><u style="single">(Item 135)</u><u style="single"> The method of item 134, wherein the label pair comprises a fluorescent probe and a quencher.</u><u style="single">(Item 136)</u><u style="single"> The method of any one of items 116-135, further comprising the step of simultaneously measuring the signals from each of the plurality of reaction vessels in the first portion of the reaction vessel.</u><u style="single">(Item 137)</u><u style="single"> In step b), the reaction vessel of the first portion of the reaction vessel is continuously and periodically moved so as to be able to operate in close proximity to a plurality of remote signal measuring devices, and each signal measuring device is heated. 136. The method of item 136, which is a fluorescent signal detector associated with the instrument so that it can operate.</u><u style="single">(Item 138)</u><u style="single"> 137. The method of item 137, wherein the fluorescence signal detector is movable relative to the heater.</u><u style="single">(Item 139)</u><u style="single"> The method according to any one of items 116 to 138, further comprising the step of simultaneously measuring a plurality of signals in the reaction vessel of the first portion of the reaction vessel, wherein the plurality of signals are used. A method, each associated with a different amplification product.</u><u style="single">(Item 140)</u><u style="single"> Prior to step a), the reagents and conditions sufficient to remove the amplification inhibitor from the reaction vessel further include an automated step of exposing the sample material present in each of the reaction vessels. , The method according to any one of items 116 to 139.</u><u style="single">(Item 141)</u><u style="single"> The above exposure steps</u><u style="single"> A step of providing a solid support material to each of the reaction vessels, wherein the solid support material can bind the first and second target nucleic acids.</u><u style="single"> In the step of isolating the solid support material in each of the reaction vessels,</u><u style="single"> The step of removing at least a part of the sample substance from each of the above reaction vessels,</u><u style="single"> The step of cleaning the solid support material with a cleaning liquid and</u><u style="single"> Further include,</u><u style="single"> The method described in item 140.</u><u style="single">(Item 142)</u><u style="single"> 141. The method of item 141, wherein the solid support material is magnetically reactive particles and the isolation step is performed by one or more magnets placed adjacent to each of the reaction vessels.</u><u style="single">(Item 143)</u><u style="single"> The method of any one of items 140-142, further comprising the step of transferring the reaction vessel to the first heater using an automated reaction vessel transfer system after the exposure step.</u><u style="single">(Item 144)</u><u style="single"> 143. The method of item 143, wherein the transfer system comprises a rotatable transfer mechanism.</u><u style="single">(Item 145)</u><u style="single"> The method according to any one of items 116 to 144, wherein each reaction vessel is one of a plurality of integrally formed reaction vessels.</u><u style="single">(Item 146)</u><u style="single"> The method according to any one of items 116 to 145, wherein all steps of the method are performed within the housing of the built-in analyzer unit.</u><u style="single">(Item 147)</u><u style="single"> A method of determining the amount of sample in question in a sample,</u><u style="single"> (a) A step of collecting data comprising a periodic measurement of the level of the signal emitted by the sample, wherein the level of the signal is associated with the presence of the sample in question in the sample. ,</u><u style="single"> (b) The step of adjusting the data collected in step (a) to eliminate the background impact on the measured level associated with sources other than the presence of the specimen in question.</u><u style="single"> (C) The step of normalizing the adjusted data in step (b) by dividing the adjusted data by the maximum level measured and adjusted to eliminate the background influence.</u><u style="single"> (d) A step of approximating a curve with a portion of the adjusted and normalized data between a given lower bound and a given upper bound.</u><u style="single"> (e) A step of determining when the curve approximated in step (d) intersects a predetermined threshold data level, and</u><u style="single"> (f) With the step of determining the amount of the sample in question by comparing the time of appearance determined in step (e) with the threshold time determined for the known amount of the sample in question. ,</u><u style="single"> Including, methods.</u><u style="single">(Item 148)</u><u style="single"> 147. The approximating step comprises applying a curve fitting procedure to the adjusted and normalized data points between the predetermined lower limit and the predetermined upper limit. Method.</u><u style="single">(Item 149)</u><u style="single"> The method of item 148, wherein the curve fitting procedure is fitting by a linear least squares method.</u><u style="single">(Item 150)</u><u style="single"> The method according to any one of items 147 to 149, wherein the collected data comprises a measurement result of the level of the emitted signal acquired at least once every 30 seconds.</u><u style="single">(Item 151)</u><u style="single"> The method according to any one of items 147 to 150, wherein the predetermined lower limit is about 0.04 and the predetermined upper limit is about 0.36.</u><u style="single">(Item 152)</u><u style="single"> The method of any one of items 147-151, wherein the normalized predetermined threshold data level is about 0.11.</u><u style="single">(Item 153)</u><u style="single"> The adjusting step includes determining the level of background impact and then reducing the determined level of background impact from the data collected in step (a), item 147. The method according to any one of 1 to 152.</u><u style="single">(Item 154)</u><u style="single"> 157. The method of item 157, wherein the step of determining the level of background impact includes a step for determining the amount of background impact.</u><u style="single">(Item 155)</u><u style="single"> A method for reducing the presence of nucleic acid amplification inhibitors in a reaction vessel.</u><u style="single"> (a) The step of providing the surface treatment agent and the nucleic acid-containing material to the reaction vessel, and</u><u style="single"> (b) A step of stirring the reaction vessel to mix the contents of the reaction vessel, and</u><u style="single"> (c) A step of exposing the contents of the reaction vessel, if present in the nucleic acid-containing material, to reagents and conditions sufficient to isolate the nucleic acid of interest in the reaction vessel.</u><u style="single"> (d) During step (c), the step of removing at least a part of the contents of the reaction vessel and</u><u style="single"> (e) The step of providing the cleaning liquid to the above reaction tank, and</u><u style="single"> (f) A step of exposing the contents of the reaction vessel, if present in the nucleic acid-containing material, to reagents and conditions sufficient to isolate the nucleic acid of interest in the reaction vessel.</u><u style="single"> (g) During step (f), the step of removing at least a part of the contents of the reaction vessel and</u><u style="single"> (h) Including the step of exposing the contents of the reaction vessel to reagents and conditions sufficient to amplify the target sequence present in the nucleic acid of interest.</u><u style="single"> The surface treatment agent is provided in the reaction vessel in an amount sufficient to reduce the presence of the nucleic acid amplification inhibitor during step (d) and step (g).</u><u style="single"> The surface treatment agent is not an inhibitor of the nucleic acid amplification reaction.</u><u style="single"> Method.</u><u style="single">(Item 156)</u><u style="single"> The method according to item 155, wherein the surface treatment agent is provided in the reaction vessel before the nucleic acid-containing material is provided in the reaction vessel.</u><u style="single">(Item 157)</u><u style="single"> The method according to item 155, wherein the surface treatment agent is provided to the reaction vessel after the nucleic acid-containing material is provided to the reaction vessel.</u><u style="single">(Item 158)</u><u style="single"> 155. The method of item 155, further comprising providing the reaction vessel with a solid support material to immobilize the nucleic acid of interest.</u><u style="single">(Item 159)</u><u style="single"> 158. The method of item 158, wherein the solid support material comprises magnetically reactive particles.</u><u style="single">(Item 160)</u><u style="single"> 159. The method of item 159, wherein the contents of the reaction vessel are exposed to a magnetic field during steps (c) and (f).</u><u style="single">(Item 161)</u><u style="single"> The method according to item 158, wherein the surface treatment agent is provided to the reaction vessel after the solid support material is provided to the reaction vessel.</u><u style="single">(Item 162)</u><u style="single"> 158. The method of item 158, wherein the surface treatment agent is provided to the reaction vessel before the solid support material is provided to the reaction vessel.</u><u style="single">(Item 163)</u><u style="single"> A method for reducing the presence of nucleic acid amplification inhibitors in a reaction vessel.</u><u style="single"> (a) The step of removing at least a part of the nucleic acid-containing material from the reaction vessel,</u><u style="single"> (b) After step (a), the step of providing the cleaning liquid and the surface treatment agent to the reaction vessel, and</u><u style="single"> (c) A step of stirring the reaction vessel to mix the contents of the reaction vessel, and</u><u style="single"> (d) A step of exposing the contents of the reaction vessel, if present in the nucleic acid-containing material, to reagents and conditions sufficient to isolate the nucleic acid of interest in the reaction vessel.</u><u style="single"> (e) During step (d), the step of removing at least a part of the contents of the reaction vessel and</u><u style="single"> (f) A step of exposing the contents of the reaction vessel to reagents and conditions sufficient to amplify the target sequence present in the nucleic acid of interest.</u><u style="single"> Including,</u><u style="single"> The surface treatment agent is provided in the reaction vessel in an amount sufficient to reduce the presence of the nucleic acid amplification inhibitor during step (e).</u><u style="single"> The surface treatment agent is not an inhibitor of the nucleic acid amplification reaction.</u><u style="single"> Method.</u><u style="single">(Item 164)</u><u style="single"> 163. The method of item 163, wherein the cleaning solution and the surface treatment agent are simultaneously provided in the reaction vessel.</u><u style="single">(Item 165)</u><u style="single"> 163. The method of item 163, wherein the cleaning solution and the surface treatment agent are separately provided in the reaction vessel.</u><u style="single">(Item 166)</u><u style="single"> 163. The method of item 163, further comprising providing the reaction vessel with a solid support material for immobilizing the nucleic acid of interest prior to step (a).</u><u style="single">(Item 167)</u><u style="single"> 166. The method of item 166, wherein the solid support material comprises magnetically reactive particles.</u><u style="single">(Item 168)</u><u style="single"> 167. The method of item 167, wherein the contents of the reaction vessel are exposed to a magnetic field during step (d).</u><u style="single">(Item 169)</u><u style="single"> The method according to any one of items 155 to 168, wherein the surface treatment agent prevents the inhibitor from adhering to the inner surface of the reaction vessel.</u><u style="single">(Item 170)</u><u style="single"> The method according to any one of items 155 to 169, wherein the surface treatment agent does not contain a detergent.</u><u style="single">(Item 171)</u><u style="single"> The method according to any one of items 155 to 170, wherein the surface treatment agent comprises silicone oil.</u><u style="single">(Item 172)</u><u style="single"> The method according to any one of items 155 to 171, wherein the reaction vessel comprises a hydrophobic material.</u><u style="single">(Item 173)</u><u style="single"> 172. The method of item 172, wherein the reaction vessel comprises polypropylene.</u><u style="single">(Item 174)</u><u style="single"> The method according to any one of items 155 to 173, wherein the inhibitor is provided by the nucleic acid-containing material.</u><u style="single">(Item 175)</u><u style="single"> 174. The method of item 174, wherein the inhibitor is hemoglobin.</u><u style="single">(Item 176)</u><u style="single"> The method according to any one of items 155 to 173, wherein the inhibitor is provided by the cleaning solution.</u><u style="single">(Item 177)</u><u style="single"> 176. The method of item 176, wherein the inhibitor is an anionic detergent.</u><u style="single">(Item 178)</u><u style="single"> The reagent for amplifying the target sequence comprises a primer or promoter primer, a nucleoside triphosphate, and a nucleic acid polymerase capable of extending the 3'end of the primer hybridized to the nucleic acid of interest. The method according to any one of 155 to 177.</u></p>

0022<figref num="1">FIG. 1 is a perspective view of a nucleic acid-based automatic diagnostic analyzer according to the present invention.</figref><figref num="2">FIG. 2 is a perspective view of the structural frame of the analyzer according to the present invention.</figref><figref num="3">FIG. 3 is a partial plan view of the assay processing deck in the analyzer according to the present invention.</figref><figref num="4">FIG. 4 is an exploded perspective view of the assay processing deck.</figref><figref num="5">FIG. 5 is a plan view of the sample ring and pipette tip wheel of the assay processing desk in the analyzer according to the present invention.</figref><figref num="6">FIG. 6 is a perspective view showing a sample ring and a pipette tip wheel.</figref><figref num="6A">FIG. 6A is a partial cross-sectional view taken along line 6A-6A in FIG.</figref><figref num="7">FIG. 7 is a perspective view of the multi-axis stirrer of the processing desk in the analyzer according to the present invention.</figref><figref num="8">FIG. 8 is a plan view of the multi-axis stirrer.</figref><figref num="9">FIG. 9 is a side view of the multi-axis stirrer.</figref><figref num="10">FIG. 10 is a plan view of the multi-axis stirrer with the container holder and the turntable cover removed.</figref><figref num="11">FIG. 11 is a cross-sectional view of a multi-axis stirrer cut in directions 11-11 of FIG.</figref><figref num="12">FIG. 12 is a perspective view of the drive assembly of the multi-axis stirrer.</figref><figref num="13">FIG. 13 is a perspective view of the transfer mechanism of the processing desk in the analyzer according to the present invention.</figref><figref num="14">FIG. 14 is a perspective view of the operating hook mounting plate and operating hook operating mechanism of the transfer mechanism, comprising an operating hook member in a retracted position that engages the reaction vessel.</figref><figref num="15">FIG. 15 is the same as FIG. 14 except that the operation hook member is in the extended position.</figref><figref num="16">FIG. 16 is an exploded perspective view of the transfer mechanism.</figref><figref num="17">FIG. 17 is a side view of the temperature gradient station in the analyzer according to the present invention.</figref><figref num="18">FIG. 18 is a front view of the temperature gradient station.</figref><figref num="19">FIG. 19 is a perspective view of a rotary incubator of a processing desk in the analyzer according to the present invention.</figref><figref num="20">FIG. 20 is an exploded view of a portion of the housing and reaching opening closing mechanism according to the first embodiment of the rotary incubator.</figref><figref num="21">FIG. 21 is a partial view of a tilted disk linear stirrer in a rotary incubator, shown in engagement with a reaction vessel used in the preferred mode of operation of the analyzer according to the invention.</figref><figref num="22">FIG. 22 is an exploded perspective view of a first embodiment of the rotary incubator.</figref><figref num="23">FIG. 23 is a perspective view of the rotary incubator according to the second embodiment of the rotary incubator.</figref><figref num="23A">FIG. 23A is an exploded perspective view of a second embodiment of the rotary incubator.</figref><figref num="23B">FIG. 23B is a partial fraction decomposition perspective view of the reaching opening closing mechanism by the second implementation of the rotary incubator.</figref><figref num="23C">FIG. 23C is an exploded view of the container transport turntable according to the second implementation of the rotary incubator.</figref><figref num="24">FIG. 24 is a perspective view of the processing desk according to the present invention in a state where the side plates of the particles are removed.</figref><figref num="25">FIG. 25 is a partial cross-sectional view of the particles.</figref><figref num="25A">FIG. 25A is a partial cross-sectional view of the tip of the suction tube that carries the tip of the antibacterial contamination prevention tip to the tip of the particles.</figref><figref num="26">FIG. 26 is an exploded perspective view of the container carrier, rotary stirrer assembly, and particle divider plate.</figref><figref num="27">FIG. 27 is a partial cross-sectional view of the cleaning solution dispenser nozzle of the particles, the suction tube having the tip of the antibacterial contamination prevention tip that engages with the tip, and the analyzer and the multi-tube to be transported to the container carrier. The multi-tube unit reaction vessel used in the preferred operation mode of the suction tube inserted into the reaction tube of the unit and the tip of the antibacterial contamination prevention tip is shown.</figref><figref num="28">FIG. 28 is a partial cross-sectional view of the cleaning solution dispenser nozzle, the suction tube, and the container carrier of the particles, and is held in the multi-tube unit transported to the container carrier and the germ contamination prevention element gripping structure of the multi-tube unit. The suction tube that engages the tip of the antibacterial contamination prevention tip is shown.</figref><figref num="29">29A-29D show a first embodiment of a chip tip removing hole in a particle tip removing plate and a partial cross-sectional view of a tip removing operation using the tip removing hole.</figref><figref num="30">30A to 30D show a second embodiment of the tip removing hole and a partial cross-sectional view of the tip removing operation using the tip removing hole.</figref><figref num="31">FIG. 31A is a plan view of a third embodiment of the chip tip removing hole in the particle tip removing plate. 31B to 31C show a third embodiment of the tip removing hole and a partial cross-sectional view of the tip removing operation using the tip.</figref><figref num="32">FIG. 32 is a perspective view of the rotary stirrer with the front plate removed.</figref><figref num="33">FIG. 33 is an exploded view of the rotary stirrer of the processing desk in the analyzer according to the present invention.</figref><figref num="34">FIG. 34 is an upper plan view of the rotary stirrer.</figref><figref num="35">FIG. 35 is a perspective view of the reagent cooling bay of the analyzer according to the present invention.</figref><figref num="36">FIG. 36 is a perspective view of the reagent cooling bay with the container tray removed.</figref><figref num="37">FIG. 37 is a bottom plan view of the reagent cooling bay.</figref><figref num="38">FIG. 38 is an exploded view of the reagent cooling bay.</figref><figref num="39">FIG. 39 is a perspective view of the modular container tray of the reagent cooling bay.</figref><figref num="40">FIG. 40 is a perspective view of a first embodiment of the illuminometer at the processing desk of the analyzer according to the present invention.</figref><figref num="41">FIG. 41 is a partial decomposition perspective view of the illuminometer in the first embodiment.</figref><figref num="42">FIG. 42A is a partial perspective view of the container transfer mechanism in the first embodiment of the illuminometer. FIG. 42B is an end view of the container transfer mechanism in the first embodiment of the illuminometer. FIG. 42C is a top view of the container transfer mechanism in the first embodiment of the illuminometer.</figref><figref num="43">FIG. 43 is a separation perspective view of a second embodiment of the illuminometer according to the present invention.</figref><figref num="44">FIG. 44 is an exploded perspective view of the multi-tube unit door assembly of the illuminometer in the second embodiment.</figref><figref num="45">FIG. 45 is an exploded perspective view of the shutter assembly for the photosensor aperture of the illuminometer in the second embodiment.</figref><figref num="45A">FIG. 45A is a perspective view of the aperture plate of the shutter assembly of the illuminometer in the second embodiment.</figref><figref num="46">FIG. 46 is a perspective view of the reaction tube positioner assembly of the illuminometer in the second embodiment, including the reaction tube positioner placed within the reaction tube positioner frame.</figref><figref num="47">FIG. 47 is a perspective view of the reaction tube positioner.</figref><figref num="48">FIG. 48 is a side view of the reaction tube positioner assembly.</figref><figref num="49">FIG. 49 is a perspective view showing the reaction tube positioner of the reaction tube positioner assembly that operably engages the multi-tube unit used in the preferred mode of operation of the analyzer.</figref><figref num="50">FIG. 50 is a perspective view of the multi-tube unit transfer mechanism of the illuminometer in the second embodiment.</figref><figref num="51">FIG. 51 is a partial perspective view showing the transfer and driving screw of the multi-tube unit in the multi-tube unit of the illuminometer.</figref><figref num="52">FIG. 52 is a perspective view of the lower chassis of the analyzer according to the present invention.</figref><figref num="53">FIG. 53 is a perspective view of the right side drawer of the lower chassis.</figref><figref num="54">FIG. 54 is a perspective view of the left drawer of the lower chassis.</figref><figref num="55">FIG. 55 is a perspective view of the sample tube tray used in the preferred operating mode of the analyzer according to the invention.</figref><figref num="56">FIG. 56 is a plan view of the sample tube tray.</figref><figref num="57">FIG. 57 is a partial cross-sectional view of the sample tube tray along lines 57-57 in FIG. 55.</figref><figref num="58">FIG. 58 is a perspective view of the multi-tube unit used in the preferred operating mode of the analyzer according to the invention.</figref><figref num="59">FIG. 59 is a side view of the tip of an anti-contact pipette tip used in the preferred mode of operation of the analyzer according to the invention and carried to the multi-tube unit shown in FIG.</figref><figref num="60">FIG. 60 is an enlarged bottom view of a part of the multi-tube unit shown in the direction of the arrow 60 in FIG. 58.</figref><figref num="61">FIG. 61 is a side view of a cross section showing a part of the photodetector module, the real-time fluorometer and the multi-tube unit.</figref><figref num="62">FIG. 62 is an exploded perspective view of the housing of the photodetector module.</figref><figref num="63">FIG. 63 is an exploded perspective view of the photodetector module.</figref><figref num="64">FIG. 64 is a plan view of a real-time fluorometer showing the preferred position of the photodetector module.</figref><figref num="65">FIG. 65 is a schematic diagram of a real-time fluorometer showing the preferred location of the photodetector module.</figref><figref num="66">FIG. 66 is a graph showing the excitation spectrum of the preferred amplification detection dye.</figref><figref num="67">FIG. 67 is a graph showing the emission spectrum of the preferred amplification detection dye.</figref><figref num="68A">FIGS. 68A to 68F are circuit diagrams of the photodetector module.</figref><figref num="68B">FIGS. 68A to 68F are circuit diagrams of the photodetector module.</figref><figref num="68C">FIGS. 68A to 68F are circuit diagrams of the photodetector module.</figref><figref num="68D">FIGS. 68A to 68F are circuit diagrams of the photodetector module.</figref><figref num="68E">FIGS. 68A to 68F are circuit diagrams of the photodetector module.</figref><figref num="68F">FIGS. 68A to 68F are circuit diagrams of the photodetector module.</figref><figref num="69">FIG. 69 is a perspective view showing a photodetector scan assembly of a scan real-time fluorometer.</figref><figref num="70">FIG. 70 is a side view of the detector scan assembly.</figref><figref num="71">FIG. 71 is a top plan view of the detector scan assembly.</figref><figref num="72">FIG. 72 is a bottom plan view of the detector scan assembly.</figref><figref num="73">FIG. 73 is a perspective view of the turntable of the real-time fluorometer.</figref><figref num="74">FIG. 74 is an exploded perspective view showing the turntable and magnetic partition of the real-time fluorometer.</figref><figref num="75">FIG. 75 is a plan view of the bottom of the turntable of the real-time fluorometer, showing a single magnetic partition attached.</figref><figref num="75A">FIG. 75A is a partial cross-sectional view cut along line AA of FIG. 75.</figref><figref num="76A">FIG. 76A is a flow chart showing the preferred real-time amplification assay protocol and some of the preferred endpoint amplification assay protocols (interrupted after exposure to amplification conditions), both assays according to the present invention.</figref><figref num="76B">FIG. 76B is a flow chart showing the rest of the preferred endpoint amplification assay protocol of Figure 76A (after exposure to amplification conditions).</figref><figref num="77">FIG. 77 is a flowchart showing the sample quantification process.</figref><figref num="78">FIG. 78 is a time plot of real-time fluorometer data.</figref><figref num="79">FIG. 79 is a plot showing how a curve is fitted to real-time fluorometer data and the fit is used to determine the threshold time.</figref>

0023The present invention may be carried out in a variety of forms, and the following description and accompanying drawings are only intended to disclose some forms as examples of the present invention. Therefore, the present invention is not limited to the forms or examples described and illustrated. The entire scope of the invention is set forth in the appended claims.

0024(Overview of analyzer) The analyzer for automatic diagnosis according to the present invention is broadly indicated by reference numeral 50 in FIGS. 1 and 2. The analyzer 50, including the housing 60 provided over the inner frame structure 62, is preferably made of steel. The analyzer 50 is preferably supported on caster wheels 64 structurally attached to the frame structure 62 to allow the analyzer to be moved.

0025The various stations and assay samples involved in performing the automated assay are housed in housing 60. In addition, the various solutions, reagents, and materials used to perform the assay should be stored in the housing 60, similar to the waste generated when the assay is performed in the analyzer 50. Is preferable.

0026Housing 60 is shown in FIG. 1 to be located on the front facing panel of housing 60, but includes a test vessel loading opening 68 that can also be located on another panel of housing 60. The pipette door 70 with the observation window 72 and the turntable door 74 with the observation window 76 are located above the substantially horizontal working surface 66. The forward protruding bow-shaped panel 78 accommodates a sample turntable, which will be described later. The flip-up bow-shaped sample door 80 is pivotally attached to the housing so that it pivots up and down with respect to the arched panel 78, providing access to the front portion of the sample turntable behind the panel 78. The sensor points out if the door is closed and the sample door 80, turntable door 74 and pipette door 70 are locked during analyzer operation. The locking mechanism of each door preferably comprises a hook attached to a DC rotary solenoid (estimated for continuous operation) with a spring return. Preferred rotary solenoids are commercially available from Lucas Control Systems, Bandaria, Ohio, under model numbers L-2670-034 and L-1094-034.

0027The stretch portion 102, preferably made of a transparent or translucent material, extends above the top of the housing 60 and provides a vertical gap for moving components within the housing 60.

0028Assays are performed primarily at the processing desk 200, which is the general location of the various assay stations of the analyzer 50 described below. For simplification of the figure, in FIG. 2, the processing desk 200 is shown without the assay station attached. The processing desk 200 includes a reference plate 8 to which various stations can be directly or indirectly mounted. The reference plate 82 preferably includes a machined aluminum plate. The processing desk 200, also known as a chemical desk, separates the inside of the housing from the chemical area or upper chassis above the reference plate 82 and the storage area or lower chassis 1100 located below the reference plate 82.

0029A certain number of fans and louvers are provided in the upper chassis portion of the housing 60, preferably to create air circulation through the upper chassis to prevent excessive temperature rise of the upper chassis.

0030Since the analyzer 50 of the present invention is computer controlled, the analyzer 50 includes a computer controller, schematically represented as Box 1000 in FIG. 2, which is known as the "assay manager program". Run the level analyzer control software. The assay manager program includes a scheduler routine that monitors and controls the movement of test samples through the Chemistry Desk 200.

0031The computer control device 1000 that controls the analyzer 50 may include a stand-alone computer system that includes a CPU, keyboard, monitor, and optionally a printing device. It may be equipped with a movable cart for storing and supporting various computer components. Alternatively, the computer hardware for executing the analyzer control software may be integrally housed in the housing 60 of the analyzer 50.

0032Low-level analyzer control, such as control of electric motors and heaters used through analyzer 50, monitoring of fluid levels in bulk fluid and waste fluid vessels, is preferably performed by an embedded controller with a Motorola 68332 microprocessor. The stepper motor used through the analyzer is also preferably controlled by a pre-programmed microprocessor chip commercially available from EM Technologies, Bara Sinwid, PA.

0033The processing desk 200 is schematically shown in FIGS. 3 and 4. FIG. 3 represents a schematic plan view of a portion of the processing desk 200, and FIG. 4 represents a schematic perspective view of the processing desk. The reference plate 82 forms the basis of the processing desk 200 to which all stations are directly or indirectly mounted.

0034The processing desk 200 includes a reaction vessel charging preparation chamber 150 extending from an opening 68 at the front of the housing 60. The plurality of reaction vessels are mounted in a stacked state in the loading preparation chamber 150. The purpose of the loading preparation chamber is to grab a predetermined number of reaction vessels and sequentially supply them to the collection position for recovery by a transfer mechanism (discussed below). A reflective sensor at the collection location confirms that the container is present at that location. The loading preparation room also includes a device that counts the number of containers present in it at any specified time.

0035The reaction vessel shuttle assembly (not shown) in the preparation chamber moves the vessel to the collection position along the vessel advance path. The light sensor points out when the shuttle assembly is in its home and fully extended positions. The preparation room contains a drawer that is pulled out to mount the container in it. However, before it can be opened, the drawer must be unlocked and the shuttle must leave the vessel forward path. When reclosed, the drawer is locked and the shuttle engages the container and moves it to the collection position. The light sensor points out when the drawer is closed and when the shuttle is engaging the container. When each container is removed from the collection position by the transfer mechanism, the container shuttle advances the container by one container width so that the next container is located at the collection position.

0036The analyzer 50 is adapted for use by a reaction vessel composed of a single reaction vessel, or an integrally formed unit containing multiple reaction vessels having any number of different shapes, sizes, and configurations. However, the reaction vessel of the present invention preferably integrally forms a multi-tube unit or a linear arrangement of reaction tubes known as the MTU. These preferred reaction vessels will be described in more detail below.

0037In a preferred embodiment, the first ring assembly with the sample ring 250 is attached to the pivot jig plate 130 at a distance above the reference plate 82. The sample ring 250 is substantially circular and preferably holds up to nine sample trays 300 in its annular fluid container carrier, and each sample tray holds 20 sample-containing containers or test tubes 320. Is preferable. The sample ring 250 is rotatably configured and arranged about a first substantially vertical axis of rotation and supplies the sample tube 320 to the sample pipette assembly 450, which is preferably an automated robot pipette system. The front part of the sample ring 250 is reachable through the flip-up turntable door 80 provided in the housing 60, and the tray 300 of the test tube 320 can be easily mounted on the sample ring 250 and also from the sample ring. Can be removed. The sample ring 250 is driven by a motor as described in detail below.

0038In a preferred embodiment, the second ring assembly with the pipette tip wheel 350 is located inside the sample ring 250 and at least a portion of the outer circumference of the pipette tip wheel 350 is located radially inward on the inner circumference of the ring 250. The wheel. The pipette tip wheel 350 carries a plurality of commercially available pipette tip packages. The pipette tip wheel 350 is driven by a motor to rotate the sample ring 250 individually about a second axis of rotation that is substantially parallel to the first axis of rotation of the sample ring 250.

0039An internal rotatable assembly configured and arranged to carry multiple fluid vessels is provided inside the pipette tip wheel 350. In a preferred embodiment, the internal rotatable assembly comprises a multiaxial stirrer 400 located radially inside the pipette tip wheel 350 (second ring assembly) and sample ring 250 (first ring assembly). The multi-axis stirrer 400 is rotatable about a third axis of rotation that is substantially parallel to the first and second axes of rotation and is fitted with four individually and eccentrically rotating container holders 406. Includes board 414. Each container holder 406 houses a container, preferably in the form of a plastic bottle, containing a fluid suspension of magnetic particles with immobilized polynucleotides and polynucleot capture probes. Each container holder 406 is substantially cylindrical and includes an axis of symmetry or an axis of rotation. The multi-axis stirrer 400 provides the container with substantially constant agitation by rotating each container eccentrically with respect to the center of the holder 406 and at the same time rotating the turntable 414 around the center. Maintains magnetic particles in the fluid.

0040The sample pipette assembly or robot 450 is mounted on the frame structure 62 (see Figure 2) above the sample ring 250 and pipette tip wheel 350. The sample pipette assembly 450 includes a pipette unit 456 with a tubular probe 457 attached to the gantry assembly to provide X, Y, Z movement. Specifically, the pipette unit 456 can move linearly in the Y direction along the track 458 formed on the lateral rail 454, and the lateral rail 454 is longitudinally along the longitudinal track 452. It can move in the direction and X direction. Pipette unit 456 provides vertical or Z-axis movement of probe 457. The drive mechanism in the sample pipette assembly 450 positions the pipette unit 456 at the exact X, Y, Z coordinates in the analyzer 50, pipettes the fluid, cleans the probe 457 of the pipette unit 456, and of the pipette unit 456. Discard the protective tip from the tip of probe 457 or house the pipette unit 456 when not in use, such as when in the "home" position. Each axis of the sample pipette assembly 450 is driven by a stepping motor in a known and conventional manner.

0041The pipette assembly is preferably off-the-shelf. Currently preferred is the Robotic Sample Processor of model number RSP9000, commercially available from Cavro Inc. (Sunnyvale, Calif.). This type has a single gantry arm.

0042The sample pipette assembly 450 is preferably connected to a syringe pump (not shown) (Cavro XP 3000 has been used) and a DC driven diaphragm fluid cleaning pump (not shown). The syringe pump of the sample pipette assembly 450 is preferably mounted in the upper position on the left side of the chemical desk 200 of the internal frame structure 62 within the housing 60 of the analyzer 50, as well as suitable tubes (not shown) or other conduits. Due to its structure, it is connected to the pipette unit 456.

0043A sample preparation opening 252 is placed on the jig plate 130 so that the sample pipette assembly 450 can reach the reaction vessel 160 in the loading preparation chamber 150 located below the jig plate 130.

0044The sample pipette assembly 450 of the analyzer 50 engages the sample tube 320 carried to the sample ring 250 through the openings 140 and 142 of the lifted cover plate 138, and also the sample ring 250 and the pipette tip wheel 350, respectively. Engage with the pipette tip carried by the pipette tip wheel 350 near the rear. As a result, during analyzer operation, the operator can reach the anterior portion of the sample ring 250 and pipette tip wheel 350 through the turntable door opening 80 without interfering with the pipette operating procedure.

0045The tip cleaning / disposal station 340 is located close to the sample ring 250 on the jig plate 130. Station 340 includes a chip disposal pipe 342 and a cleaning station basin 346. During sample preparation, the pipette unit 456 of the sample pipette assembly 450 is moved through the probe 457 to a position above the wash station basin 346 where the tubular probe 457 can be washed with pumped distilled water, but the basin of the wash station 346. Is preferably connected to the waste liquid container of the lower chassis 1100 by a flexible hose (not shown).

0046The chip disposal pipe 342 includes an upright tubular member. While moving the sample from the sample tube 320 to the reaction vessel 160, the elongated pipette tip is rubbed onto the tip of the tubular probe 457 of the pipette unit 456 so that the sample material is removed from the sample tube 320 into the elongated pipette tip. When this is done, it does not come into contact with the tubular probe 457 of the pipette unit 456. After the sample has been moved from the sample tube 320, it is important that the pipette tip used to move the sample is not reused for another unrelated sample. Therefore, after sample transfer, the pipette unit 456 moves above the tip disposal tube 342 and into the tip disposal tube 342 that connects the used disposable pipette tip to the solid waste container carried to the lower chassis 1100. Discharge.

0047The elongated pipette tip is also preferably frictionally secured to probe 457 in order to move the target capture reagent from the vessel transported to the multiaxial stirrer 400 to the reaction vessel 160. After the reagent is transferred, the pipette tip is discarded.

0048As mentioned above, the sample ring 250, pipette tip wheel 350, and multiaxial stirrer 400 shall be mounted on a hinged jig plate 130 (see Figures 5 and 6) supported above the reference plate 82. Is preferable. The jig plate 130 is hinged at the rear end 132 (see FIG. 6), whereby the plate and the rings 250, wheels 350, stirrer 400 attached therein can be pivoted upwards and the jig Allows access to the chemical desk area below the plate.

0049The first or right transfer mechanism 500 is attached to the jig plate 130 and the reference plate 82 below the sample ring 250 on substantially the same surface as the loading preparation chamber 150. The transfer mechanism 500 includes a rotating body portion 504 that defines the container transport assembly, and an extendable operating hook 506 that is mounted within the body 504 and is extendable and retractable with respect to the body by a power hook member drive assembly. Each reaction vessel 160 preferably comprises an operating structure that can be engaged by an extendable operating hook 506. Thereby, during assaying in the reaction vessel 160, when the reaction vessel is sequentially moved from one station to another, the transfer mechanism 500 engages and operates the reaction vessel 160 to operate the processing desk. You can move from one position of 200 to another.

0050A second or left transfer mechanism 502, which has substantially the same configuration as the first transfer mechanism 500, is also provided on the processing desk 200.

0051The plurality of container stop stations 210 are also located below the jig plate 130. The stop station 210, as the name suggests, is a structure for gripping the sample-containing reaction vessel until the station performing the assay on the processing desk 200 of the analyzer 50 is ready to accept the reaction vessel. If necessary, the reaction vessel is withdrawn from the stop station 210 by the transfer mechanism 500 and inserted into the stop station 210.

0052The right rotary stirrer 550 is attached to the reference plate 82 and houses the reaction vessel 160 inserted therein by the right transfer mechanism 500. A rotary stirrer is provided to mix the contents of the reaction vessel 160. When mixing is complete, the right transfer mechanism 500 removes the reaction vessel from the right rotary stirrer 550 and moves it to another location on the processing desk.

0053Multiple incubators 600, 602, 604, and 606 with substantially the same configuration are provided. Incubators 600, 602, 604, and 606 are preferably rotary incubators. The particular assay performed and the desired throughput will determine the desired number of incubators required, but it is preferred that the analyzer 50 be equipped with four incubators.

0054As described in detail below, each incubator (600,602,604,606) may also include a first container reach opening and a second container reach opening through which the transfer mechanism 500 or 502 reacts. The vessel 160 can be inserted into the incubator or the reaction vessel 160 can be withdrawn from the incubator. Rotating vessel carrier turntables within each incubator (600, 602, 604, 606) grip multiple reaction vessels 160 within individual vessel stations while the vessels are being incubated. In a nucleic acid-based diagnostic assay preferably performed on the analyzer 50 of the present invention, the first rotary incubator TC is the TC incubator (also known as the "TC incubator") and the second rotary incubator. Reference numeral 602 is an active temperature pre-reading cooling incubator (also known as "AT incubator"), and third rotary incubator 604 is an amplification incubator (also known as "AMP incubator") and a fourth rotation. Formula incubator 606 is a hybridization incubator (also known as a "HYB incubator"). (The names given to the incubators are for convenience only to indicate their use in one preferred endpoint amplification assay and are not considered to limit other possible uses of these incubators. ) Further details on the configuration, function, and role of the incubator in performing the general assay will be described later.

0055The processing desk 200 preferably also includes a plurality of temperature gradient stations 700. The two such stations 700 are shown in FIG. 3 as mounted between the incubators 602 and 604 of the reference plate 82. Additional gradient stations may be located at other locations on the processing desk 200, which are reachable by one of the transfer mechanisms 500 and 502.

0056The reaction vessel 160 may be installed at the temperature gradient station 700 or removed from the temperature gradient station 700 by either the transfer mechanism 500 or 502. Each gradient station 700 raises or lowers the temperature of the reaction vessel and its contents to the desired temperature before the vessel is installed in an incubator or other temperature sensitive station. Temperature fluctuations within the incubator are minimized by bringing the reaction vessel and its contents to the desired temperature prior to insertion into one of the incubators (600, 602, 604, 606).

0057The processing desk 200 includes a magnetic separation station for magnetic separation cleaning procedures. Each magnetic separation station 800 can accommodate and carry out one reaction vessel 160 cleaning procedure at a time. Therefore, it is preferred that five magnetic separation stations work in parallel to achieve the desired throughput. The container 160 is inserted into and removed from the magnetic separation station by the left transfer mechanism 502.

0058The reagent cooling bay 900 is mounted approximately halfway between the incubators 604 and 606 of the reference plate 82. The reagent cooling bay 900 comprises a turntable structure with a plurality of container receivers for holding bottles of temperature sensitive reagents. The turntable is provided within a rejected housing structure with a lid in which a pipette access hole is formed.

0059The second or left rotary stirrer 552, which is substantially identical to the right rotary stirrer 550, is located between the incubators 606 and 604. The left rotary stirrer 552 includes a dispenser nozzle and a line for dispensing fluid into a reaction vessel provided within the left rotary stirrer 552.

0060The reagent pipette assembly or robot 470 includes a double gantry structure attached to frame structure 62 (see Figure 2) and is located approximately above incubators 604 and 606 on the left side of the processing desk 200. Specifically, reagent pipette assembly 470 includes pipette units 480 and 482. The pipette unit 480 contains a tubular probe 481 and is mounted for linear motion along track 474 of a lateral rail in approximately X direction, and a pipette unit 482 containing a tubular probe 483 also contains a tubular probe 483 in approximately X direction. Installed for linear motion along track 484 of lateral rail 478. The lateral rails 476 and 478 can be translated along the longitudinal track 472 in approximately Y direction. Each pipette unit 480 and 482 provides individual vertical or Z-axis movement of the respective probes 481 and 483. The drive mechanism within the assembly 470 positions the pipette units 480 and 482 at the exact X, Y, Z coordinates within the analyzer 50, pipettes the fluid and cleans the tubular probes 481 and 483 of the pipette units 480 and 482, respectively. Or to accommodate pipette units 480 and 482 when not in use, such as when in the "home" position. Each axis of pipette assembly 470 is driven by a stepping motor.

0061The reagent pipette assembly 470 is preferably off-the-shelf. The currently preferred unit is the Cavro Robotic Sample Processor of model number RSP9000 with two gantry arms.

0062Each pipette unit 480 and 482 of the reagent pipette assembly 470 is preferably coupled to a respective syringe pump (not shown) (Cavro XP 3000 has been used) and a DC driven diaphragm fluid cleaning pump. The syringe pump of the reagent pipette assembly 470 is preferably mounted in the upper position on the left side of the chemical desk 200 of the internal frame structure 62 within the housing 60 of the analyzer 50, as well as suitable tubing (not shown) or other conduit. The structure connects to pipette units 480 and 482, respectively.

0063Each pipette unit 480 and 482 is preferably equipped with a volume level detection capability. Capacitive level detection, widely known in the technical field of medical equipment, is formed by the pipette unit as one of the plates of the capacitor and as the opposite plate as the structure and hardware surrounding the container engaged with the pipette unit. Utilizing the capacitance change when the dielectric of the formed capacitor changes from air to fluid, it detects when the probe of the pipette unit invades the fluid in the container. The level of fluid in the vessel engaged by the pipette unit may be determined by ascertaining the vertical position of the probe of the pipette unit, which may be perceived by monitoring the stepping motor driving the vertical movement of the pipette unit.

0064Pipette unit 480 moves reagents from reagent cooling bay 900 into a reaction vessel located in HYB incubator 606 or rotary stirrer 552, and pipette unit 482 moves reagent material from reagent cooling bay 900 to AMP incubator 604. Alternatively, it is moved into a reaction vessel arranged in the rotary stirrer 552.

0065Pipette units 480 and 482 use volume level detection to check the fluid level in the container, submerge only a small portion of the probe tip of the pipette unit, and pipette the fluid out of the container. The pipette units 480 and 482 are preferably tilted as the fluid is pipetted into the tubular probes 481 and 483, respectively, to keep the probe tips submerged to a certain depth. After removing the reagent into the tubular probe of pipette unit 480 or 482, the pipette unit creates a minimum movement gap of 10 μl at the tip of each probe 481 or 483 and the pipette unit moves to another position above the chemical desk 200. Ensure that no water droplets fall from the probe tip when forced.

0066The results of the assay, preferably performed on the analyzer 50 of the present invention, are confirmed by the amount of chemiluminescence or light emitted from the reaction tube 162 at the end of the appropriate preparation step. Specifically, the result of the assay is determined by the amount of light emitted by the label associated with the hybridized polynucleotide probe at the end of the assay. Therefore, the processing desk 200 includes detection and / or quantification of the amount of light emitted by the contents of the reaction vessel. Simply put, the illuminometer 950 includes a housing through which the reaction vessel moves under the influence of transfer mechanisms, photomultiplier tubes, and related electronics. Details of examples of various illuminometers will be described later.

0067The processing desk 200 also preferably includes a deactivation preparation room 750. The assay performed on analyzer 50 involves isolation and amplification of nucleic acids belonging to at least one organism or cell of interest. Therefore, it is usually preferred to inactivate the contents of the reaction vessel 160 by dropping a bleach-based reagent into the reaction vessel 160 at the end of the assay. This deactivation takes place in the deactivation preparation room 750.

0068After deactivation, the contents of the deactivated reaction vessel 160 are stored in one of the waste liquid containers of the lower chassis 1100, and the used reaction vessel is placed in the dedicated solid waste container in the lower chassis 1100. Will be discarded. The reaction vessel is preferably not reused.

0069(Analyzer operation) The operation of the analyzer 50, as well as the configuration, coordination, and interaction of the stations, components, and modules described above, may be performed by the analyzer 50 on a single test sample in one type of assay run. Explained by describing 50 operations. Other diagnostic assays that require the use of one or more of the stations, components, and modules described herein may also be performed by the analyzer 50. The specific assay procedures described herein are for, but are not limited to, exemplifying the operation and interaction of the various stations, components, and modules of the analyzer 50. It is clear to experts in the field of diagnostic testing technology that the analyzer 50 of the present invention allows a variety of chemical and biological assays to be performed by automated means.

0070The analyzer 50 is configured to perform the assay by first loading the bulk fluid into the bulk fluid storage bay of the lower chassis 1100 and connecting the bulk fluid vessel to a suitable hose (not shown).

0071The analyzer is preferably powered on in a sequential process. First power on the stations or modules that are needed early in the process, then power on the stations that are not needed until later in the process. This saves energy and avoids large power surges that could activate circuit breakers as the entire analyzer is powered on. The analyzer also employs a "sleep" mode when not in use. During sleep mode, a minimum amount of power is supplied to the analyzer, avoiding the large surge required to power on the analyzer from a completely stopped state.

0072A number of reaction vessels 160, preferably in the form of a plastic integrally formed multi-tube unit (MTU) (details below), are loaded into the loading preparation chamber 150 through the opening 68. From now on, the reaction vessel 160 will be MTU to be consistent with the preferred method of using the analyzer 50.

0073The reaction vessel shuttle assembly (not shown) in the loading preparation chamber 150 moves the MTU 160 from the loading opening 68 to the collection position at the end of the preparation chamber 150. The right transfer mechanism 500 picks up the MTU 160 from the end of the preparation chamber 150, moves it to a barcode reader (not shown), is on the MTU, and reads its own barcode label that identifies the MTU. The MTU is moved from the barcode reader to the available sample transfer station 255 below the opening 252.

0074(Multi-tube unit) A preferred MTU is an example of a multi-vessel reaction vessel disclosed by Homer et al., US Pat. No. 6,086,827, "Reaction Receptacle MFP." As shown in FIG. 58, the MTU 160 comprises a plurality of separate reaction tubes 162, preferably five. Reaction tubes 162, preferably in the form of a cylindrical tube with an open top and a closed bottom, are connected to each other by a connecting rib structure 164 that defines a downward step extending longitudinally along either side of the MTU 160. Be connected. In one embodiment, the dimensions of each reaction tube 162 of the MTU 160 are 12 x 75 mm, but the analyzer 50 houses reaction vessels of different dimensions provided independently or as part of a multi-vessel reaction vessel. It can be easily adapted to.

0075The MTU160 is preferably formed from injection molded polypropylene. The most preferred polypropylene is polypropylene with product number PD701NW, sold by Montell Polyolefins in Wilmington, Delaware. Montell's material is easy to mold, chemically compatible with the preferred mode of operation of the analyzer 50, and has a small number of electrostatic discharge events that can interfere with the accurate detection or quantification of chemiluminescence. used.

0076The bow shield structure 169 is provided at one end of the MTU 160. The MTU operating structure 166 engaged by one of the transfer mechanisms 500 and 502 extends from the shielding structure 169. The MTU operating structure 166 comprises a laterally extending plate extending from a shielding structure 169 having a vertically extending component 167 at the opposite end of the plate 168. The gusseted wall 165 extends downward from the side plate 168 between the shielding structure 169 and the vertical component 167.

0077As shown in FIG. 60, the shielding structure 169 and the vertical component 167 have convex surfaces facing each other. The MTU160 moves the engaging member laterally (in the "A" direction) into the space between the shielding structure 169 and the vertical component 167, as described below, to transfer mechanisms 500 and 502, and other configurations. Engaged by the element. The convex surfaces of the shielding structure 169 and the vertical component 167 provide a wider entrance for engaging members that make lateral relative movements into the space. The convex surfaces of the vertical component 167 and the shielding structure 169 include ridges 171 and 172 formed in the center, respectively. The purposes of the ridges 171 and 172 will be described later.

0078A labeling structure 174 with a flat labeling surface 175 is provided on the opposite side of the shielding structure 169 and the MTU operating structure 166 at one end of the MTU 160. Labels, such as scannable barcodes, are placed on the surface 175 to provide identification and operational information about the MTU160.

0079The MTU160 preferably includes a tip tip gripping structure 176 near the opening of each reaction tube 162. Each tip tip gripping structure 176 includes a cylindrical mouth for accommodating the contact prevention tip tip 170 inside. The configuration and function of the tip tip 170 will be described later. Each grip structure 176 prevents the tip tip 170 from falling out of the grip structure 176 when the MTU 160 is inverted, while the tip 170 is removed from the grip structure 176 when engaged with the pipette. structure to house the tip 170 frictionally are formed and arranged.

0080As shown in FIG. 59, the tip tip 170 comprises a substantially cylindrical structure having a peripheral flange 177 and an upper collar 178, which is generally larger in diameter than the lower 179 of the tip tip 170. The tip tip 170 is preferably made of conductive polypropylene. When the tip tip 170 is inserted into the opening of the grip structure 176, the flange 177 contacts the top of the structure 176 and the collar 178 fits snugly between the tip tip 170 and the grip structure 176. Provides a releaseable tightening fit.

0081The through hole 180 extending in the axial direction passes through the tip of the insert. The hole 180 includes an outwardly extending tip 181 above the tip tip 170, which facilitates insertion of a pipette tubular probe (not shown) into the tip tip 170. The two annular ridges 183 draw a line on the inner wall of the hole 180. The ridge 183 provides a frictional fit between the tip tip 170 and the tubular probe inserted into the tip tip 170.

0082The lower end of the tip tip 170 preferably includes a slope portion 182. When the tip tip 170 is used at the tip of an aspirator that is inserted to the bottom of the reaction vessel, such as the reaction tube 162 of the MTU160, the slope 182 creates a vacuum between the end of the tip tip 170 and the bottom of the reaction tube. Avoid being formed.

0083(Lower chassis) Examples of the lower chassis according to the present invention are shown in FIGS. 52 to 54. The lower chassis 1100 includes a steel frame 1101 with a black polyurethane powder coating, a pull-out water drop tray 1102 located below the chassis, a right side drawer 1104, and a left side drawer 1106. The left drawer 1106 is effectively centered within the lower chassis 1100. The left side of the lower chassis 1100 is preferably mounted on various power system components, such as the seven syringe pumps 1152 mounted on the mounting platform 1154, the vibration isolation device (not shown) on the floor of the lower chassis 1100. Houses other analyzer mechanisms such as pump 1162, power supply unit 1156, power filter 1158, and fan 1160.

0084Different syringe pumps 1152 are designated for each of the five magnetic separation stations 800, one for the left rotary stirrer 552 and the other for the deactivation preparation chamber 750. Syringe pumps are preferred, but peristaltic pumps may be used as an alternative.

0085Vacuum pump 1162 engages each magnetic separation station and deactivation preparation room 750. The preferred rating of the vacuum pump is 5.3 to 6.5cfm at 0 Hg and 4.2 to 5.2cfm at 5 Hg. A preferred vacuum pump is commercially available from Thomas Industries, Inc. (Sheboygan, Wisconsin) under model number 2750CGHI60. The capacitor 1172 is sold with the pump 1162.

0086As the power supply unit 1156, ASTEC model number VS1-B5-B7-03, which is commercially available from ASTEC America, Inc. (Carlsbad, CA), is preferable. The power supply unit 1156 receives power from a 220 volt, or regular 220 volt wall outlet, in the 50-60 Hz range. The power filter 1158 is preferably a Corcom model number 20MV1 filter commercially available from Corcom, Inc. (Libertyville, Illinois). The fan 1160 is preferably a Whisper XLDC fan commercially available from Comair Rotron, San Isidro, CA. Each fan is powered by a 24VDC motor and has an output of 75cfm. As shown in FIG. 52, the fan 1160 is preferably located adjacent to the left outer wall of the lower chassis 1100. The fan 1160 is preferably directed outwards to draw air from the entire lower chassis from right to left, thereby drawing excess heat from the lower chassis.

0087Other power system components are housed on the rear left side of the lower chassis 1100. To do this, use Eaton's Cutler-Hammer Division (Cleveland, Ohio) commercially available Eaton circuit break switch, 2-pole, JA / S series preferred power switch 1174, and analyzer 50 as external power sources. Includes a power supply port module 1176 to which the power cord to be connected (not shown) is connected. The power system for the analyzer 50 also includes a terminal block (not shown), which is preferably a plurality of electrical terminals, Crydom Series 1 model number D2425, commercially available from Cal Switch (Carson City, Calif.). It can be fitted with a solid-state switch (not shown) that switches between different circuits, and a 9-pin RS232 connection port that connects the analyzer 50 to the external computer controller 1000.

0088The right and left drawer bays are preferably closed by the assay manager program during the operation of the analyzer, preferably behind one or two doors in front of the analyzer. It is preferable that a microswitch for verifying the closed state of the door is provided. The leftmost bay is covered by the front panel. End panels are provided on opposite ends of the lower chassis to surround the chassis.

0089The four level legs 1180 extend from the four corners of the chassis 1100. The standard foot 1180 includes a threaded shaft with a pad at the lower end. When the analyzer is in the desired position, the foot 1180 can be lowered until the pad engages the floor to level and stabilize the analyzer. You can also raise your foot and move the analyzer with the casters.

0090Bulk fluid, typically contained in the container of the lower chassis 1100, is a wash solution (to wash the immobilized object), distilled water (fixed pipette tip), diagnostic test reagents, silicon oil (on test reagents and samples). It may contain a bleach-based reagent (used for sample deactivation), as well as a suspended fluid to form a layer.

0091The right drawer 1104 is detailed in FIG. 53. The right drawer 1104 includes a box-shaped drawer structure with a front drawer handle 1105. The drawer handle 1105 is shown as a conventional pull-type drawer handle, but in a preferred embodiment of the analyzer 50, the handle 1105 is a T-handle such as that commercially available from Southco, Inc. (Concordville, PA). It is a latch. The drawer 1104 is attached to a slide bracket (not shown) on the lower chassis, which allows the drawer 1104 to be pulled in and out of the lower chassis. It is preferable to include a sensor (not shown) to verify that the drawer 1104 is closed. The front part of the drawer is bottle 1128 (shown in Fig. 52), which is a bottle containing waste liquid for cleaning with a dedicated pipette, and bottle 1130 (shown in Fig. 52), which is a dedicated waste bottle for storing waste from magnetic cleaning, which is the target capture procedure. Includes a bottle container 1122 for gripping (shown). Bottle 1130 is preferably emptied.

0092Analyzer 50 does not start processing the assay if any of the bottles required for the lower chassis 1100 are missing. The bottle container 1122 preferably includes a bottle presence sensor (not shown) that verifies the presence of the bottle in each container 1122. The bottle presence sensor is preferably a diffuse reflection type optical sensor of model number EX-14A commercially available from SUNX / Ramco Electric, Inc. (West Des Moines, Iowa).

0093The right drawer 1104 further includes a waste box 1108 for holding the used MTU and sample chips. The waste box 1108 is preferably an open box with a sensor mount 1112 for mounting the sensor on top of it, preferably a 24VDC diffuse reflector switch for detecting whether the waste box 1108 is full. It is a structure. Another diffuse light sensor (not shown) is installed in the right drawer 1104 to verify that the waste box 1108 is in the correct position. The diffuse reflection optical sensor of model number EX-14A commercially available from SUNX / Ramco Electric, Inc. (West Des Moines, Iowa) is also preferred here.

0094The deflector 1110 extends diagonally from the side of the waste box 1108. The deflection plate 1110 is placed directly under the chute that passes when the used MTU is dropped into the waste box 1108, deflects the dropped MTU toward the center of the waste box 1108, and the MTU deflects the dropped MTU toward the center of the waste box 1108. Prevents accumulation in one corner. The deflector 1110 is preferably mounted pivotally so that it can pivot upwards to a substantially vertical position, thereby covering the interior of the waste box 1108 and the waste bag covering the deflector 1110 is waste. When removed from the box 1108, the deflection plate 1110 pivots upward with the bag being pulled out so that it does not tear the bag.

0095The printed circuit board (not shown) and cover 1114 can be mounted on the front of the waste box 1108. The sensor mount and 1117 are also mounted on the front of the waste bin 1108. The sensors 1118 and 1119 are mounted on the sensor mount 1116, and the sensors 1120 and 1121 are mounted on the sensor mount 1117. The sensors 1118, 1119, 1120, and 1121 are preferably DC capacitive proximity sensors. Upper sensors 1118 and 1119 point out that bottles 1128 and 1130 are full, and lower sensors 1120 and 1121 point out that bottles are empty. The sensors 1118 to 1121 are preferably those of model number C2D45AN1-P commercially available from Stedham Electronics Corporation (Rino, Nevada). The reason for this selection is that due to its relatively flat physical shape, the lower chassis 1100 occupies less space within a narrow range and the Stedham sensor provides a preferred detection distance of 3-20 mm.

0096It is preferred that the analyzer 50 not initiate any assay if the assay manager program detects that any of the waste fluid vessels in the right drawer 1104 is not empty from the beginning.

0097Capacitive proximity sensors 1118 to 1121 and right drawer 1104 bottle presence, waste box presence, waste box full light sensor are connected to a printed circuit board (not shown) on the back of cover 1114, and the printed circuit board is analyzed. Connected to the built-in controller of vessel 50.

0098The right drawer 1104 cannot be fully pulled out of the lower chassis 1100, so it must be possible to pull the waste bin 1108 forward so that it can reach the waste bin to install or remove the waste bag liner. is there. For this purpose, when the handle 1126 is attached to the front of the waste box 1108, the Teflon® piece 1124 is placed on the bottom floor of the right drawer 1104, and the bottles 1128 and 1130 are removed, inside the drawer 1104. Facilitates sliding back and forth of the waste box 1108.

0099Details of the left drawer 1106 are shown in FIG. The left drawer 1106 includes a box-like structure with a front mounting handle 1107 and is mounted on a slide bracket (not shown) on the lower chassis 1100. The handle 1107 is shown as a conventional pull-out drawer handle, but in a preferred embodiment of the analyzer 50, the handle 1107 is a T-handle latch such as that commercially available from Southco, Inc. (Concordville, PA). Is. It is equipped with a sensor and verifies that the drawer 1106 is closed.

0100The left drawer 1106 includes a tip tip waste box 1134 having a mounting structure 1135 for mounting a tip tip waste box full sensor (not shown). It is preferred that the tip tip waste box presence sensor be provided in the left drawer 1106 to verify that the tip tip waste box 1134 is installed correctly. The diffuse reflection type optical sensor of model number EX-14A commercially available from SUNX / Ramco Electric, Inc. (West Des Moines, Iowa) is preferred for both the chip tip waste box full sensor and the chip tip waste box presence sensor.

0101The bundling structure 1132 is provided to secure and grip various pipes and / or wiring (not shown) within the lower chassis 1100. A preferred bundling structure to use is Energy Chain Systems, manufactured and sold by Igus, Inc. (East Providence, Rhode Island).

0102The printed circuit board 1182 is mounted behind the panel 1184 located behind the chip tip waste box 1134. The solenoid valve mounting panel 1186 is located below the tip waste box 1134.

0103The left drawer 1106 includes a forward vessel gripping structure for gripping six bottles of uniform size. The vessel structure includes partition walls 1153, 1155, 1157, and 1159, and a vessel block 1151 with a curved bottle matching front end, which together define six vessel grip areas. The lower sensor 1148 and the upper sensor 1150 (6 each) are mounted on the partition walls 1155, 1157, and 1159. The upper and lower sensors 1148 and 1150 are preferably DC capacitive proximity sensors (selected from flat shape and detection range, commercially available from Stedham Electronics Corporation, Reno, Nevada), model number C2D45AN1-P. sensor). The upper sensor 1150 points out that the bottle gripped by the container structure is full, and the lower sensor 1148 points out that the bottle is empty. In a preferred arrangement, the two left bottles 1146 contain a detector (Detection I), the two central bottles 1168 contain silicone oil, and the two right bottles 1170 contain another detector (Detection II). ) Is preferably included.

0104To verify the presence of bottles in each container grip area, a bottle presence sensor (not shown) is provided in each container grip area defined by the container block 1151 and the bulkheads 1153, 1155, 1157, and 1159. Is preferable. As the bottle presence sensor, a diffuse reflection type optical sensor of model number EX-14A commercially available from SUNX / Ramco Electric, Inc. (West Des Moines, Iowa) is preferable.

0105The centrally located large container receiver 1164 grips the bottle 1140 (shown in FIG. 52), which preferably contains deionized water. Container receiver 1166 (shown in FIG. 54, only one) grips bottles 1142 and 1144 (shown in FIG. 52), which preferably contain a wash solution. The partition 1143 between the containers 1164 and 1166 is fitted with sensors such as the sensor 1141 for monitoring the fluid levels of bottles 1140, 1142, and 1144. The sensor, such as sensor 1141, is preferably a DC capacitive proximity sensor (sensor model number C2D45AN1-P, commercially available from Stedham Electronics Corporation, Reno, Nevada).

0106The container receivers 1164 and 1166 preferably include a bottle presence sensor (not shown) to verify that the bottles are properly placed in their respective containers. As the bottle presence sensor, a diffuse reflection type optical sensor of model number EX-14A commercially available from SUNX / Ramco Electric, Inc. (West Des Moines, Iowa) is preferable.

0107Analyzer 50 does not initiate any assay if the assay manager program determines that any of the bulk fluid vessels in the left drawer 1106 is empty from the beginning.

0108Capacitive proximity fluid level sensors, various bottle presence sensors, chip tip waste box full sensors, and chip tip waste box presence sensors are all connected to printed circuit board 1182, which is connected to the built-in controller of analyzer 50. Will be done.

0109The four solenoid valves (not shown) are mounted below the solenoid valve mounting panel 118. The solenoid valve is a bulk fluid bottle in which the fluid is stored in a pair of bottles, namely bottles 1140 and 1142 containing the cleaning solution, two bottles 1146 containing the "Detection I" agent, two bottles 1168 containing the oil, and Connect two bottles 1170 containing the "Detection II" agent. The solenoid valve responds to signals from each capacitive proximity sensor and replaces the bottle from which the fluid is taken out when one of the two bottles containing the same fluid is empty. In addition, the solenoid valve may be bottle-replaced after performing a specified number of tests. A preferred solenoid valve is Beco Manufacturing Teflon (registered trademark) solenoid valves of model numbers S313W2DFRT and M223W2DFRLT commercially available from Co. Inc. (Laguna Hills, CA). These two different model numbers correspond to solenoid valves adapted for the use of two different sized tubes. Teflon (registered trademark) solenoid valves are preferred because they are less likely to contaminate the fluid flowing through the valve and are not damaged by the corrosive fluid flowing through the valve.

0110Bottle 1136 (see FIG. 52) is a vacuum trap held by the vacuum trap bracket 137, which contains an inactive agent such as a bleach-containing reagent. Again, it is preferred that a bottle presence sensor be provided to verify the presence of bottles 1136 and 1138.

0111A handheld barcode scanner 1190 may be provided on the lower chassis 1100 to scan the information provided on the scannable container label into the assay manager program. The scanner 1190 is preferably corded to the printed circuit board 1182 of the left drawer 1106 and housed in a bracket (not shown) attached to the bulkhead 1143. LS2100 series scanners commercially available from Symbol Technologies, Inc. (Holtsville, NY) are preferred.

0112(Sample ring and sample tube tray) The sample contained in the sample tube 320 and the tube 320 are loaded into the tube tray 300 outside the analyzer 50. The tray 300 carrying the sample tube 320 is installed on the sample ring 250 through the reach opening provided by opening the flip-up turntable door 80.

0113Referring to FIGS. 5 and 6, the first ring assembly or sample ring 250 is formed from uncured ground aluminum and extends through a groove 251 around the outer periphery of a ring 250 having a plurality of raised radial dividers 254. Includes a raised ring structure that defines the annular groove 251. Preferably, nine dividers 254 divide the groove 251 into nine bowed sample tube tray storage wells 256. Grooves 251 and wells 256 define an annular fluid container carrier that is configured and arranged to carry a plurality of containers, as described below.

0114The sample ring 250 has three 120 ° spaced V-grooves that engage a continuous V-ridge 262 formed on the inner circumference of the ring 250, as shown in FIGS. 5, 6, and 6A. It is preferred that the ring 250 be rotationally supported by rollers 257, 258, and 260 so that the ring 250 is rotatable about a first central axis of rotation. Laura is preferably model number W1SSX manufactured by Bishop-Wisecarver Corp. in Pittsburg, California. The rollers 257 and 260 are rotationally attached to a fixed shaft, which springs around a vertical axis and spring-biased to urge the rollers 258 radially outward with respect to the inner circumference of the ring 250. Attached to the bracket to be. With two fixed rollers and one radially movable roller, the three rollers can accommodate the non-circular inner circumference of the ring 250.

0115The sample ring 250 is a stepper motor via a continuous belt 270 extending around the outside of the ring 250 on the guide rollers 266 and 268 (preferably model number A6R3M444080 commercially available from SDP / SI (New Hyde Park, NY)). It is driven by 264 (Preferably VEXTA stepping motor of model number PK266-01A, which is commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan)). Home and sector sensors (not shown), preferably slotted light sensors, are provided adjacent to the ring 250, in a position corresponding to one of the rotating home position and sample tube tray storage well 256. Ring 250 includes a home flag (not shown) located in the home position on the wheel and nine equidistant sector flags (not shown) corresponding to each of the nine sample tube tray storage wells 256 positions. The home flag and sector flag, in cooperation with the home and sector sensors, provide ring position information to the assay manager program and also control the ring 250 for user reloading and arrival by the pipette unit 450. Stop at nine discrete positions corresponding to the coordinates specified in. The preferred sensor as the home sensor and sector sensor is the Optek slotted optical sensor of model number OPB857, commercially available from Optek (Carolton, Texas).

0116The sample cover is arranged over the annular fluid container carrier or portion of the groove 251 and comprises an arched cover plate 138 that is secured in an elevated position with respect to the wheels 250 of the three mounting columns 136. The plate 138 has a bow shape that substantially matches the curved surface of the groove 251. The first opening 142 is formed in the plate 138, and the second opening 140 is such that the radial distance of the plate 138 from the axis of rotation of the ring 250 is larger than that of the opening 142 and circumferentially from the opening 142. It is formed at spaced positions.

0117With reference to FIGS. 55-57, each sample tube tray 300 comprises a tester standing structure that curves to match the curvature of the ring 250. Each tray 300 comprises a central wall structure 304 having side end walls 303 and 305 located at any end of the wall 304. Floor 312 extends across the bottom of tray 300. The main purpose of the sample tube tray 300 is to grip the sample tube into the sample ring 250 for the sample pipette assembly 450 to reach, and to facilitate loading and unloading of multiple sample tubes into the analyzer.

0118The plurality of Y-shaped partitions 302 are equidistantly spaced along the ends facing the tray 300. Each of the two adjacent dividers 302 defines a test tube storage area 330. The end wall 303 includes inward bending flanges 316 and 318, and the end wall 305 includes inward bending flanges 326 and 328. The inwardly bent flanges of the end walls 303 and 305, together with the ends of the partition 302, define the end tube storage area 332. Storage areas 330 and 332 are opposite to the central wall structure 304 and are bowed along two bow rows.

0119With reference to FIG. 57, the leaf spring element 310 is attached to the central wall 304 within each of the tube storage areas 330, 332. The leaf spring element 310, preferably formed of stainless spring steel, elastically deflects when inserted into the tube storage area 330 or 332 by the examiner 320, prompting the tube 320 outward with respect to the partition 302. .. As a result, the pipe 320 is secured in the upright direction. The shape of the partition 302 and the elasticity of the leaf spring element 310 allow the tray 300 to accommodate sample tubes of various shapes and sizes, such as tubes 320 and 324. Each tray 300 contains 9 dividers 302 along their respective ends and 10 tube storage areas on each side of the central wall structure 304 (20 in total on one tray) along the end walls 303 and 305. It is preferable to form 330 and 332. A mark for designating the tube storage areas 330 and 332, such as a convex number notation, may be provided on the central wall 304 of the tray or the like.

0120Each tray 300 may also include a boss structure 308, which is shown integrally with the end partition 302 in the illustration of the embodiment. A vertical inverted U-shaped (not shown) handle (not shown) may be attached to the tray boss structure 308 or other suitable location. The vertical handle facilitates operation of the tray 300 when loading and unloading the tray 300 through the bow turntable door 80, but is not always preferred.

0121A gap is provided between the adjacent dividers 302 so that the barcode label 334 on the tube 320, or other readable or scannable information, is reachable when the tube is installed in the tray 300. Become. As the tray 300 carried to the wheel 250 passes under the plate 138 of the sample cover, one tube 320 in a curved row, radially inward with respect to the wall structure 304, is the first. Another tube 320, alongside the opening 142, in a curved row, located radially outward with respect to the wall 304, is aligned with the second opening 140. The ring 250 is instructed to sequentially move each tube 320 under the openings 140 and 142 to allow access to the tube.

0122With reference to FIG. 5 again, the barcode scanners 272 and 274 are located close to the ring 250. The Opticon, Inc. scanner of model number LHA2126RR1S-032, commercially available from Opticon, Inc. (Orangeburg, NY), is preferred. The scanner 272 is located outside the ring 250 and the scanner 274 is located inside the ring 250. Scanners 272 and 274 are positioned to scan the bar code data label of each sample tube 320 that is transported to sample tube tray 300 as the ring 250 rotates tray 300 of sample tube 320 and passes through scanners 272 and 274. Be done. In addition, scanners 272 and 274 scan the bent flanges 316 and 318 outer bar code labels 337 (see Figure 55) of the end walls 303 of each tray 300 as the trays 300 reach the sample preparation area. Various information, such as sample and assay identification, can be provided on the tubes and / or trays 300, which can be scanned by scanners 272 and 274 and stored in a central processing unit. In the absence of a sample tube, tray 300 presents a special code 335 (see Figure 55) that is read by scanners 272 and 274.

0123Preferred sample tube holders are disclosed in Knight et al., US Provisional Application No. 60 / 672,609, "Sample Tube Holder," which enjoys co-ownership. Knight discloses a sample tube holder with multiple sample tube compartments with a series of side-by-side finger springs for gripping the sample tube in a fixed vertical direction. If the sample tube is capped with a penetrating lid, the sample tube holder includes a fixture to keep the sample tube within the sample tube during the sampling procedure. For example, see US Pat. No. 6,893,612, "Penetrable Cap," by Kacian et al. (A sample tube capped with a cap with a fragile seal or filter to limit the spray of contaminants or air bubbles. Disclose).

0124(Pipette tip wheel) The second ring assembly of the preferred embodiment was a pipette tip wheel 350, primarily as shown in FIGS. 5 and 6, with a circular ring 352 at the bottom, a circular inner circumference and five circumferential spacings. The upper panel 374, which defines the radially protruding area 370, and the upper panel 374 are held in place by a mechanical fastener 356 that separates the upper panel 374 from the ring 352 and extends into the riser 354 through the upper panel 374 and the ring 352. A plurality of substantially rectangular risers 354 are provided. Five rectangular openings 358 are formed in close proximity to each of the areas 370 of the upper panel 374, and one rectangular box 376 is placed under the panel 374, one for each opening 358. The top panel 374, ring 352, and riser 354 are preferably made of machined aluminum, and the box 376 is preferably made of stainless steel plate material.

0125The opening 358 and the associated box 376 are configured and arranged to accommodate a tray 372 that holds a plurality of disposable pipette tips. The pipette tip tray 372 is manufactured and sold by TECAN (TECAN US Inc., Research Triangle Park, North Carolina), and the brand name "Disposable Tips for GENESIS Series" is desirable. Each chip has a capacity of 1000 μl and is conductive. Each tray holds 96 elongated disposable tips.

0126Horizontal slots 378 and vertical slots 380 are formed in the upper panel 374 along the lateral and longitudinal edges of the openings 358, respectively. Slots 378 and 380 accommodate downwardly extending flanges (not shown) arranged along the lateral and longitudinal edges of tray 372. The flanges of slots 378 and 380, and the associated tray 372, serve to accurately align the tray 372 with the opening 358 and grip the tray 372 in the correct position on the panel 374.

0127The pipette tip wheel 350 engages three 120 ° spaced V-grooves that engage a continuous V-ridge 362 formed on the inner circumference of the ring 352, as shown in FIGS. 5, 6, and 6A. It is preferred that the pipette tip wheel 350 be rotationally supported by rollers 357, 360, and 361 and be rotatable about a second central axis of rotation that is approximately parallel to the first axis of rotation of the sample ring 250. .. Laura is Bishop-Wisecarver The model number W1SSX manufactured by Corp. (Pittsburg, CA) is preferred. The rollers 357 and 360 are rotationally attached to a fixed shaft, which springs around a vertical axis and spring-biased to urge the rollers 361 radially outward with respect to the inner circumference of the ring 352. Attached to the bracket to be. With two fixed rollers and one radially movable roller, the three rollers can accommodate the non-circular inner circumference of the ring 352. In addition, the wheel 350 simply propels the pivot roller 361 inward in the radial direction, moves the ring 352 laterally, and disengages the continuous V ridge 362 from the fixed V-groove rollers 357 and 360. Can be installed and removed.

0128The pipette tip wheel 350 is driven by a motor 364 with spur gears mounted on a shaft that meshes with meshing gear teeth formed on the outer circumference of the ring 352. As the motor 364, a VEXTA gear head stepping motor of model number PK243-A1-SG7.2, which has a 7.2: 1 gear reduction and is commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan), is preferable. A gearhead stepper motor with 7.2: 1 gear reduction is preferred because the spur gear of the motor 364 engages directly with the ring 352 to provide smooth operation of the pipette tip wheel.

0129Home and sector sensors (not shown), preferably slotted light sensors, are provided in one of the rotating home position and box 376, adjacent to the pipette tip wheel 350. The pipette tip wheel 350 includes a home flag (not shown) located in the home position on the wheel and five equidistant sector flags (not shown) corresponding to each position of the five boxes 376. The home and sector flags work with home and sector sensors to provide wheel position information to the assay manager program and to control the pipette tip wheel 350 for user reloading and reach by pipette unit 450. Stop at five discrete positions corresponding to the coordinates specified for. The preferred sensor as the home sensor and sector sensor is the slotted optical sensor of Opter Technology, Inc., model number OPB980, commercially available from Optek Technology, Inc. (Carolton, Texas).

0130(Multi-axis stirrer) Referring to FIGS. 7-12, the multi-axis stirrer 400 is secured to the jig plate 130 using a mechanical fastener (not shown) extending through an opening 419 formed around the outer periphery of the fixed base 402. Includes a turntable structure 414 (see Figure 10) that is rotatably attached to the center shaft 428, supported by the center bearing 430 of the base 402. The cover member 404 is attached to the turntable 414 and rotates together.

0131The turntable 414 consists of three rectangular arms 444 of the same length, spaced 90 ° apart, extending radially outward from the center of the turntable 414, and an extension that makes the arm 445 slightly longer than the arm 444. It is preferably a right-angled cross with a fourth arm 445 having a portion 417. As shown in FIGS. 10 to 12, the central portion of the turntable 414 is connected to the central shaft 428 by a screw 429.

0132The four container holders 406 are located at the tips of arms 444 and 445 of the turntable frame 414. Each container holder 406 is attached to one of four vertical shafts 423 rotatably supported by the container holder bearing 415. The container holder bearing 415 is pushed into the arms 444 and 445 of the turntable 414 and is located at a uniform radial distance from the shaft 428.

0133The cover member 404 includes four circular openings having an upwardly rotated peripheral flange 401 through which the shaft 423 extends. The upward flange 401 advantageously prevents spilled liquid from flowing into the opening.

0134The container holder 406 comprises a substantially cylindrical member having a bottom opening and a top opening for storing and gripping the container 440 of the target capture reagent, preferably a plastic bottle.

0135Target capture reagents used in preferred assays include immobilized polynucleotides, polynucleode capture probes, and magnetically reactive particles with sufficient reagents to lyse cells containing the nucleic acid of interest. After cytolysis, the target nucleic acid comprises one or more capture probes, each of which has a nucleotide base configuration region and is capable of hybridizing the nucleotide base region contained in at least one of the target nucleic acids. It can be used for hybridization under the first predetermined hybridization condition group. Under the second defined hybridization condition group, the homopolymeric ends of the immobilized polynucleotide (such as oligo (dT)) hybridize with the complementary homopolymeric ends (such as oligo (dA)) contained in the capture probe. , It is possible to immobilize the nucleic acid of interest. Various target capture methods and dissolution procedures are well known in the art and are readily adapted for use with the analyzer 50 of the present invention. This preferred two-step capture method for capturing and immobilizing the nucleic acid of interest in magnetically reactive particles is disclosed by Weisburg et al. In US Pat. No. 6,534,273.

0136The container fixture spring 408 extends the container 440 by propelling the container 440 over a lateral slot formed in the wall of each container holder 406 toward a portion of the inner peripheral wall of the holder 406 opposite the spring 408. Assists in gripping inside the container holder 406.

0137Each container holder 406 is secured to the associated vertical shaft 423 by a shaft block structure 432. The shaft block structure 432 includes a curved end that matches the interior of the cylindrical container holder 406, which is secured to the block 432 by fasteners 434. The substantially circular opening 449 accommodates the shaft 423. Slot 438 extends from the opening 449 to the tip of block 432, which does not extend into the interior of the container holder 406, second slot 436 extends from the tip of block 432 approximately at right angles to slot 438 and cantilevered arm. Define 435. The machine screw 437 extends through a through hole 441 formed laterally through the block 432 and into a threaded hole 447 formed laterally through the arm 435. When the screw 437 is tightened, the arm 435 deflects, thereby tightening the opening 449 around the shaft 423.

0138The shaft block structure 432, shaft 423, and container holder bearing 415 associated with each container holder 406 attach the container holder 406 to the turntable 414, allowing the container holder 406 to rotate about the axis 412 of the shaft 423. Define a preferred container holder mounting structure associated with each container holder 406, which is configured and arranged to do so.

0139The container holder planetary gear 422 is attached to the opposite end of the shaft 423. The planetary gear 422 operably engages the fixed sun gear. The drive pulley 418 is attached to the central shaft 428 and is connected to the drive motor 420 by a drive belt (not shown). The drive motor 420 is preferably mounted so as to extend through an opening (not shown) of the jig plate 130 below the pedestal 402. The drive motor 420 is preferably a stepping motor, and most preferably a VEXTA stepping motor of model number PK264-01A, which is commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan). The drive motor 420 rotates the central shaft 428 and the turntable 414 attached thereto via the drive belt and the drive pulley 418. When the turntable frame 414 rotates around the center line of the center shaft 428, the planetary gear 422 that engages with the sun gear 416 attaches the shaft 423 and the container holder 406 attached to it to the tip of the arm 444 of the turntable frame 414. Rotate. Each container holder 406 is preferably mounted so that its axis 410 is offset from the axis 412 of the associated shaft 423. Thereby, each container holder 406 rotates eccentrically about the axis 412 of the associated shaft 423. As a result, the planetary gear 422 and the sun gear 416 are configured and arranged so that when the turntable 414 rotates about the axis of rotation of the shaft 428, the container holder 406 rotates about the respective axis of rotation of the shaft 423. It constitutes a motion connecting element.

0140The barcode scanner device 405 is preferably attached to the bracket 403 and reads the barcode information of the container 440 through a scanner slot 407 formed in each container holder 406. A preferred scanner is the scanner model number NFT1125 / 002RL, commercially available from Opticon, Inc. (Orangeburg, NY).

0141The multiaxial stirrer 400 typically rotates during the operation of the analyzer 50 to agitate the fluid contents of the vessel 440, thereby keeping the target capture reagent in suspension and the pipette unit 456 in the vessel. Stop only for a short time so that a certain amount of mixture can be taken out from one. The pipette unit 456 removes the mixture of bottles in the same position each time. Therefore, it is preferable to monitor the position of the bottle so that the bottle from which the mixture is drawn can be identified each time.

0142Four slotted light sensors 426, each containing a photodetector and a detector, are deployed around the fixed base 402 at 90 ° intervals. An optical sensor of model number OPB490P11, commercially available from Optek Technology, Inc. (Carolton, Texas), is preferred. The sensor tab 424 extends downward from the extension 417 at the tip of the arm 445 of the turntable 414. When the sensor tab 424 passes through the sensor 426, communication between the ejector and the detector is cut off, thus giving a "container presence" signal. Tab 424 is provided in only one place, such as the first container position. By knowing the position of the first container, the position of the remaining containers fixed to the first container can also be known.

0143Power and control signals are provided to the multi-axis stirrer 400 via the power and data connectors. The multi-axis stirrer 400 performs mixing by rotation and eccentric rotation, but other mixing methods such as vibration and rotation may also be used.

0144(Sample preparation procedure) To initiate sample preparation, the pipette unit 456 is transported from container 440 to the multiaxial stirrer 400 to move the target capture reagent, which is preferably a magnetic oligo reagent, into each reaction tube 162 of the MTU 160. Move to. The Target Capture Reagent comprises a supporting material that can bind and immobilize the Target Specimen. The supporting material preferably comprises magnetically reactive particles. At the beginning of the sample preparation procedure, the pipette unit 456 of the right pipette assembly 450 moves laterally and longitudinally until the probe 457 is operably positioned on the pipette tip in one of the trays 372.

0145The tip tray 372 is transported to the pipette tip wheel 350 so that it is placed in the correct position to achieve proper alignment of the pipette tip of the pipette unit 456 with the tubular probe 457. The pipette unit 456 moves downward to insert the free end of the tubular probe 457 into the open end of the pipette tip and frictionally engage the pipette tip. The Cavro processor preferably used for the pipette unit 456 includes colors (not shown) specific to the Cavro processor. This collar moves slightly upwards as the pipette tip frictionally engages the tip of the tubular probe 457, and the displaced collar activates the electrical switch on the pipette unit 456 to verify the presence of the pipette tip. .. If tip collection is unsuccessful (due to lack of tip in tray 372, aiming error, etc.), a tip shortage signal is generated and the pipette unit 456 moves to another tip position to engage the tip. Retry.

0146The assay manager program stops the rotation of the multi-axis stirrer 400 for a short time so that the pipette unit 456 has a pipette tip 457 attached to the tubular probe 457 of the pipette unit 456 on one of the stationary vessels 440. You can move to a lined up position. The pipette unit 456 lowers the pipette tip attached to the tubular probe 457 into the container 440 and removes the desired amount of target capture reagent into the pipette tip. The pipette unit 456 then moves the probe 457 out of the container 440, the multiaxial stirrer 400 resumes rotation, and the pipette unit 456 moves above the opening 252 and the sample transfer station 255. The pipette unit 456 then descends while moving the pipette tip and tubular probe 457 through the opening 252, dropping the required amount of subject capture (typically 100-500 μl) onto one or more of the reaction tubes 162 of the MTU 160. To do. It is preferred that the target capture reagent is incorporated only into the pipette tip and not into the probe 457 itself. In addition, the pipette tip preferably has a volume sufficient to grip sufficient reagents for all five reaction tubes 162 of the MTU 160.

0147After moving the target capture reagent, the pipette unit 456 moves to the "tip discard" position above the tip discard tube 342, and the disposable pipette tip is extruded or ejected from the tip of the tubular probe 457 of the pipette unit 456 and tube 342 It falls through and into a solid waste container. An optical sensor (not shown) is placed in close proximity to tube 342 and the sample pipette assembly 450 moves the pipette unit 456 to the sensor's detection position before the tip is discarded. The sensor detects if the tip is engaged with the tip of the tubular probe 457 and verifies that the tip is gripped by the tubular probe 457 of the pipette unit 456, thereby throughout sample preparation. , Make sure the tip is on the tubular probe 457. A preferred sensor is a wide-gap slotted optical sensor of model number OPB900W, commercially available from Optek Technology, Inc. (Carolton, Texas).

0148The pipette tip is preferably ejected by a collar (not shown) on the tubular probe 457 of the pipette unit 456. The collar engages the hard stop as the tubular probe 457 rises, resulting in the collar retaining and engaging the top edge of the pipette tip as the probe 457 continues to rise, pushing it out of the tubular probe 457. ..

0149After pipetting the subject capture and discarding the pipette tip, the probe 457 of the pipette unit 456 can be washed with running distilled water through the tubular probe 457 at the tip wash station basin 346. The tip wash water is collected and drains into the waste liquid container below.

0150Following the reagent dropping procedure, the pipette unit 456 of the right pipette assembly 450 moves laterally and vertically until the tubular probe 457 of the pipette unit 456 is centered on a new pipette tip in one of the tip trays 372. To do. After successful tip engagement, the pipette unit 456 moves back to close to the sample preparation opening 252 on the sample ring 250 and test samples from the sample tube 320 alongside one of the openings 140 and 142 of the cover plate 138. Take out (about 25-900 μl). Note that both openings 140 and 142 include a peripheral flange that extends upward to prevent any fluid spilling onto the plate 138 from flowing into the openings 140 and 142. The pipette unit 456 then travels over the MTU160 of the sample transfer station 255, moves down through the opening 252, and drops the test sample into one of the reaction tubes 162 of the MTU160 containing the target capture reagent. The pipette unit 456 is then moved to the "tip disposal" position above the tip disposal tube 342, where the disposable pipette tip is ejected into the tube 342. The pipette unit 456 then collects a new disposable pipette tip from the pipette tip wheel 350, the sample ring 250 directs the new sample tube to be reachable by the pipette unit 456, and the unit 456 moves to the sample tube. The sample fluid is removed into a disposable pipette tip, and the pipette unit 456 is then moved above the sample transfer station 255 to drop the sample fluid into a different reaction tube 162 containing the target capture reagent. This process is preferably repeated until all five reaction tubes 162 contain a combination of fluid sample and target capture reagent.

0151Alternatively, depending on the assay protocol or protocol performed by analyzer 50, the pipette unit 456 may drop the same test sample material into two or more of the reaction tubes 162, the analyzer being similar to each partial sample. Alternatively, different assays can be performed.

0152As mentioned above for pipette units 480 and 482, pipette unit 456 also has volume level sensing capability. The pipette tip used at the tip of the tubular probe 457 is preferably made of a conductive material, which allows the pipette unit 456 to detect the volume level even when the tip is transported to the tip of the tubular probe 457. It will be possible. When the pipette unit completes the test sample dropping procedure, the pipette unit 456 moves the tubular probe 457 downwards back into the reaction tube 162 until a change in volume detects the highest fluid level. Note the vertical position of the tubular probe 457 to determine if the reaction tube 162 contains the appropriate amount of fluid material. Due to the solidification of the test sample, the reaction tube 162 may not contain sufficient material, which causes the tip of the tubular probe 457 to solidify, preventing proper suction of the test sample material to the tip, and / or It may prevent proper dropping of the test sample from the chip.

0153After moving the sample, the pipette tip is discarded in the tip disposal tube 342 as described above. Again, the tubular probe 457 of the unit's pipette can be washed with distilled water if desired, but in the preferred method of operation, washing the probe is usually not necessary as the sample material only contacts the disposable pipette tip. Absent.

0154The assay manager program includes pipette unit control logic that controls the operation of pipette units 456, 480, and 482. Also, this is never to the pipette unit 456, except when the pipette unit 456 positions the tubular probe 457 on the sample tube 320, or when the sample tube 320 is below the plate 138 of the sample cover, to retrieve the test sample. It is preferable to move the sample ring 250 so as not to pass over the sample tube 320. This method avoids the entry of fluid droplets from the tubular probe 457 of the pipette unit 450 into another sample tube, which can also cause secondary contamination.

0155Following sample preparation, the MTU160 is moved from the sample transfer station to the right rotary stirrer 550 by the right transfer mechanism 500 to mix the sample / reagent mixture. Further details of the rotary stirrer 550 and 552 will be described later.

0156After the MTU160 is removed from the sample transfer station by the right transfer mechanism 500, the reaction vessel shuttle assembly in the loading preparation chamber 150 advances the next MTU to the collection position by the right transfer mechanism 500, and the right transfer mechanism 500 , Move the next MTU to the sample transfer station. The sample preparation procedure is repeated for this next MTU.

0157(Transfer mechanism) Here, the details of the right and left transfer mechanisms 500 and 502 will be described. Referring to FIGS. 13-16, the right transfer mechanism 500 (similar to the left transfer mechanism 502) is a hook mount that is slidably displaceable in the radial direction of slot 510 on the plate 512 in the illustrated embodiment. It has an operating hook member that includes an extendable distribution hook 506 that extends from structure 508. The housing 504 at the top of the plate 512 has an opening 505 configured to house the top of the MTU 160. A stepper motor 514 mounted on the plate 512 rotates the threaded shaft 516, which, in conjunction with the lead thread mechanism, is a component of the motor 514 and threaded shaft 516 of the preferred hook member drive assembly, the distribution hook 506. Is moved from the extension position shown in FIGS. 13 and 15 to the contraction position shown in FIG. The stepping motor 514 is preferably a modified HIS series 46000. HIS stepper motor is Haydon Switch and Commercially available from Instrument, Inc. (Waterbury, Connecticut). The HIS motor is modified by cutting a thread from one end of the threaded shaft 516 so that the shaft 516 can accommodate the hook mounting structure 508.

0158The housing 504, motor 514, and plate 512 are preferably covered with a matching shroud 507.

0159As shown in FIG. 16, the stepping motor 518 rotates the pulley 520 via the belt 519. (Preferably VEXTA stepper motors with model number PK264-01A, commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan), and SDP timing belts with model number A6R51M200060, commercially available from SDP / SI (New Hyde Park, NY). The pulley 520 is preferably a custom-made pulley having 162 axial grooves arranged around it. Using a uniquely molded mounting block 523, the main shaft 522 fixed and mounted to the plate 512 extends downward through the base 524 and is secured to the pulley 520. The base 524 is attached to the reference plate 82 using a mechanical fastener that extends through an opening 525 formed around the outer periphery of the base 524. The flex circuit 526 provides power and control signals to the hook mounting structure 508 and motor 514, while at the same time fully pivoting the plate 512 (and the components carried to the plate) and rotating it 340 ° with respect to the base 524. .. The transfer mechanism 500 and 502 assemblies preferably include a hard stop (not shown) at any end of the unit's moving rotation path.

0160The arm position encoder 531 is attached to one end of the main shaft 522. The arm position encoder is preferably an absolute encoder. The A2-series encoder model number A2-SK-315-H by US Digital in Seattle, Washington is preferred.

0161The assay manager program supplies control signals to motors 518 and 514 and the hook mounting structure 508 and commands the distribution hook 506 to engage the MTU operating structure 166 of the MTU 160. With the hook 506 engaged, the motor 514 can be powered to rotate the shaft 516, thereby pulling the hook 506 and MTU 160 back into the housing 504. The slide engagement by the connecting rib structure 164 of the MTU 160, which has the opposing ends 511 of the plate 512 in close proximity to the slot 510, allows the MTU 160 to be securely gripped by the transfer mechanisms 500 and 502. The plate 512 thereby constitutes an element of a preferred vessel transport assembly that is rotatable about a axis of rotation (such as the shaft of shaft 522) and is configured and arranged to house and transport a reaction vessel (such as MTU160). To do. Motor 518 can rotate pulley 520 and shaft 522 via belt 519, thereby rotating plate 512 and housing 504 with respect to base 524. Thus, the rotation of the housing 504 changes the orientation of the MTUs engaged, thereby aligning the MTUs with different stations on the processing desk.

0162Sensors 528 and 532 are provided on the opposite side of the housing 504 and point to the position of the distribution hook 506 on the housing 504. Sensor 528 is a mobile termination sensor and sensor 532 is a home sensor. Sensors 528 and 532 are Optek A slotted optical sensor of model number OPB980T11, commercially available from Technology, Inc. (Carolton, Texas), is preferred. For the home sensor 532, when the hook 506 is in the fully contracted position, the sensor beam is blocked by the home flag 536 extending from the hook mounting structure 508.

0163The MTU presence sensor 530 attached to the side surface of the housing 504 detects the presence of the MTU 160 in the housing 504. Sensor 530 is SUNX / Ramco It is preferably a SUNX infrared sensor commercially available from Electric, Inc. (West Des Moines, Iowa).

0164(Temperature gradient station) One or more temperature gradient stations 700 are preferably located below the jig plate 130 and the sample ring 250 (the figure does not show the temperature gradient stations located below the sample ring 250). After mixing the contents of the MTU 160 in the rotary stirrer 550, the right transfer mechanism 500 may move the MTU 160 from the right rotary stirrer 550 to the temperature gradient station 700 by assay protocol.

0165The purpose of each gradient station 700 is to adjust the temperature of the MTU 160 and its contents up and down as desired. The MTU and its contents may be adjusted to a temperature close to that of the incubator before inserting the MTU into the incubator to avoid large temperature fluctuations in the incubator.

0166As shown in FIGS. 17-18, the temperature gradient station 700 includes a housing 702 into which the MTU 160 can be inserted. Housing 702 includes mounting flanges 712 and 714 for mounting the gradient station 700 to the reference plate 82. A thermoelectric module 704 (also known as a Peltier element) that thermally contacts the heat sink structure 706 is attached to the housing 702, preferably the bottom 710. The preferred thermoelectric module is preferably of model number CP1.4-127-06L, commercially available from Melcor, Inc. (Trenton, NJ). Although one thermoelectric module 704 is shown in FIG. 17, the gradient station 700 preferably includes two such thermoelectric modules. Alternatively, the outer surface of the housing 702 can be covered with a Mylar film resistance heating foil material (not shown) to heat the gradient station. Suitable Mylar Membrane Heated Foil is Minco Etched foil commercially available from Products, Inc. (Minneapolis, Minnesota) and from Heatron, Inc. (Levenworth, Kansas). In the case of a rising station (such as a heater), a resistance heating element is preferably used, and in the case of a falling station (such as a cooler), a thermoelectric module 704 is preferably used. The housing 702 is preferably covered with a heat insulating coating structure (not shown).

0167The heat sink structure used in conjunction with the thermoelectric module 704 preferably includes an aluminum block with extending radiating fins 708.

0168Two thermal sensors (not shown) (preferably a thermistor rated at 25 ° C and rated at 10 KΩ) are preferably located on or inside the housing 702 to monitor temperature. The YSI44036 series thermistors commercially available from YSI, Inc. (Yellow Springs, Ohio) are preferred. YSI thermistors are preferred due to their high accuracy and the ± 0.1 ° C compatibility between thermistors provided by the YSI thermistors. One of the thermal sensors is for primary temperature control, sending a signal to the built-in controller to control the temperature inside the gradient station, and the other thermal sensor is the primary temperature control heat. As an auxiliary confirmation of the sensor, it is for monitoring the temperature of the gradient station. The built-in controller monitors the thermal sensor and controls the heating foil or thermoelectric module of the gradient station to keep the desired temperature within the gradient station 700 approximately uniform.

0169The MTU160 can be inserted into the housing and supported on the MTU support flange 718 that engages the connecting rib structure 164 of the MTU160. The notch 720 is formed at the front end of the lateral panel of the housing 702. The notch 720 allows the distribution hook 506 of the transfer mechanism 500 or 502 to engage or disengage the MTU operating structure 166 of the MTU 160, which is inserted all the way into the temperature gradient station 700, by lateral movement to it. And.

0170(Rotary incubator) Following a sufficient temperature rise at the gradient station 700 and continuing to outline the assay procedure, the right transfer mechanism 500 retrieves the MTU from the gradient station 700 and places the MTU 160 inside the TC incubator 600. In the preferred mode of operation of the analyzer 50, the TC incubator 600 incubates the contents of the MTU 160 at about 60 ° C. In some tests, it is important that the annealing incubation temperature does not fluctuate above ± 0.5 ° C and the amplified incubation (discussed below) temperature does not fluctuate above ± 0.1 ° C. Therefore, the incubator is designed to provide a consistent and uniform temperature.

0171Details about the structure and operation of the two embodiments of the rotary incubators 600, 602, 604, and 606 are described here. With reference to FIGS. 19-23C, each incubator has a housing with a substantially cylindrical portion 610 properly mounted within the insulating coating 612 and insulating cover 611 of the reference plate 82.

0172The cylindrical portion 610 is preferably made of nickel-plated cast aluminum, and the metal portion of the cover 611 is preferably cut aluminum. The cylindrical portion 610 is preferably mounted on the reference plate 82 on three or more resin "feet" 609s. The foot 609 is preferably formed of Ultem (R) -1000 supplied by General Electric Plastics. Due to the low thermal conductivity of this material, the foot 609 functions to separate the incubator from the reference plate. The insulation for the insulation 612 and cover 611 is preferably made of 1/2 inch thick polyethylene supplied by Boyd Corporation (Pleasanton, CA).

0173The container reach openings 614 and 616 are formed in the cylindrical portion 610 and the cooperating container reach openings 618 and 620 are formed in the coating 612. For incubators 600 and 602, one of the reachable openings is positioned to be reachable by the right transfer mechanism 500 and the other reachable opening is positioned to be reachable by the left transfer mechanism 502. Incubators 604 and 606 need to be reachable only by the left transfer mechanism 502 and therefore have only one container reach opening.

0174The closing mechanism with swivel doors 622 and 624 is rotatably positioned within openings 614 and 616. Each swivel door 622 and 624 has an MTU slot 626 extending through a solid cylinder. The MTU slot 626 is configured to closely match the outer shape of the MTU160, with the top wider than the bottom. Door rollers 628 and 630 are mounted on the top of each door 622 and 624, respectively. The swivel doors 622 and 624 are actuated by solenoids (not shown) controlled by instructions from the assay manager program to open and close the doors 622 and 624 at appropriate times. Doors 622 and 624 are opened by rotating doors 622 and 624 so that their MTU slots 626 line up with their respective container reach openings 614 and 616, and their MTU slots 626 are opened with reachable openings 614 and 616. It is closed by rotating the doors 622 and 624 so that they extend laterally to. Cylindrical portions 610, covers 611, doors 622 and 624, and floor panels (not shown) form an enclosure that defines the incubation chamber.

0175Doors 622 and 624 are opened for insertion or recovery of the MTU into the incubator, otherwise they are always closed to minimize heat loss in the incubator through the reachable openings 614 and 616.

0176The centrally positioned radial fan 632 is driven by an internal fan motor (not shown). Commercially available from ebm / Papst (Farmington, Connecticut), the Papst model number RER 100-25 / 14 centrifugal fan with a 24VDC motor and a rating of 32cfm is ideal for use in incubators. preferable.

0177Next, referring to FIG. 22, the MTU turntable assembly 671 is a preferred container carrier for transporting a plurality of radially oriented, circumferentially arranged MTU 160s in an incubator. The MTU turntable assembly 671 is carried by an upper plate 642 supported by a cylindrical portion 610 of the housing, but is powered by a rotating motor 640, preferably a stepper motor, supported by the peripheral end of the upper plate 642. It is preferable to be done. The rotary motor 640 is preferably a VEXTA stepping motor of model number PK246-01A, which is commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan).

0178The MTU turntable 671 includes a hub 646 located below the upper plate 642 and connected to the pulley 644 via a shaft 649 extending through the upper plate 642. The pulley 644 is preferably a custom pulley with 162 axial grooves arranged around it and is connected to the motor 640 through a belt 643 so that the motor 640 can rotate the hub 646. The belt 643 is preferably a GT (R) series timing belt commercially available from SDP / SI (New Hyde Park, NY). The hub 646 has a plurality of evenly spaced internal ventilation slots 645, which are optionally divided by a radially oriented, circumferentially arranged partition wall 647. Although only three partition walls 647 are shown in the figure, it is clear that partition walls may be provided all around the hub 646. In a preferred embodiment, the partition wall 647 is omitted. The support disk 670 is attached to the hub 646 and is arranged substantially parallel below the top plate 642. A plurality of MTU grip members 672 extending radially and arranged circumferentially are attached to the bottom of the support disk 670 (for clarity, only three MTU grip members 672 are shown). The MTU grip member 672 has a support ridge 674 extending along its opposite side. Radially oriented MTUs are transported to the MTU turntable assembly 671 within station 676 defined by the circumferentially adjacent MTU grip members 672, and support ridges 674 are transported by the MTU turntable assembly 671. Supports the connecting rib structure 164 of the MTU160.

0179The MTU turntable assembly rotates on a turntable drive shaft to which a drive pulley (644 in the illustrated embodiment) is mounted. The turntable position encoder is preferably attached to the outer tip of the turntable drive shaft. The turntable position encoder preferably includes a combination of a slotted wheel and an optical slot exchange (not shown). The slotted wheels can be connected to the turntable assembly 671 to rotate together, and the optical slot exchange can be fixed stationary to the cylindrical portion 610 or top plate 642 of the housing. The slotted wheel / slot exchange combination can be adopted to point out the rotational position of the turntable assembly 671 and can point out the "home" position (eg, MTU station 676 specifies # 1 station). The position to be is is in front of the reach opening 614). The A2-series encoder model number A2-SK-315-H by US Digital in Seattle, Washington is preferred.

0180The heat source is provided to communicate heat with an incubation chamber defined within the housing of the incubator with the cylindrical portion 610 and cover 611. In a preferred embodiment, an electrical resistance heating foil 660 wrapped in a Mylar membrane surrounds the housing 610, which may also be attached to the cover 611. Preferred Mylar film heating foils are etched foils commercially available from Minco Products, Inc. (Minneapolis, Minnesota) and from Heatron, Inc. (Levenworth, Kansas). Alternative heat sources include internally mounted resistance heating elements, thermoelectric heating chips (Peltiers), or remote heating mechanisms that are thermally connected to the housing by means of conduits or the like.

0181As shown in FIGS. 19 and 22, pipette slots 662 extend through the incubator cover 611, radially arranged pipette holes 663 extend through the top plate 642, and pipette slots 664 each MTU station 676. Formed on the upper support disc 670, the reagent can be pipetted and dropped onto the MTU placed in the incubator. In a preferred embodiment of the analyzer 50 for a preferred mode of operation, only two incubators, the AMP incubator 604 and the hybridization protection assay incubator HYB incubator, include pipette holes 663 and pipette slots 662 and 664. This is because in the preferred mode of operation, fluid is dropped onto the MTU160 while in the incubator only in these two incubators.

0182The two temperature sensors 666, preferably thermistors (10 KΩ at 25 ° C), are located on the top plate 642. The YSI44036 series commercially available from YSI, Inc. (Yellow Springs, Ohio) is preferred. YSI thermistors are preferred due to their high accuracy and the ± 0.1 ° C compatibility between thermistors provided by the YSI thermistors. One of the sensors 666 is for primary temperature control, sending a signal to the built-in controller to control the temperature in the incubator, and the other sensor is an auxiliary to the primary temperature control sensor. As a confirmation, it is for monitoring the temperature of the incubator. The embedded controller monitors the sensor 666 and controls the heating foil 660 and fan 632 to keep the desired temperature inside the incubator housing 610 uniform.

0183When the transfer mechanisms 500 and 502 are ready to load the MTU 160 into the incubator 600, 602, 604, or 606, the motor 640 rotates the hub 646 to open the empty MTU station 676 to the container reach opening 614 ( Or line up with 616). When this is done, the door actuating solenoid correspondingly rotates the swivel door 622 (or 624) a quarter turn and aligns the door's MTU slot 626 with the MTU station 676. Reach opening 614 is exposed to allow installation or removal of the MTU 160. The transfer mechanism 500 or 502 then advances the distribution hook 506 from the retracted position to the extended position, pushing the MTU 160 from the housing 504 through the reach opening 614 into the MTU station 676 in the incubator. When the distribution hook 506 is removed, the motor 640 rotates the hub 646, moves the already inserted MTU 160 through the reach opening 614, and the swivel door 622 is closed again. This arrangement is repeated for subsequent MTUs inserted into the rotary incubator. Incubation of each loaded MTU continues while that MTU advances (counterclockwise) around the incubator towards exit slot 618.

0184The MTU sensor of each MTU station 676 (preferably an infrared light reflecting sensor) detects the presence of MTU160 in the station. Optek Technology, Inc. sensors of model number OPB770T, commercially available from Optek Technology, Inc. (Carolton, Texas), are preferred because they have the ability to withstand the high temperature environments of the incubator and also have these. This is because the sensor has the ability to read the bar code data fixed to the labeling surface 175 of the labeling structure 174 of the MTU 160. In addition, each door assembly (swivel doors 622 and 624) preferably includes a slotted light sensor (not shown) for pointing out the open / closed position of the door. Optek The sensor of model number OPB980T11, commercially available from Technology, Inc. (Carolton, Texas), is preferred because the relatively high resolution it provides allows accurate monitoring of door position. The tilted disk linear stirrer (also known as the wobbler plate) 634 is provided in close proximity to the MTU turntable assembly 671 within the housing 610 and acts as a vessel stirrer. The stirrer 634 comprises a disc that is tilted and mounted on the shaft of the motor 636 that extends into the housing 610 through the opening 635. The motor is preferably a VEXTA stepping motor of model number PK264-01A, which is commercially available from Oriental Motors Corporation (Tokyo, Japan), similar to the motor preferably used for the MTU turntable assembly 671. It is preferable to attach a viscous harmonic damper 638 to the motor 636 to reduce the harmonic speed of the motor, which can stall the motor. A preferred harmonic damper is a VEXTA harmonic damper commercially available from Oriental Motor Co., Ltd. The operation of the tilt disk linear stirrer 634 will be described below.

0185Only two incubators, the AMP incubator 604 and the HYB incubator 606, are equipped with a tilted disk linear stirrer 634, but in the preferred mode of operation, only in these two incubators, fluid drips onto the MTU160 while in the incubator. Because it is done. Therefore, it is only necessary to provide linear agitation of the MTU 160 with the AMP incubator 604 and HYB incubator 606 by the tilt disk linear stirrer 634.

0186To achieve linear agitation of the incubator MTU160 by the linear stirrer 634, the MTU turntable assembly 671 moves the MTU160 alongside the tilted disc linear stirrer 634, and the tilted disc of the tilted disc linear stirrer 634 Engage the MTU operating structure 166 of the MTU160. Motor 636 rotates the tilt disc of the tilt disc linear stirrer 634, and the portion of the tilt disc structure engaged with MTU 160 moves in and out of the radial direction with respect to the wall of the housing 610, thereby the MTU operating structure 166 and the shield Alternately engages with the vertical component 167 of structure 169. As a result, the MTU 160 engaged to the tilt disk linear stirrer 634 is moved in and out of the radial direction, preferably at high speeds, to provide linear stirring of the contents of the MTU 160. In the amplification incubation step of the preferred mode of operation performed in the AMP incubator 604, a stirring speed of 10 Hz is preferred. In the probe incubation step of the preferred mode of operation performed in the HYB incubator 606, a stirring speed of 14 Hz is preferred. Finally, in the preferred mode of operation selective incubation step performed in the HYB incubator 606, a stirring speed of 13 Hz is preferred.

0187The raised bows 171 and 172 are provided between the convex surfaces of the vertical parts 167 and the shielding structure 169 of the MTU160 (see Figure 60) to minimize friction between the MTU160 and the tilted disk linear stirrer 634. In addition, surface contact between the tilted disk linear stirrer 634 and the MTU 160 may be minimized.

0188In a preferred embodiment, the tilted disc linear stirrer 634 is equipped with a sensor to ensure that the tilted disc linear stirrer 634 stops rotating at the "home" position shown in FIG. As the 671 rotates, the MTU operating structure 166 can engage and disengage the tilted disk linear stirrer 634. A preferred "home" sensor is a tilted disc linear stirrer structure and a pin extending laterally from and a slotted optical switch that verifies the orientation of the tilted disc linear stirrer assembly when the pin blocks the optical switch beam.

0189Alternative MTU turntable assemblies and turntable drive mechanisms are shown in Figures 23A and 23C. As shown in FIG. 23A, alternative incubators are generally for cylindrical portions 1610 composed of nickel-plated cast aluminum, cover 1676, which is preferably formed with a machined aluminum shell, cover 1676. Includes insulation 1678, and housing assembly 1650 with an insulating coating 1651 surrounding the cylindrical portion 1610. Similar to the incubator embodiment described above, the incubator may include a linear stirrer mechanism including a linear stirrer motor 636 with a harmonic damper 638. The closing mechanism 1600 (discussed below) operates to close or to allow reach through the container reach opening 1614. Similar to the embodiments described above, the incubator may include one or two reach openings 1614, depending on the placement of the incubator or its function within the analyzer 50.

0190The centrifugal fan 632 is mounted on the bottom of the housing 1650 and is driven by a motor (not shown). The fan cover 1652 is mounted over the fan and includes sufficient openings to allow airflow generated by the fan 632. The turntable support shaft 1654 includes a lower shaft 1692 and an upper shaft 1690, which are split by a support disc 1694. The support shaft 1654 is supported by a downward shaft 1692 extending downward into the fan cover 1652, and is rotatably supported and fixed in the fan cover by bearings (not shown).

0191The MTU turntable 1656 includes an upper disk 1658 with a central 1696. The top surface of the support disc 1694 is mounted engaged with the bottom surface of the central portion 1696 of the upper disc 1658 so that the weight of the turntable 1656 can be supported from below. As shown in FIG. 23C, a plurality of radially spaced station dividers 1660 are mounted under the upper disk 1658. The lower disc 1662 contains a plurality of radial flanges 1682 extending from the annular interior 1688. The radial flange 1682 corresponds in number and spacing to the turntable station divider 1660, and the lower disk 1662 is attached to the bottom of the turntable station divider 1660 with each flange 1682 fixed to one of the associated dividers 1660. It is fixed.

0192The radial flange 1682 defines a plurality of radial slots 1680 between adjacent pairs of flanges 1682. As is clear from FIG. 23C, the circumferential width of each flange 1682 at the inner end 1686 is smaller than the circumferential width at the outer end 1684 of the flange 1682. The tapered shape of the flange 1682 ensures that the opposing sides of slot 1680 are substantially parallel to each other.

0193When the lower disk 1662 is mounted under the turntable station divider 1660, the width along at least a portion of the overall length of each of the flanges is greater than the width of each divider 1660, which is also from the outer edge to the inner edge. It may taper toward. Flange 1684 defines side shelves along adjacent pairs of partitions 1660 and supports MTU160 connecting rib structures 164 inserted into each MTU station 1663 defined between adjacent pairs of dividers 1660. ..

0194The pulley 1664 is secured to the top of the center 1696 of the upper disc 1658, and the motor 1672 is provided on the mounting bracket 1670 over the diameter of the housing 1650, with the opposing tips secured to the cylindrical portion 1610 of the housing. The motor is preferably a Vexta PK264-01A stepper motor, which is connected to the pulley (having a 9: 1 ratio to the motor) by a belt 1666, which is preferably supplied by the Gates Rubber Company. Will be done. The position encoder 1674 is secured to the upper center of the mounting bracket 1672 and is connected to the upper shaft 1690 of the turntable support shaft 1654. Encoder 1674 (preferably A2 series absolute encoder by US Digital Corporation (Vancouver, WA)) points to the rotation position of turntable 1656.

0195The incubator cover is defined by an incubator plate 1676, which is preferably formed from cut aluminum, and a matching cover insulating element 1678. The cover plate 1676 and the insulating element 1678 include an opening suitable for accommodating the encoder 1674 and the motor 1672, and also drip the fluid carried in the incubator as described in connection with the above embodiment. It may also include radial slots formed for the purpose.

0196An alternative preferred closing mechanism 1600 is shown in Figure 23B. The cylindrical portion 1610 of the incubator housing has at least one container reach opening 1614 having outwardly projecting walls 1616 and 1618 extending integrally along the opposing sides of the reach opening 1614 from the cylindrical portion 1610. including.

0197The revolving door 1620 is operably attached to the reach opening 1614 using a door mounting bracket 1636 mounted on top of the reach opening 1614 of the cylindrical portion 1610 of the housing. Door 1620 provides the operation of the vessel through the reach opening 1614 and the first part in which the bow closure panel 1622, and the bow closure panel 1622 cooperate with the protruding walls 1616 and 1618 to close the reach opening 1614. Door mounting between a second portion rotated outward with respect to the reaching opening 1614 to allow a hole 1634 for accommodating mounting columns (not shown) of the reaching opening 1614. Includes a laterally extending hinge plate portion 1628. The internal arched surface of the arched panel 1622 coincides with the arched surface 1638 of the door mounting bracket 1636 and the arched surface 1619 located below the container reach opening 1614, with respect to the surfaces 1638 and 1619. By providing a minimum gap between each surface, the heat loss from it is minimized.

0198The door 1620 is driven by a motor 1642 mounted in the incubator housing with a motor mounting bracket 1640 secured to the bottom of the container reach opening 1614 in the cylindrical portion 1610 of the housing. The motor shaft 1644 is connected to the lower actuating plate 1626 of the revolving door 1620, whereby the rotation of the shaft 1644 is transmitted to the rotation of the revolving door 1620. The motor 1642 is preferably a HIS 7.5 ° / step motor commercially available from Haydon Switch and Instrument, Inc. (Waterbury, Connecticut). The reason for choosing this HIS motor is that it is relatively low cost and the closed assembly 1600 does not require a robust motor with high torque.

0199Door sensors 1646 and 1648 (preferably slotted light sensors) are operably mounted on opposite sides of the door mounting bracket 1636. Sensors 1646 and 1648 can be configured to point out the relative position of the revolving door 1620 and, for example, the open / closed state of the door, in cooperation with the sensor tabs 1632 and 1630 on the hinge plate 1628 of the door 1620.

0200The door cover element 1612 is secured to the outside of the cylindrical portion 1610 of the housing so as to cover part of the door mounting bracket 1636 and the revolving door 1620. The cover element 1612 includes a reach opening 1613 alongside a reach opening 1614 of the incubator housing, further including a container bridge 1615 extending laterally from the lower end of the reach opening 1613. The vessel bridge 1615 facilitates insertion and removal of vessels (such as MTU160) into the incubator.

0201While in the TC incubator 600, the MTU160 and test sample should be kept at a temperature of about 60 ° C ± 0.5 ° C for a time sufficient to allow hybridization of the capture probe with the nucleic acid of interest. Under these conditions, the capture probe preferably does not hybridize directly to the immobilized polynucleotide on the magnetic particles.

0202Following the subject capture incubation in the TC incubator 600, the MTU160 is rotated by the incubator turntable to the right side or to the entrance door 622, also known as the first distribution door. The MTU160 is recovered from its MTU station 676 in the TC incubator 600 and moved by the right transfer mechanism 500 to a temperature drop station (not shown) below the sample ring 250. At the drop station, the MTU temperature is lowered to the next incubator level. The lowering station that precedes the AT incubator 602 is theoretically a heater as opposed to a cooler, but this is the temperature at which the MTU is lowered, about 40 ° C, which is still about 30 of the ambient analyzer temperature. This is because it is higher than ° C. Therefore, the lowering station preferably uses a resistance heating element as opposed to a thermoelectric module.

0203The MTU160 is moved from the lowering station to the AT incubator 602 by the right movement mechanism 500. The design and operation of the AT Incubator 602 is similar to the TC Incubator 600 described above, except that the AT Incubator 602 incubates at 40 ± 1.0 ° C.

0204In AT incubator 602, hybridization conditions allow the polytimidine (poly (dT)) tail of the immobilized polynucleotide to hybridize with the polyadenyl (poly (dA)) tail of the capture probe. Is. When the target nucleic acid hybridizes with the capture probe in the TC incubator 600, a hybridization complex is formed between the immobilized polynucleotide which is the capture probe and the target nucleic acid in the AT incubator 602, thereby immobilizing the target nucleic acid. To do.

0205During the active temperature coupling incubation, the turntable assembly 1656 (or 671) of the AT incubator 602 rotates the MTU to the exit door 624, also known as the second or left distribution door, from which the left transfer mechanism 502 brings the MTU 160. Can be removed. The left transfer mechanism 502 removes the MTU160 from the AT incubator 602 and installs it at the available magnetic separation station 800.

0206The temperature gradient station 700 can be a bottleneck when processing a large number of MTUs through the chemical desk 200. It may be possible to use the underutilized MTU station 676 in one or more of the incubators where temperature sensitivity is less of an issue. For example, within the AT incubator 602, the active temperature binding process at about 40 ° C is not as temperature sensitive as other incubators, with up to 30 MTU stations 676 in up to 15 incubators at any given time. May be unused. As currently considered, the chemistry desk has only about eight ascent stations or heaters. Therefore, considerably more MTUs can be preheated in the unused slots of the AT incubator 602 compared to in the ascending station 700. Furthermore, by using an unused incubator slot instead of a heater, it is possible to remove some or all of the heater, freeing up space on the chemistry desk.

0207(Magnetic separation station) Referring to FIGS. 24-25, each magnetic separation station 800 includes a module housing 802 having an upper 801 and a lower 803. Mounting flanges 805 and 806 extend from the bottom 803 for mounting the magnetic separation station to the reference plate 82 with appropriate mechanical fasteners. Locator pins 807 and 811 extend from the bottom of bottom 803 of housing 802. Pins 807 and 811 meet the openings (not shown) formed in the reference plate 82 to assist in positioning the magnetic separation station on the reference plate 82 before the housing 802 is secured by fasteners.

0208The loading slot 804 extends through the front wall of the lower 803, and a transfer mechanism (such as 502) allows the MTU160 to be installed and removed from the magnetic separation station 800. The tapered slot extension 821 surrounds a portion of the loading slot 804 to facilitate MTU insertion into slot 804. The partition 808 separates the upper 801 from the lower 803.

0209The pivot magnet operating structure 810 is mounted inside the lower 803 so that it can be pivoted around point 812. The magnet operating structure 810 carries a permanent magnet 814 located on any side of the MTU slot 815 formed in the magnet operating structure 810. It is preferred that five magnets be gripped in an aligned arrangement on any side of the magnet operating structure 810 so that one corresponds to each individual reaction tube 162 of the MTU 160. The magnet is preferably made from neodymium iron boron (NdFeB), which is the lowest grade-35 and has the preferred dimensions of 0.5 inches wide, 0.3 inches high and 0.3 inches deep. The electric actuator, roughly represented by the 816, pivots the magnet operating structure 810 up and down, thereby moving the magnet 814. As shown in FIG. 25, it is preferable that the actuator 816 includes a rotary stepping motor 819 that rotates a driving screw mechanism connected to the magnet operating structure 810 to selectively raise and lower the magnet operating structure 810. Motor 819 is Haydon Switch and A HIS linear stepping actuator of model number 26841-05, commercially available from Instrument, Inc. (Waterbury, Connecticut), is preferred.

0210The sensor 818, which is preferably a sensor with an optical slot, is located inside the lower 803 of the housing and indicates the lower or "home" position of the magnet operating structure 810. The sensor 818 is preferably Optek Technology, Inc. model number OPB980T11, commercially available from Optek Technology, Inc. (Carolton, Texas). Similarly, another sensor 817, preferably a sensor with an optical slot of Optek Technology, Inc. model number OPB980T11, is preferably provided to point out the upper or engaging position of the magnet operating structure 810.

0211The MTU carrier 820 is located below the adjacent partition 808 of the loading slot 804 to operably support the MTU 160 located within the magnetic separation station 800. Referring to FIG. 26, the MTU carrier 820 has a slot 822 for accommodating the upper end of the MTU 160. The lower branch plate 824 attaches to the bottom of the carrier 820 and supports the bottom of its connecting rib structure 164 when the MTU 160 is slid into the carrier 820 (see FIGS. 27 and 28). The spring clip 826 is attached to the carrier 820, but its opposing protrusions 831 and 833 extend into slot 822 and grip the MTU so that it can be released into the carrier 820.

0212The rotary stirrer assembly 828 is coupled to the carrier 820 to rotationally mix the contents of the MTU gripped by the MTU carrier 820. The rotary stirrer assembly 828 includes a stepper motor 830 mounted on a mounting plate 832, a drive pulley 834 with an eccentric pin 836, an idler pulley 838 with an eccentric pin 840, and a belt 835 connecting the drive pulley 834 to the idler pulley 838. .. The stepping motor 830 is preferably VEXTA of model number PK245-02A, which is commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan), and the belt 835 is commercially available from SDP / SI (New Hyde Park, NY). Model number A It is preferably a timing belt of 6G16-170012. As shown in FIGS. 25 and 26, the eccentric pin 836 fits into the slot 842 formed longitudinally in the MTU carrier 820. The eccentric pin 840 fits into a circular opening 844 formed at the opposite end of the MTU carrier 820. As the motor 830 rotates the drive pulley 834, the idler pulley 838 also rotates through the belt 835, and the MTU carrier 820 engages with the openings 842 and 844 formed in the carrier 820, respectively, the eccentric pins 836 and 840. Moves on the horizontal rotation path. The rotating shaft 839 of the idler pulley 838 preferably has a lateral slot 841 extending upward and formed through it. The sensor 843 with an optical slot is arranged in the same hierarchy as the slot 841 and measures the rotation speed of the idler pulley 838 via a sensor beam that is intermittently directed through the slot 841 as the shaft 839 rotates. Sensor 843 is commercially available from Optek Technology, Inc. (Carolton, Texas), Optek. It is preferably a sensor of Technology, Inc. model number OPB980T11.

0213The drive pulley 834 also includes a locator plate 846. The locator plate 846 passes through the slotted light sensors 847 and 848 that are mounted on the sensor mounting bracket 845 that extends from the motor mounting plate 832. Optical sensors 847 and 848 are preferably sensors of Optek Technology, Inc. model number OPB980T11, commercially available from Optek Technology, Inc. (Carolton, Texas). The locator plate 846 is formed with a number of circumferentially spaced axial openings that fit one or both of the sensors 847 and 848 and the location of the rotary stirrer assembly 828 or the MTU. Point out the location of the carrier 820.

0214Referring to FIG. 24, the cleaning solution supply tube 854 is connected to the fitting 856 and extends through the top surface of the module housing 802. The cleaning solution supply pipe 854 extends through the partition 808 via the connecting fitting 856 to form a cleaning solution supply network.

0215As shown in FIGS. 27 and 28, the cleaning solution dispenser nozzle 858 extending from the fitting 856 is located within the partition 808. Each nozzle is installed above each reaction tube 162 of the MTU 160 in a laterally eccentric position with respect to the reaction tube 162. Each nozzle includes a laterally oriented bottom 859 for directing the wash solution from an eccentric position to each reaction tube. By dropping the fluid onto the reaction tubes 162 in the direction of having the lateral components, it is possible to suppress the bounce when the fluid flows down the side surfaces of the respective reaction tubes 162. In addition, the laterally directed fluid can wash away the material on the sides of each reaction tube 162.

0216As shown in FIGS. 24 and 25, the suction tube 860 extends through the tube holder 862 to which the tube 860 is fixedly attached and through the opening 861 of the partition 808. The tube guide yoke 809 (see FIG. 26) is attached to the side of the partition 808, below the opening 861 by a mechanical fastener. The suction hose 864 connected to the suction tube 860 extends to the vacuum pump 1162 (see FIG. 52) in the analyzer 50, and the sucked fluid is removed into the fluid waste container carried to the lower chassis 1100. Each suction tube 860 has a preferred length of 12 inches and an inner diameter of 0.041 inches.

0217The tube holder 862 is attached to the drive screw 866 and is actuated by the lift motor 868. The lift motor 868 is preferably VEXTA of model number PK245-02A, which is commercially available from Oriental Motor Co., Ltd. (Tokyo, Japan), and the driving screw 866 is commercially available from Kerk Motion Products, Inc. (Horis, New Hampshire). ZBX series threaded loosening prevention lead threads are preferred. The tube holder 862 is attached to the screw-in sleeve 863 of the drive-in screw 866. The rod 865 and slide rail 867 serve as guides for the tube holder 862. Z-axis sensors 829 and 827 (optical sensors with slots) point out the vertical stroke position of the suction tube 860 in coordination with the tabs extending from the threaded sleeve 863. The Z-axis sensor is preferably a sensor of Optek Technology, Inc. model number OPB980T11 commercially available from Optek Technology, Inc. (Carolton, Texas).

0218The cable supplies power and control signals to the magnetic separation station 800 via connector 870.

0219When the MTU160 is inserted through the insertion opening 804 into the magnetic separation station, the MTU carrier 820, the magnet operating structure 810 is initially in the lower position (shown in the phantom in Figure 25) verified by the sensor 818. .. When the magnet operating structure 810 is in the lower position, the magnetic field of the magnet 814 has virtually no effect on the magnetically reactive particles contained in the MTU 160. In this context, "substantially unaffected" means that the attractive force of the magnetic field of the magnet 814 does not pull out the magnetically reactive particles from the suspension. The rotary stirrer assembly 828 moves the MTU carrier 820 only part of the entire orbit so that the carrier 820 and MTU 160 are moved laterally, so that each tip 170 transported to the tip gripping structure 176 of the MTU 16 , Lined up with each suction tube 860, as shown in FIG. The location of the MTU carrier 820 can be verified by the locator plate 846 and one of the sensors 847 and 848. Alternatively, the stepper motor 830 can be moved by a known number of steps to place the MTU carrier 820 in the desired position, omitting one of the sensors 847 and 848.

0220The tube holder 862 and suction tube 860 are lowered by a lift motor 868 and a driving screw 866, and each suction tube 860 frictionally engages the tip tip 170 gripped by the associated transport structure 176 on the MTU 160.

0221As shown in FIG. 25A, at the lower end of each suction tube 860, the tube 860 has a first portion 851 along most of the total length of the tube, a second portion 853, which is smaller in diameter than the first portion 851. And characterized by a tapered step configuration with a third portion 855, which is smaller in diameter than the second portion 853. The diameter of the third portion 855 allows the tip of the tube 860 to be inserted into the wide mouth 181 of the through hole 180 of the tip tip 170 and a line on the outer surface of the third portion 855 and the inner wall of the hole 180 of the tip tip 170. A friction tightening fit is formed between the two annular ridges 183 (see FIG. 59) to be drawn. The annular step 857 is defined at the transition between the second portion 853 and the third portion 855. The step portion 857 limits the extent to which the tube 860 is inserted into the tip tip 170, whereby the tip tip is removed after use, as described below.

0222Since the tip tip 170 is at least partially conductive, the presence of the tip tip 170 on the suction tube 860 is as a suction tube 860 as a half of the capacitor and as the other half of the capacitor. It can be verified by the capacitance of the capacitor with the hardware surrounding the magnetic separation station. The capacitance changes when the tip tip 170 engages the tip of the suction tube 860.

0223In addition, five sensors with optical slots (not shown) can be strategically positioned above the partition 808 to verify the presence of a chip tip 170 on each suction tube 860. A preferred "chip tip presence" sensor is a sensor of Optek Technology, Inc. model number OPB930W51, commercially available from Optek Technology, Inc. (Carolton, Texas). The tip tip 170 on the tip of the suction tube 860 blocks the beam of the associated sensor and verifies the presence of the tip tip 170. After the tip tip collecting operation, the MTU160 must be discontinued if the tip tip presence sensor does not verify tip tip engagement for all five suction tubes 860. The discontinued MTU is collected from the magnetic separation station, sent to the deactivation preparation room 750, and finally discarded.

0224After successful tip tip engagement, the rotary stirrer assembly 828 moves the MTU carrier 820 and the fluid movement position verified by one or both of the locator plate 846 and sensors 847 and 848 (shown in Figure 27). Return to).

0225The magnet operating structure 810 is raised to the upward position shown in FIG. 24, whereby the magnet 814 is placed on opposite sides in close proximity to the MTU 160. When the contents of the MTU are exposed to the magnetic field of the magnet 814, the magnetically reactive particles indirectly bound to the nucleic acid of interest are attracted to the sides of the individual reaction tubes 162 in the vicinity of the magnet 814. The residual material in the reaction tube 162 should be substantially unaffected, thereby isolating the nucleic acid of interest. Magnetic particles are attached to the sides of each reaction tube 162 by maintaining the magnet operating structure 810 in an ascending position with an appropriate residence time defined by the assay protocol and controlled by the assay manager program.

0226The suction tube is then lowered into the reaction tube 162 of the MTU 160 to attract the fluid contents of the individual reaction tubes 162, while at the same time magnetic particles adhere to the sides of the reaction tube 162 near the magnet 814 and the reaction tube. Remains in 162. The tip tip 170 at the tip of the suction tube 860 ensures that the contents of the reaction tube 162 do not come into contact with the sides of the suction tube 860 during the suction procedure. The tip tip 170 is discarded before the next MTU is processed at the magnetic separation station 800, minimizing the possibility of secondary contamination by the suction tube 860.

0227The conductive tip tip 170 can be used in a known manner for detecting the fluid volume level in the MTU's reaction tube 162. The suction tube 860 and the conductive tip tip 170 have half of the capacitor, the conductive structure surrounding the inside of the particles has the second half of the capacitor, and the fluid medium between these two halves of the capacitor is a dielectric. Configure. Capacitive changes due to changes in the properties of the dielectric can be detected.

0228The capacitive circuit configuration of the suction tube 860 can be arranged so that all five suction tubes 860 can operate as a single group capacitance detection mechanism. As a group capacitance detection mechanism, the circuit configuration can be determined only when the fluid level of any one of the reaction tubes 162 is high, and cannot be determined when the fluid level of one of the reaction tubes is low. That is, when either the suction tube 860 or the associated tip tip 170 comes into contact with the fluid material in the reaction tube, the capacitance of the system changes due to changes in the dielectric. If the Z position of the suction tube 860 where the volume change occurs is too high, high fluid levels in at least one reaction tube are pointed out, thus suggesting suction failure. On the other hand, if the Z position of the suction tube where the capacitance change occurs is correct, the circuit configuration suction tubes cannot be distinguished from each other, and therefore one or more of the other tubes are still above the fluid due to the low fluid level. If not in contact, low fluid levels will not be detected.

0229Alternatively, the suction tube capacitive circuit configuration can be arranged so that each of the five suction tubes 860 operates as an individual capacitance detection mechanism.

0230With five individual capacitance detection mechanisms, the capacitance level detection circuit configuration can detect fluid suction failure in one or more of the reaction tubes when the fluid level of one or more of the reaction tubes 162 is high. The individual capacitance detection circuit configurations can detect the failure of fluid dripping into one or more of the reaction tubes when the fluid level of one or more of the reaction tubes 162 is low. Further, the capacitance level detection circuit configuration can be used for volume verification to determine whether the volume in each reaction tube 162 is within a predetermined range. Volume verification stopped the suction tube 860 descent above the expected fluid level, for example 110% of the expected fluid level, and confirmed that none of the reaction tubes had such a high level. This is done by stopping the descent of the suction tube 860 below the expected fluid level, for example 90% of the expected fluid level, and making sure that each reaction tube has a fluid level of at least that height. Can be done.

0231Following suction, the suction tube 860 is raised, the magnet operating structure 810 is lowered, and a defined volume of wash solution is dropped into each reaction tube 162 of the MTU 160 through the wash solution dispenser nozzle 858. Air suction after a short period of dripping is preferred to prevent dripping of the cleaning solution dispenser nozzle 858.

0232The rotary stirrer assembly 828 then moves the MTU carrier 820 at high speeds on the horizontal rotation path to mix the contents of the MTU 160. It is preferable to operately mix or stir the MTU in a horizontal plane to avoid splashing of the fluid contents of the MTU and to avoid the formation of sprays. Following mixing, the rotary stirrer assembly 828 stops the MTU carrier 820 at the fluid movement position.

0233To further purify the nucleic acid of interest, the magnet operating structure 810 is raised again and maintained in the raised position for a defined residence period. After magnetic retention, the suction tube 860 with the engaged tip tip 170 is lowered to the bottom of the reaction tube 162 of the MTU 160 to suck the test sample fluid and wash solution in a suction procedure essentially similar to the procedure described above. To do.

0234A wash cycle, each with a drop, mix, magnetic retention, and suction procedure, may be performed one or more additional times, as defined by the assay protocol. Nucleic acid-based diagnostic test experts can determine the appropriate magnetic residence time, number of wash cycles, wash solution, etc. for the desired target capture procedure.

0235Depending on the desired throughput, the number of magnetic separation stations may vary, but the analyzer 50 preferably includes 5 magnetic separation stations 800, thereby performing magnetic separation cleaning procedures in parallel with 5 different MTUs. Can be carried out.

0236After the final cleaning step, the magnet operating structure 810 is moved downwards and the MTU160 is removed from the magnetic separation station by the left transfer mechanism 502 and installed in the left rotary stirrer 552.

0237After the MTU160 is removed from the cleaning station, the tip tip 170 is removed from the suction tube 860 by a removal plate 872 located at the bottom of the lower 803 of the housing 802.

0238The removal plate 872 has a number of aligning removal holes 871 corresponding to the number of suction tubes 860, which is five in the preferred embodiment. As shown in FIGS. 29A-29D, each removal hole 871 includes a first portion 873, a second portion 875 smaller than the first portion 873, and a bevel 877 surrounding the portions 873 and 875. Since the removal plate 872 is located at the bottom of the housing 802, the small portion 875 of each removal hole 871 is substantially aligned with each of the associated suction tubes 860, as shown in FIG. 29A. The suction tube 860 is lowered so that the tip tip 170 at the tip of each suction tube 860 engages the removal hole 871. Since the small portion 875 is too small to accommodate the diameter of the tip tip 170, the bevel 877 orients the tip tip 170 and the suction tube 860 towards the large portion 873, as shown in FIG. 29B. The suction tubing 860 is made of an elastically flexible material, preferably stainless steel, whereby the slope 877 deflects each suction tubing 860 laterally as the suction tubing 860 continues to descend. The small portion 875 of the removal hole 871 can accommodate the diameter of the suction tube 860, whereby each suction tube 860 due to its own elasticity after the peripheral edge 177 of the tip tip 170 passes under the removal hole 871. , As shown in FIG. 29C, fits into the small portion 875 of the removal hole 871. The suction tube 860 is then raised and the peripheral edge 177 of each tip tip 170 engages the bottom peripheral end of the small portion 875 of the removal hole 871. As the suction tube 860 rises further, the tip tip 170 is pulled out of the suction tube 860 by the removal hole 871 (see Figure 29D). The removed tip 170 is directed by a chute into a solid waste container such as the tip tip waste box 1134.

0239The capacity of the suction tube 860 is tested to verify that all tip tips 170 have been removed and discarded. The removal step can be repeated as needed.

0240Alternative removal plates 882 are shown in Figures 31A-31C. The removal plate 882 includes a number of removal holes 881 corresponding to the number of suction tubes 860, which is five in the preferred embodiment. Each removal hole 881 includes a through hole 883 surrounded by an oblique countersunk hole 887. The pair of protrusions 885 extend laterally from a diametrically opposed position below the through hole 883. The protrusion 885 is preferably made of spring steel and has a V-shaped notch at the tip.

0241When the suction tube 860 with the tip tip 170 located at the tip is lowered towards the removal hole 881, the bevel portion 887 ensures that all misaligned tubes are oriented into the through hole 883. The distance between the tips of the opposing protrusions 885 is smaller than the diameter of the tip 170, which lowers the suction tube 860 and the tip 170, causing the tip to engage the protrusion 885 and the tip 170 to protrude. When pushed between objects 885, the protrusions are deflected downward. When the suction tube 860 is raised, the notch 886 of the protrusion 885 grabs the relatively soft material of the tip tip 170, thereby preventing the tip tip 170 from moving upward relative to the protrusion 885. As the tube continues to rise, the protrusion 885 pulls the tip 170 out of the tube 860. When the suction tube 860 is subsequently lowered and the subsequent series of tip tips is removed, the tip tips gripped between the protrusions from the previous removal are pushed through the protrusions by the next tip tip, resulting in five tips. Oriented towards waste box 1134 (see Figure 52) located in the lower chassis 1100, approximately below the magnetic separation station 800.

0242Yet another alternative, currently preferred removal plate 1400 is shown in Figures 30A-30D. Removal plate 1400 contains five removal cavities 1402, each containing a conical portion 1404. The conical portion 1404 tapers towards the neck 1406, which connects to the enlarged straight portion 1408. Since the straight portion 1408 is corrected with respect to the center of the neck 1406, one side of the straight portion 1408 is flush with the side of the neck 1406 and the other side of the straight portion 1408 is corrected from the side of the neck 1406. The bottom of the side is cut off, thereby forming the ledge 1414. Following the straight section 1408, an inclined portion 1410 is provided on the side of the removal cavity 1402 on the opposite side of the ledge 1414. The ramp 1410 tapers inward towards the bottom opening 1412.

0243The suction tube 860 with the tip tip 170 at its end is moved towards the removal cavity 1402, and the conical portion 1404 directs the tip tip 170 and the tube 860 towards the neck 1406. The suction tube 860 continues to descend and the tip tip 170 enters the straight section 1408 as the edge 177 of the tip tip 170 crosses the bottom of the conical portion 1404 and passes through the neck 1406.

0244If the suction tube 860 and the removal cavity 1402 are in proper and favorable alignment, a portion of the edge 177 of the tip tip 170 will be removed as the tip tip 170 moves through the neck 1406 to the straight section 1408. It will be placed under the ledge 1414 of 1402. The tip is further lowered to ensure that part of the edge 177 is directly below the ledge 1414, prompting the suction tube to laterally orient the tip 170 below the ledge 1414. Tip 170 engages downward slope 1410.

0245An annular step 857 (see Figure 25A) formed at the bottom of the suction tube 860 ensures that the tube 860 is further pushed into the through hole 180 of the tip tip 170 as it descends into the removal cavity 1402. It disappears. The suction tube 860 then rises and the ledge 1414 locks the edge 177 and removes the tip tip 170 from the tube 860. The removed tip tip 170 falls into the waist bin 1134 in the lower chassis 1100 through the bottom opening 1412 (see Figure 52).

0246When using each of the removal plates described above, the positions of the tip tip removal elements are not all the same. For example, the ledge 1414 of the removal cavity 1402 of the removal plate 1400 is not at the same height across all cavities. It is preferred that the three tip removal elements are at one height and the two tip removal elements are at slightly different heights above or below the other three elements. As a result of correcting the tip removal element, it is no longer necessary to overcome or block the static friction of the tip tip 170 at the end of the suction tube 860 for all five tubes 860 at the same time. When the suction tube 860 starts to rise, the static friction of the tip tip 170 is cut off for one set (2 or 3) of suction tubes 860, and then as the suction tube 860 continues to rise, the remaining tubes 860. The static friction of the tip 170 is cut off. By not simultaneously blocking the static friction of the tip 170 for all five suction tubes 860, the load on the tube holder 862, drive screw 866, screw sleeve 863, and lift motor 868 is kept at a low level.

0247(Rotary stirrer) As shown in FIGS. 32 to 34, the left rotary stirrer 522 (and the right rotary stirrer 550) is configured and similarly configured as the lower housing station 803 and rotary stirrer assembly 828 of the magnetic separation station 800 described above. Works on. Specifically, the rotary stirrer 550 (552) includes a front plate 551, a back plate 559, and mounting flanges 555, 556 for mounting the rotary stirrer 550 (552) to the reference plate 82, housing 554. including. The insertion opening 557 is formed within the front edge of the housing 554. The MTU carrier 558 has an MTU fastening clip attached to the rear of the carrier 558, along with a branch plate 560 attached to the bottom and opposing protrusions of a clip 562 extending into the internal cavity of the carrier 558 that houses the MTU. Has 562 and. The rotary stirrer assembly 564 includes a drive motor 566 mounted on a motor mounting plate 567, a drive wheel 568 with an eccentric pin 570, a floating wheel 572 with an eccentric pin 573, and a belt 574. The drive motor 566 is preferably a stepping motor, most preferably Oriental Motors Ltd. VEXTA with model number PK245-02A, which is commercially available from (Tokyo, Japan). Belt 574 is preferably a timing belt of model number A6G16-170012, commercially available from SDP / SI (New Hyde Park, NY). The rotary stirrer assembly 564 is coupled to the MTU carrier 558 via eccentric pins 570, 573 to move the MTU carrier 558 in the rotation path and agitate the contents of the MTU. The drive wheel 568, in conjunction with the sensor 578 attached to the sensor mounting bracket 579, is suitable for the MTU carrier 558 to insert the MTU160 into the rotary stirrer 552 (550) and remove the MTU160 from the rotary stirrer. Includes locator plate 576 to verify positioning. The sensor 578 is preferably a sensor of Optek Technology, Inc. model number OPB980T11 commercially available from Optek Technology, Inc. (Carolton, Texas).

0248The top plate 580 is attached on top of the housing 554. The upper plate 580 of the counterclockwise stirrer 552 has a similar number of flexible dropping tubes (not shown) for dropping fluid from the bulk fluid vessel through the dispensing nozzle 583 to the MTU 160 located within the stirrer. Includes multiple, preferably five, tube connection fittings 582 connected. The top plate 580 also includes a plurality, preferably five pipette openings 581, to correspond to the number of individual reaction tubes 162 with a single MTU 160.

0249Using the MTU160 fixedly held in the counterclockwise stirrer 552, the pipette unit 480 of the left pipette assembly 470 transfers a specified amount of amplification reagent from the container in the reagent cooling bay 900 through the pipette opening 581. Transfer to each reaction tube 162. The amplification reagent contains at least one amplification oligonucleotide such as a primer, a promoter primer, and / or a promoter oligonucleotide, a nucleoside triphosphate, and a cofactor such as magnesium ion in a suitable buffer. However, the specific components of the amplification reagent will depend on the amplification procedure performed. See, for example, Kacian et al., US Pat. No. 5,399,491. Other amplification procedures for nucleic acid testing are known to those of skill in the art, some of which have been identified in the "Background of the Invention" section above, but may be adapted for use in the analyzer 50 of the present invention.

0250The contents of the MTU are then mixed by the rotary stirrer assembly 564 of the rotary stirrer 552 to ensure proper exposure of the nucleic acid of interest to the amplification reagent. For any special amplification procedure, one of ordinary skill in the art will be able to determine the appropriate components and amounts of amplification reagents, as well as the frequency and duration of mixing.

0251After pipetting the amplification reagent to the MTU 160, the pipette unit 480 is washed by moving the pipette unit 480 to a rinsing tank (discussed below) on the processing desk 200 and running distilled water through the probe 481. Distilled water is pumped from bottle 1140 in the lower chassis 1100 and purged water is collected in the liquid waste container 1128 in the lower chassis 1100.

0252After mixing the contents of MTU160, a layer of silicone oil is dispensed into each reaction tube 162 via the dispensing nozzle 583. A layer of oil pumped from bottle 1168 in the lower chassis 1100 helps prevent evaporation and splashing of the fluid contents of MTU160 during post-operation and incubation of MTU160 and its contents.

0253(Reagent cooling bay) Here, the reagent cooling bay 900 will be described.

0254See Figures 35-39 , the reagent cooling bay 900 includes an insulating coating 902, preferably made of aluminum, fitted around a cylindrical housing 904. The cover 906 is preferably made of Delrin and sits on top of the housing 904 along with a registration tab 905 of the cover 906 that fits into the slot 907 within the housing 904 to ensure proper orientation of the cover 906. An optical sensor may be provided near or within slot 907 to verify that tab 905 is mounted in slot 907. Alternatively, the light sensor assembly 909 may be secured to the edge of the upper edge of the housing 904 to verify cover placement. The optical sensor assembly 909 works with a sensor trip structure (not shown) on the cover 906 to verify that the cover is in place. The optical sensor assembly 909 is preferably commercially available from Optek Technology, Inc. (Carolton, Texas), Optek. Technology, Inc. Includes slotted optical sensor with model number OPB980T11. The cover 906 also includes a pipette opening 908, through which pipette units 480, 482 can reach the reagent vessel in the cooling bay 900.

0255The housing 904 is attached to the floor plate 910, which extends through an opening formed within the mounting flange 911 at intervals around the perimeter of the floor plate 910 with suitable mechanical fasteners. Used and attached to the reference plate 82. Two cooling units 912 are preferably attached to the floor plate 910. Each cooling unit 912 includes a thermoelectric module 914 attached to the bottom surface of the floor plate 910 with the cooling side facing up. Commercially available from Melcor, Inc. (Trenton, NJ), the thermoelectric module model number CP1.4-127-06L provides the desired cooling capacity. The heat sink 916, which includes the plurality of heat dissipation fins 915, may be attached to or integrated with the thermoelectric module 914 on the bottom surface of the floor plate 910. The fan unit 918 is attached at a position for exhausting heat from the heat sink 916. The fan unit 918 is preferably Oriental Motors. It is an Orix fan of model number MD825B-24, which is commercially available from Ltd. (Tokyo, Japan). The cooling unit 912 also cools the interior of the housing 904 to a specified temperature for the benefit of temperature sensitive reagents (eg, enzymes) stored in the bay 900.

0256Within the cooling bay 900 housing 904 are two temperature sensors (showing only one temperature sensor 920) for monitoring and controlling its internal temperature. The temperature sensor is preferably a thermistor (10 KOhm at 25 ° C), most preferably the YSI44036 series thermistors commercially available from YSI, Inc. (Yellow Springs, Ohio). YSI thermistors are preferred due to their high accuracy and the ± 0.1 ° C compatibility that YSI thermistors provide between one thermistor and another. One of the sensors is the main temperature control sensor and the other is the temperature monitoring sensor. Based on the temperature display from the main control sensor, the embedded controller adjusts the power to the thermoelectric module 914 and / or the power to the fan unit 918 to control the cooling bay temperature. The temperature monitoring sensor provides verification confirmation of the main temperature control sensor.

0257As shown in FIG. 38, the container tray 922 is an integrated turntable structure with a bottle gripping cavity 924 sized and shaped for storing and gripping a particular reagent bottle 925. The drive system for the container tray 922 includes a motor 926, a small pulley 931 on the shaft of the motor 926, a belt 928, a pulley 930 and a shaft 932 (commercially available from Oriental Motors Ltd. (Tokyo, Japan). VEXTA stepping motors with model number PK265-02A and GT (R) series SDP timing belts commercially available from SDP / SI (New Hyde Park, New York) are preferred). The motor 926 and the cooling unit 912 extend through an opening (not shown) formed in the reference plate 82 and extend beneath the floor plate 910.

0258The container tray 922 may include a central upright handle 923 for mounting the tray 922 in the housing 904 and removing the tray 922 from the housing 904. The tip 933 of the shaft 932 extends through the floor plate 910 and is received by a joint opening (not shown) formed at the bottom of the tray 922. The sensor 940, which extends through the floor plate 910 to the housing 904, verifies that the tray 922 is in place within the housing 904. The sensor 940 is preferably a capacitive proximity sensor of model number FCP2 commercially available from Advanced Controls, Inc. (Bradington, FL).

0259The position of the container tray 922 can be detected using a position encoder 934 (preferably a slotted disc) that works with the light sensor 935, so that the pipette opening 908 in the 906 covers a particular reagent bottle 925. Can be placed below.

0260As shown in FIG. 37, a preferred alternative to the position encoder 934 and the optical sensor 935 is a slotted optical sensor 937 (in FIG. 36) provided within the housing 904 with a flag pin (not shown) extending from the bottom of the container tray 922. Only two sensors are visible). One sensor is provided for each quadrant of the container tray 922, and the flag is set to one of the four sensors to indicate in which quadrant of the container tray 922 the pipette opening 908 is located. Start. The sensor 937 is preferably an Optek Technology, Inc. sensor of model number OPB980T11, commercially available from Optek Technology, Inc. (Carolton, Texas).

0261A preferred alternative to the integrated container tray 922 shown in FIG. 38 is the modular tray 1922 shown in FIGS. 35 and 39. Tray 1922 includes a circular substrate 1926 and an upright handle post 1923 attached to its center. Modular parts 1930 with bottle gripping cavities 1924 are preferably connected to each other and to substrate 1926 by pins 1928 and screws (not shown) to form a circular tray 1922. As an alternative to the pins 1928 and screws, other means of fixing the modular component 1930 may be used. Since the modular part 1930 shown in the figure is a quarter circle of one circle, it is naturally required that the four corresponding parts 1930 complete the tray 1922. A quadrant is preferred, but the modular component may be sensors of various sizes, for example 1/2 of a circle or 1/8 of a circle.

0262It is preferred to provide an alphanumeric bottle position label 1940 on the substrate 1926 to locate the reagent container within the tray 1922. Preferred labeling schemes include circled letter-number pairs, including leading letters A, E, P, or S with trailing letters 1, 2, 3, or 4. The letters A, E, P, and S specify the amplification reagent, enzyme reagent, probe reagent, and selection reagent, respectively, corresponding to the preferred mode of use of the analyzer 50, and the numbers 1 to 4 are trays. It specifies the 1922 quadrant. Each modular component 1930 includes a round hole 1934 at the bottom of each bottle gripping cavity 1924. Hole 1934 is aligned with the bottle position label 1940, so the label 1940 is visible if the modular component 1930 is in place on the board 1926.

0263The modular part 1930 of the container tray 1922 houses reagent containers of different sizes, corresponding to sufficient reagent volumes to perform 250 assays or 500 assays. It is configured as follows. Four 250-pass assay modular quadrants can stockpile reagent cooling bays for 1000 assays, and four 500-assay modular quads allow reagent cooling for 2000 assays. You can stockpile the bay. Modular quadrants for 250 or 500 assay reagent kits can be mixed and adapted to form a container tray for accommodating a large number of single assay types or a large number of different assay types. ..

0264An insulating pad 938 is placed between the container tray 922 and the floor plate 910. A cable (not shown) connected to connector 936 and the built-in controller of analyzer 50 provides power, control, temperature, and position signals to / from reagent cooling bay 900.

0265The barcode scanner 941 is mounted on the upright scanner mounting plate 939 attached to the floor plate 910 in front of the opening 942 formed in the side wall of the cooling bay 900. The barcode scanner 941 can scan barcode information from each of the reagent containers transported to the container tray 922. As shown in FIG. 39, a vertical slot 1932 is formed along the bottle gripping cavity 1924, and the barcode information arranged on the side surface of the reagent container gripped in the bottle gripping cavity 1924 is provided by the barcode scanner 941. It may be lined up with slot 1932 so that barcode information can be scanned. A preferred barcode scanner is commercially available from Microscan, Inc. (Newbury Park, CA) under model number FTS-0710-0001.

0266Pipette rinsing tanks 1942 and 1944 are attached to the sides of the housing 904. Each of the rinse tanks 1942 and 1944 is provided with probe storage openings 1941 and 1945 formed in the upper panel thereof and waste liquid pipes 1946 and 1948 connected to the bottom thereof, respectively, in the housing structure. The probe of the pipette unit can be inserted into the rinsing tanks 1942, 1944 through the probe housing openings 1941, 1945, and the wash and / or rinse liquid can be placed in the tank through the probe. The fluid in the rinse tanks 1942 and 1944 is directed by the waste pipes 1946 and 1948, respectively, to the appropriate waste container in the lower chassis 1100. In the preferred placement and mode of operation of the analyzer 50, the probe 481 of the pipette unit 480 is rinsed in the rinse tank 1942 and the probe 483 of the pipette unit 482 is rinsed in the rinse tank 1944.

0267After adding the amplification reagent and oil to the reaction tube 162 of the MTU160 in the counterclockwise rotary stirrer 552, the left transfer mechanism 502 removes the MTU160 from the counterclockwise rotary stirrer 552 and is reachable to the left rotary stirrer 502, ie the chemical desk. Move the MTU160 to the available temperature rise station 700 on the left side of the 200 to raise the temperature of the MTU160 and its contents to about 60 ° C.

0268After a sufficient climb time at the climb station 700, the left transfer mechanism 502 moves the MTU 160 to the TC incubator 600. The MTU turntable assembly 671 in the TC incubator 600 presents an empty MTU station 676 so that the left distribution door 624 of the TC incubator 600 opens and the left transfer mechanism can insert the MTU into the TC incubator. Subsequently, the MTU160 and its contents are incubated at about 60 ° C for a specified incubation time. During incubation, the MTU turntable assembly 671 may rotate continuously within the TC incubator 600 as the other MTU 600 is removed from and inserted into the TC incubator 600.

0269By incubating at 60 ° C in the TC incubator 600, the capture probe / nucleic acid hybridization complex can be dissociated from the immobilized polynucleotide present in the assay solution. At this temperature, the amplified oligonucleotide (eg, primer, promoter primer, or promoter oligonucleotide) introduced from the reagent cooling bay 900 hybridizes with the nucleic acid of interest, facilitating subsequent amplification of the target nucleotide sequence. Can be done.

0270Following incubation, the MTU turntable assembly 671 in the TC incubator 600 rotates the MTU 160 to the left distribution door 624, the left side distribution door 624 opens, and the left transfer mechanism 502 from the TC incubator 600 MTU turntable assembly 671 to MTU 160. Take out. The left transfer mechanism 502 then moves the MTU 160 to an available temperature drop station 700 that can reach the left transfer mechanism 502 and inserts the MTU 160 there. At the lowering station, lower the temperature of MTU160 and its contents to about 40 ° C. Subsequently, the MTU160 is taken out from the lowering station by the left transfer mechanism 502 and moved to the AT incubator 602. The MTU turntable assembly 671 in the AT incubator 602 presents an empty MTU station 676 so that the left distribution door 624 of the AT incubator 602 opens and the left transfer mechanism 502 can insert the MTU into the AT incubator 602. Incubate the MTU in the AT incubator 602 at about 41 ° C for the time required to stabilize the temperature of the MTU.

0271The MTU is transferred from the AT incubator 602 to the AMP incubator 604, where the MTU temperature stabilizes at 41.5 ° C, by the transfer mechanism 502. The MTU turntable assembly 671 in the AMP incubator 604 rotates to place the MTU in the pipette station below the pipette opening 662 formed in the cover 611 (see, eg, FIG. 19). The container tray 922 in the reagent cooling bay 900 rotates to place the enzyme reagent container under the pipette opening 908, and the pipette unit 482 of the pipette assembly 470 contains one or more polymerases required for enzyme synthesis. The enzyme reagent is transferred from the reagent cooling bay 900 to each of the reaction tubes 162 of the MTU 160.

0272As mentioned above, pipette units 480, 482 use volume level detection to check the liquid level in the container, and a small portion of the ends of probes 481, 483 of pipette units 480, 482 are pipette solution in the container. Soak in. The pipette units 480 and 482 preferably descend as the fluid is sucked into the probes 481 and 483 in order to keep the ends of the probes immersed to a certain depth. After pipetting the reagent into pipette unit 480 or 482, the pipette unit should have a minimum movement gap of 10 μl at the end of each probe 481 or 483 to ensure that no drops fall from the end of the probe. create.

0273After the enzyme reagent is added to each reaction tube 162, the MTU turntable assembly 671 of the AMP incubator 604 rotates the MTU 160 to the tilted disk linear stirrer 634 in the AMP incubator 604 and of the nucleic acid of interest for the added enzyme reagent. To facilitate exposure, MTU160 and its contents are mixed at about 10 Hz as described above. Rinse probe 483 by moving pipette unit 482 to rinsing tank 1942 and passing distilled water through it.

0274The MTU160 is then incubated in the AMP incubator 604 for a defined incubation time at approximately 41.5 ° C. The incubation time must be long enough to allow sufficient amplification of at least one target nucleotide sequence contained in one or more target nucleic acids that may be present in the reaction tube 162. Preferred embodiments are devised to facilitate amplification following the TMA procedure, but those modifications necessary for practitioners to perform other amplification procedures using the analyzer 50. Will fully understand. Also, preferably, an internal control sequence is added at the start of the assay to ensure that the amplification conditions and reagents were suitable for amplification. Internal controls are known in the art and need not be considered further herein. For example, Wang et al., U.S. Pat. No. 5,476,774, "Quantitation of Nucleic See Acids Using the Polymerase Chain Reaction.

0275Following amplification incubation, the left transfer mechanism 502 moves the MTU 160 from the AMP incubator 604 to an available ascent station 700 accessible to the left transfer mechanism 502, bringing the temperature of the MTU 160 and its contents to approximately 60 ° C. To do. Subsequently, the MTU160 is moved to the HYB incubator 606 by the left transfer mechanism 502. Rotate the MTU160 to the pipette station in the HYB incubator 606 and pipette the probe reagents from the reagent cooling bay 900 into each reaction tube 162 through the opening 662 inside the cover of the HYB incubator 606 by pipette unit 480. .. In a preferred embodiment, the probe reagent is a chemiluminescent detection probe, and preferably an Acridinium ester that can be detected in a Hybridization Protection Assay (HPA). ester; AE) Includes label probe. Acridinium ester label probes and HPA methods are known in the art. See, for example, Arnold et al., U.S. Pat. Nos. 5,639,604, 4,950,613, 5,185,439, and 5,585,481, and Campbell et al., U.S. Pat. No. 4,946,958. AE-labeled probes and HPAs are preferred, but the analyzer 50 may be adapted to adapt to a variety of detection methods and associated probes, which may or may not be labeled. Good. Confirmation that the detection probe has been added to the reaction tube 162 is a probe reagent other than the detection probe that binds to the target sequence existing in the reaction tube 162 in the HYB incubator 606 or its complement under HPA conditions. It can be achieved using an internal control that can hybridize with the probe inside (or a complement of the internal control that can hybridize). The label on this probe must be distinguishable from the label on the detection probe. See, for example, Nelson et al., US Pat. No. 5,827,656, "Compositions and Methods for the Simultaneous Detection and Quantitation of Multiple Specific Nucleic Acid Sequences."

0276After dispensing the probe reagent into each of the reaction tubes 162 of the MTU160, the pipette unit 480 is moved to the pipette rinsing tank 1944 and the probe 481 of the pipette unit is rinsed with distilled water.

0277The MTU turntable assembly 671 rotates the MTU160 to a tilted disk linear stirrer 634 to facilitate exposure of the amplification product containing the target sequence or its complement to the added detection probe, the MTU160 and its contents. Mix things at about 14 Hz as described above. The MTU160 is then incubated for a time sufficient to allow hybridization of the detection probe to the target sequence or its complement.

0278After hybridization incubation, the MTU turntable assembly 671 rerotates the MTU 160 in the HYB incubator 606 to the pipette position below the pipette opening 662. The selective reagent stored in the container in the reagent cooling bay 900 is pipette-added to each reaction tube 162 by the pipette unit 480. Selective reagents are used in HPA assays and specifically include alkaline reagents that hydrolyze the acridinium ester label associated with the unhybridized probe, which disrupts or inhibits its chemiluminescent ability, while targeting. The acridinium ester label associated with the probe hybridized with the amplification product containing the sequence or complement thereof is not hydrolyzed and can chemiluminescent in a detectable manner under appropriate detection conditions. ..

0279Following the addition of selective reagents to each reaction tube 162 of the MTU160, the pipette probe 481 of the pipette unit 480 is rinsed with distilled water in the pipette rinse tank 1944. The MTU turntable assembly 671 in the HYB incubator 606 rotates the MTU 160 to a tilted disk linear stirrer 634 and mixes at about 13 Hz as described above to facilitate exposure of the amplification product to the added selection reagents. The MTU is then incubated in the HYB incubator 606 for a time sufficient to complete the selection process.

0280After the selective incubation is complete, the left transfer mechanism 502 transfers the MTU 160 to an available lowering station 700 accessible to the left transfer mechanism 502 to cool the MTU 160. After cooling the MTU160, it is removed from the lowering station by the left transfer mechanism 502 and moved into the AT incubator 602 by the transfer mechanism 502 to stabilize the temperature of the MTU160 at 40 ° C.

0281After sufficient time to stabilize the temperature of the MTU160, the MTU turntable assembly 671 inside the AT incubator 602 rotates to present the MTU160 at the right distribution door of the AT incubator 602. The right distribution door 622 opens and the right transfer mechanism 500 removes the MTU 160 from the AT incubator 602.

0282The right transfer mechanism 500 moves the MTU to a bar code scanner (not shown) that scans the MTU bar code information posted on the label sticking surface 175 of the label sticking structure 174 of the MTU 160. The barcode scanner is preferably attached to the outer wall of the housing of the illuminometer 950. A preferred barcode scanner is commercially available from Opticon, Inc. (Orangeburg, NY) under stock number LHA1127RR1S-032. The scanner validates the total assay time before entering the illuminometer 950 by limiting the exact MTU at the exact assay time. The right transfer mechanism 500 moves the MTU 160 from the barcode reader to the illuminometer 950.

0283In a preferred mode of operation, the right transfer mechanism 500 allows the MTU 160 to be available at the MTU reduction station, in order to reduce the temperature of the MTU 160 to 24 ± 3 ° C before the right transfer mechanism 500 moves the MTU 160 to the illuminometer 950. In other words, it can be put in the refrigerator. MTU contents have been found to exhibit more consistent chemiluminescence "light emission" at this low temperature.

0284(Illuminance meter) With reference to FIGS. 40-42C, the first embodiment of the illuminometer 950 includes an electronic unit (not shown) within the housing 950. A photomultiplier tube (PMT) 956 connected to an electronic unit extends through the PMT plate 955 into the housing 954, with the front end of the PMT 956 aligned with the opening 953. The preferred PMT is commercially available from Hamamatsu Corp. (Bridgewater, NJ) as model number HC135. Signal measurements using the preferred PMT are based on known photon counting systems.

0285The opening 953 is in the center of the opening box 958 on the front of the PMT plate 955. The opening 953 and opening box 958 are completely sealed by a housing defined by a floor plate 964, an upper plate 966, a PMT plate 955, and a back frame 965 and a back plate 967, the housing having stray light in the opening 953. It prevents entry and is attached to the reference plate 82. The MTU transfer path passes through the housing in front of the opening 953 and extends approximately laterally with respect to the optical axis of the opening. The MTU160 passes through the illuminometer 950 via the MTU transfer path. The back rail 991 and front rail 995 provide a parallel horizontal flange that is located on the opposite side of the MTU transfer path and supports the connecting rib structure 164 of the MTU 160 that is located within the illuminometer 950. The swivel door 960 is supported for rotation within the associated door housing 961 located at the opposite end of the MTU transfer path and is rotated by a door motor 962, which may include a stepper motor or a DC gear motor.

0286The door housing 961 provides an opening through which the MTU 160 enters and exits the illuminometer 950. The MTU160 enters the illuminometer 950 using a right transfer mechanism 500 that inserts the MTU160 through one of the door housings 961. The MTU160 is described below for its various embodiments, but is influenced by the MTU transfer assembly that moves the MTU through the MTU transfer path and ultimately exits the photometer through the other door housing 961. , Exit the illuminometer.

0287The swivel door 960 is substantially cylindrical and includes a notch 963. Each swivel door 960 has an open position where the notch 963 is generally aligned with the opening of the door housing 961 associated with it so that the MTU 160 can pass through the opening, and neither the MTU 160 nor the light can pass through the opening. The side of the swivel door opposite the notch 963 can rotate to and from a closed position extending beyond the opening of the associated door housing 961. Except when the MTU 160 is in and out of the illuminometer 950, the swivel door 960 is preferably in its respective closed position to prevent stray light from entering the illuminometer. Since the test results are confirmed by the amount of light detected by the PMT956, stray light from sources other than the sampled container 160 may give erroneous results.

0288As shown in FIGS. 40-42C, the MTU transfer assembly may include an MTU forward motor 972 that drives a lead thread 974 by a timing belt (not shown) or a bevel gear (not shown). The thread splicing 976 engaged to the lead thread 974 is coupled to the MTU bracket 977 extending away from the lead thread 974 to engage the MTU 160. The MTU bracket 977 has a guide flange 978 in which an elongated, slightly bowed guide hole 979 is formed. The guide rod 980 extends through an illuminometer 950 adjacent to and parallel to the lead screw 974. The guide rod 980 extends through the guide hole 979.

0289To advance the MTU bracket 977 (from bottom to top in Figure 42C), rotate the lead thread 974 counterclockwise, as shown in Figure 42B. Due to system friction, the threaded 976 and MTU bracket 977 will also rotate counterclockwise with the lead thread 974 until the guide rod 980 touches the left side of the guide hole 979. When the guide rod 980 comes into contact with the side surface of the guide hole 979, the MTU bracket 977 and the threaded joint 976 cannot rotate with the lead thread 974 any more, and when the lead thread 974 rotates further, the MTU bracket 977 and the threaded joint 976 It will move forward along the lead screw 974. The arm 981 extending from the MTU bracket 977 will also rotate counterclockwise over a limited arc as the lead screw 974 rotates to engage the MTU160 and advance it through the illuminometer 950. ..

0290After the MTU160 has passed the PMT956, the MTU is pushed out of the illuminometer 950 and can be attracted to the next MTU through the illuminometer 950. The MTU bracket 977 moves toward the MTU inlet end of the MTU transfer path by rotating the lead screw 974 clockwise. Due to system friction, the threaded joint 976 and MTU bracket 977 rotate counterclockwise until the guide rod 980 touches the right side of the guide opening 979, after which the threaded joint 976 and MTU bracket 977 continue to rotate. The MTU bracket 977 will retract along the lead thread 974. This clockwise movement of the MTU bracket 977 causes the arm 981 to rotate clockwise over a limited arc and withdraw from the MTU, thus causing the MTU bracket 977 to retract without contacting the MTU. be able to. That is, when the MTU bracket 977 retracts, the arm 981 passes through the tip of the MTU.

0291As shown in FIG. 41, the blinder 982 driven by the blinder actuator 993 moves vertically and vertically in line with the opening 953. The blinder 982 is attached to slide with respect to the opening box 958 and includes a substantially rectangular opening (not shown) formed therein, which opening may be aligned with the opening 953. Includes front panel 983. The tip of the front panel 983 blocks the opening 953 when the opening formed in the panel 983 is not aligned with the opening 953 and thus acts as a shutter for the opening 953. The blinder 982 is placed parallel to the opposite side of the opening and extends over the two side walls 987, which are approximately perpendicular to the front panel 983, and the rear end of the side wall 987 opposite the front wall 983. Includes a rear wall 988 that is approximately parallel to 983. The side wall 987 and rear wall 988 are partially rectangular housings sized to accommodate one MTU160 reaction tube 162 as the blinder 982 is raised below one of the MTU160 reaction tubes 162 by the blinder actuator 993. Define the body. The blinder actuator 993 may be a linear stepping actuator including a stepping motor 992 and a lead thread 994. A HIS linear stepping actuator commercially available from Haydon Switch and Instrument, Inc. (Waterbury, Connecticut) was used.

0292After the MTU 160 is placed in the illuminometer 950 by the right transfer mechanism 500, the motor 972 is energized to pull the first reaction tube of the MTU in line with the opening 953. The blinder 982 is typically stored outside the MTU transport path, with the side wall 987 and rear wall 988 of the blinder 982 surrounding the reaction tube 162, and the opening formed in the front panel 983 of the blinder 982 with the opening 953. Lifted by blinder actuator 993 until lined up. Since the blinder 982 substantially prevents light from sources other than the reaction tube 162 in front of the opening 953 from reaching the opening 953, the PMT956 only emits light from the reaction tube directly in front of the opening 953. Is detected.

0293When the PMT shutter opens, different detection reagents (Detect I and Detect II) taken from containers 1146, 1170 of the lower chassis 1100 subsequently extend to reagent port 984 at the tip of the photometer 950 (not shown). ) Is dropped into the arranged reaction tube 162. The Detect I and Detect II reagents are hydrogen peroxide-containing reagent and sodium hydroxide-containing reagent, respectively, and when combined, form a basic hydrogen peroxide solution that enhances the chemiluminescence of the unhydrolyzed acridinium ester label. To do. Due to the instability of basic hydrogen peroxide, the Detect I and Detect II reagents are preferably combined in the reaction tube 162 immediately prior to detection on the photometer 950.

0294After the addition of DetectII, the PMT956 is used to detect the light emitted from the contents of the reaction tube 162, followed by the PMT shutter closing. The PMT956 converts the light emitted by the chemiluminescent label into an electrical signal, which is processed by the electronic unit and then connected to the controller 1000 or connector 986 via a cable (not shown). Sent to other peripherals.

0295If the required sensitivity is low, it may be possible to use an optical sensor instead of a photomultiplier tube. An example of an acceptable optical sensor that can be used with the illuminometer 950 is a diode. An optical sensor may also be suitable if the material of the MTU160 is a preferred polypropylene material with a relatively transparent appearance rather than translucent. When choosing materials for MTU160, care should be taken to avoid materials that emit naturally or have the property of accumulating static electricity, both of which can be likely to interfere with false positives and quantification measurements. ..

0296The process described above is repeated for each reaction tube 162 of MTU160. After measuring the chemiluminescent signal from each reaction tube 162 of the MTU160, the motor 972 advances and moves the MTU160 through the exit door 961 through the photometer 950 and into the amplicon deactivation station 750.

0297The currently preferred alternative illuminometer is generally the one specified by reference number 1360 in FIG. The luminometer 1360 includes a housing 1372 with a bottom wall 1370, a door assembly 1200 opposite the bottom wall 1370 defining the ends of the housing 1372, an optical sensor shutter assembly 1250 defining the front wall of the housing 1370, and a housing. Complete 1370, including an upper wall (not shown) and a rear wall (not shown) defining the housing. The right door assembly 1200 defines the container inlet opening 1374, and the left door assembly 1200 defines the container outlet opening 1376 through which the MTU 160 can enter and exit housing 1370. Each door assembly 1200 controls access through its opening 1374 or 1376, and is a revolving door rotatably located between the end wall 1202, the cover plate 1232, and the end wall 1202 and the cover plate 1232. Equipped with 1220. The photosensor aperture shutter assembly 1250 controls a photosensor (not shown in FIG. 43), for example, the light entering a photomultiplier tube. The illuminometer 1360 has an optical receiver mounting wall 1250 and a cover plate 1290 with an opening 1292 formed therein.

0298A barcode scanner 1368 is attached to the front of the housing 1372 to scan the MTU before entering the illuminometer 1360.

0299The container transfer assembly 1332 moves the container (eg, MTU160) from the inlet opening 1374 to the outlet opening 1376 via the illuminometer 1360. Assembly 1332 includes a transfer 1342 that is movably transported to a threaded lead screw 1340 that is rotated by a motor 1336 connected to a lead thread 1340 by a belt (not shown).

0300The dispensing nozzle 1362 is attached within the upper wall (not shown) and is connected by conduit tubes 1364 and 1366 to the pump and ultimately to bottles 1146 and 1170 in the lower chassis 1100. Nozzle 1362 dispenses "Detect I" and "Detect II" reagents into the reaction vessel 162 of MTU160 in housing 1372.

0301The reaction tube positioner assembly 1300 is located within the housing 1372 and is configured to hold each reaction tube 162 of the MTU 160 in front of the opening 1292 and optionally isolate each retained reaction tube from an adjacent reaction tube. Due to the placement, only light from one reaction tube at a time enters the opening 1292. The positioner assembly 1300 comprises a container positioner 1304 rotatably mounted within a positioner frame 1302 fixed to floor 1370 of housing 1372.

0302A door assembly 1200 for the MTU entrance opening 1374 and exit opening 1376 of the illuminometer 1360 is shown in FIG. The door assembly 1200 includes an illuminometer end wall 1202 that forms the end wall of the illuminometer housing 1372. The end wall 1202 includes a first recessed area 1206, and a second circular recessed area 1208 is superposed on the first recessed area 1206. A circular groove 1207 extends around the periphery of the circular recessed area 1208. Within the circular recessed area 1208, a slot 1204 having a shape that substantially matches the vertical profile of the MTU 160 is formed toward one side of its center. A short center pillar 1209 extends from the center of the circular recessed area 1208.

0303The revolving door 1220 has a circular shape and includes a shaft wall 1222 extending around the periphery of the revolving door 1220. The shaft wall 1222 is located at a short radial distance from the outer peripheral edge of the revolving door 1220, thus defining an annular step 1230 around the outer outermost peripheral edge of the shaft wall 1222. Off-center positions of the revolving door 1220 form a slot 1226 having a shape that closely matches the vertical profile of the MTU 160.

0304The revolving door 1220 is installed in the circular recessed area 1208 of the end wall 1202. The central opening 1224 receives the middle column 1209 of the end wall 1202, and the circular groove 1207 receives the shaft wall 1222. The annular step 1260 is on the flat surface of the recessed area 1206 that surrounds the circular recessed area 1208.

0305The end wall 1202 includes a drive gear recess 1210 that houses the drive gear 1212 attached to the drive shaft of the motor 1213 (see FIG. 43 showing only the motor 1213 for the right door assembly 1200). The motor 1213 is preferably a DC gear motor. Preferred DC gear motors are commercially available from Micro Mo Electronics, Inc. (Clearwater, Florida) under model number 1524TO24SR16 / 7 66: 1. The outer circumference of the shaft wall 1222 of the revolving door 1220 has gear teeth formed on it that mesh with the drive gear 1212 when the shutter is installed in the circular recess 1208.

0306The cover plate 1232 has a substantially rectangular shape and includes a raised area 1234 having a size and shape that substantially matches the recessed area 1206 of the end wall 1202. The cover plate 1232 forms an opening 1236 in it that has a shape that closely matches the longitudinal profile of the MTU, and if the cover plate 1232 is installed on the end wall 1202, the rectangular raised area 1234 is rectangular. Housed in the recessed area 1206, the opening 1236 is generally in line with the opening 1204. Thus, the revolving door 1220 is sandwiched between the cover plate 1232 and the end wall 1202, and the openings 1236 and 1204 both define the entrance opening 1374 and the exit opening 1376.

0307When the drive gear 1212 is rotated by the motor 1213, the revolving door 1220 meshing with the drive gear 1212 is rotated around the center column 1209. If the opening 1226 is aligned with the openings 1204 and 1236, the MTU 160 can pass through the opening 1374 (1376) of the door assembly 1200. The revolving door 1220 located in the circular recessed area 1208 and the raised area 1234 of the cover plate 1232 located in the recessed area 1206 of the end wall 1202 have realized a structure that virtually does not leak light. As a result, if openings 1226 are not aligned with openings 1204 and 1236, there is very little or no light coming in through the door.

0308Slotted light sensors are located in slots 1214 and 1216 located at opposite positions on the outer edge of the circular recessed area 1208. A preferred sensor, model number OPB857, is commercially available from Optek Technology, Inc. (Carolton, Texas). Slotted sensors located in slots 1214 and 1216 detect the presence of a notch 1228 formed in the shaft wall 1222 and signal the door open and door closed states.

0309The light sensor aperture shutter assembly 1250 is shown in Figure 45. An optical receiver such as a photomultiplier tube 956 is connected to an optical receiver opening 1254 formed in the optical receiver mounting wall 1252. The optical receiver mounting wall 1252 includes a substantially rectangular two-layer raised area 1256 that defines a substantially rectangular step 1257, with a circular recessed area 1258 superimposed on the rectangular raised area 1256. .. A circular groove 1261 extends around the periphery of the circular recessed area 1258. A central pillar 1259 is held in the center of the circular recessed area 1258. An optical receiver opening 1254 is formed in the circular recessed area 1258. In the illustrated embodiment, the optical receiver opening 1254 is located under the center column 1259, but the optical receiver opening 1254 may be held at any position within the circular recessed area 1258.

0310Aperture shutter assembly 1250 includes a rotary shutter 1270 having a shaft wall 1274 with gear teeth formed on its outer periphery. The shaft wall 1274 is formed not on the outer periphery of the shutter 1270 but in the vicinity thereof, thereby defining the annular step 1276. The rotary shutter 1270 is installed in a circular recess area 1528, along with a center column 1259 housed in a central opening 1272 formed in the rotary shutter 1270 and a shaft wall 1274 housed in a circular groove 1261. The drive gear 1262, located in the gear recess 1260 and connected to the drive motor 1263, meshes with the outer gear teeth formed on the shaft wall 1274 of the rotary shutter 1270 to rotate the rotary shutter 1270 around the center column 1259. .. The preferred drive motor 1263 is a DC gear motor commercially available from Micro Mo Electronics, Inc. (Clearwater, Florida) under model number 1524TO24SR16 / 7 66: 1. Micro Mo gear motors are preferred because they provide high quality, low resilience motors. An opening 1280 is formed in the rotary shutter 1270, and the opening can be moved in line with and out of line with the optical receiver opening 1254 when the rotary shutter 1270 is rotated.

0311A shutter 1270 installed in the circular recessed area 1258 installs a cover plate or sensor opening wall 1290 in the sensor mounting area 1252. As shown in FIG. 45A, the sensor opening wall 1290 defines a substantially rectangular step 1297 and is approximately rectangular in size and shape to accommodate the rectangular ridge area 1256 of the sensor mounting portion 1252. Includes a layered recessed area 1296. The sensor opening 1292 is formed by the opening wall 1290 and generally aligns with the light receiver opening 1254 formed within the sensor mounting portion 1252. The sensor aperture 1292 is generally an elongated oval shape having a width substantially corresponding to the width of the individual reaction tubes 162 of the MTU 160 and a height corresponding to the height of the desired viewing area. In the illustrated embodiment, the opening 1280 of the shutter 1270 is shown as circular, but the opening 1280 is a rectangle with a width corresponding to the width of the reaction tube 162, or an elongated shape similar to the sensor opening 1292. It may have other shapes such as an ellipse. Rotation of the rotating shutter 1270 to a position where the opening 1280 is aligned with the optical receiver opening 1254 and the sensor opening 1292 allows light to reach the PMT956, where the opening 1280 is the optical receiver opening 1254 and Rotating the rotary shutter 1270 to a position not aligned with the sensor aperture 1292 prevents light from reaching the PMT956.

0312Slotted light sensors are placed in slots 1264 and 1266 to detect the notch 1278 formed in the shaft wall 1274 of the shutter 1270 to detect the open and closed positions of the shutter 1270. A preferred slotted light sensor is commercially available from Optek Technology, Inc. (Carolton, Texas) under model number OPB857.

0313The opening wall 1290 includes an upward step 1294 extending along its width. The downward step of the MTU160, defined by the connecting rib structure 164 of the MTU160 (see Figure 58), is supported by the step 1294 as the MTU160 slides through the illuminometer.

0314Reaction tube positioner assembly 1300 is shown in Figures 46 and 48-49. The reaction tube positioner 1304 is arranged so as to be operable in the reaction tube positioner frame 1302. The reaction tube positioner 1304 is mounted within the reaction tube positioner frame 1302 so that it rotates around the shaft 1308. The shaft 1308 operates with a rotary solenoid, or more preferably a gear motor 1306, to selectively rotate the reaction tube positioner 1304 between the retracted position shown in FIG. 46 and the fully extended position shown in FIG. Connected as possible. A preferred gear motor drive is commercially available from Micro Mo Electronics, Inc. (Clearwater, Florida) as model number 1724T024S + 16/7 134: 1 + X0520.

0315As shown in FIG. 47, the reaction tube positioner 1304 contains a V-block structure 1310 that defines two parallel walls 1312. The reaction tube positioner 1304 further includes an area at its lower end from which a portion of the thickness of the reaction tube positioner 1304 has been removed, thereby defining a relatively thin arched flange 1314.

0316When the MTU160 is inserted into the illuminometer 1360, the reaction tube positioner 1304 is in the retracted position shown in FIG. If the individual reaction tubes 162 are located in front of the sensor opening 1292 (see Figure 45A) so that the chemiluminescent sensor reading of the contents of the reaction tube 162 is performed, the reaction tube positioner 1304 is shown in Figure 49. Rotate forward to the indicated engagement position. At the engagement position shown in FIG. 49, the V block 1310 engages the reaction tube 162, thereby gripping the reaction tube in an appropriate position in line with the photometer opening 1292. As shown in FIG. 45, the opening wall 1290 includes a protrusion 1298 extending from the back surface of the wall 1290 to the MTU passage of the illuminometer. The protrusion 1298 is aligned with the opening 1292 so that when the reaction tube positioner 1304 engages the reaction tube 162, the reaction tube is pushed laterally and collides with the protrusion 1298 as a hard stop, so that the reaction tube positioner 1304 Prevents the reaction tube 162 in the MTU passage from being tilted significantly. The parallel side wall 1312 of the V block 1310 prevents stray light from the adjacent reaction tube 162 of the MTU 160 from reaching the optical receiver while the reaction tube 162 located directly in front of the opening 1292 is being read. To do.

0317The slotted light sensor 1318 is attached to the lower part of the frame 1302, along with an arched flange 1314 held operably with respect to the sensor 1318. A preferred slotted light sensor is commercially available from Optek Technology, Inc. (Carolton, Texas) under model number OPB930W51. An opening 1316 is formed in the flange 1314. If the reaction tube positioner 1304 engages the reaction tube 162 and the reaction tube 162 and the protrusion 1298 prevent further rotation of the reaction tube positioner 1304, the opening 1316 is properly aligned with the sensor 1318. If the reaction tube 162 is not properly held in front of the reaction tube positioner 1304, the reaction tube positioner 1304 will rotate forward to the position shown in FIG. 48, in which case the opening 1316 will not line up with the sensor 1318 and will be erroneous. A signal will be generated.

0318When using a gear motor 1306 to rotate the reaction tube positioner 1304, the positioner retract, or "home," to stop the gear motor when the reaction tube positioner 1304 is completely retracted, as shown in FIG. It is necessary to provide a second sensor (not shown) to generate the signal. A preferred sensor is commercially available from Optek Technology, Inc. (Carolton, Texas) under model number OPB900W.

0319The MTU transfer assembly 1332 is shown in Figure 50. The MTU transfer assembly 1332 is held operably adjacent to the top edge of the intermediate wall 1330 (not shown in Figure 43) of the illuminometer 1360. The intermediate wall 1330 defines one side of the MTU transfer path through the photometer housing 1372 and includes a rectangular opening 1334. The reaction tube positioner frame 1302 (see, eg, FIG. 48) is attached to an intermediate wall 1330 near the opening 1334, and the reaction tube positioner 1304 rotates through the opening 1334 until it engages the MTU 160.

0320The MTU transfer 1342 includes a threaded joint 1344 that is transported to a threaded lead thread 1340 and has a thread that meshes with the threaded thread of the lead thread 1340, and an MTU yoke 1346 that is integrally formed with the threaded lead thread 1344. As shown in FIG. 51, the MTU yoke 1346 includes a longitudinally extending portion 1356 and two laterally extending arms 1348 and 1350, from which a longitudinal extension 1352 extends. The stepping motor 1336 drives the lead screw 1340 via the drive belt 1338. The preferred stepper motor is the VEXTA stepper motor of model number PK266-01A, commercially available from Oriental Motors Ltd. (Tokyo, NY), and the preferred drive belt is commercially available from SDP / SI (New Hyde Park, NY). ing.

0321When the MTU 160 is inserted into the MTU transfer path of the illuminometer 950 by the right transfer mechanism 500, the first reaction tube 162 of the MTU 160 is preferably placed directly in front of the sensor opening 1292 and is therefore suitable for the first reading. Is held in. The width of the yoke 1346 between the lateral arms 1348 and 1350 corresponds to the length of a single MTU160. The transfer 1342 is moved between the first position and the second position shown in the phantom of FIG. 50 by the rotation of the lead screw 1340. Slotted light sensors 1341 and 1343 indicate whether the transfer 1342 is in the first or second position, respectively. Friction between the lead thread 1340 and the thread splicing 1344 causes the MTU transfer 1342 to tend to rotate with the lead thread 1340. However, the rotation of the MTU transfer 1342 with the lead screw 1340 engages with the tip of the intermediate wall 1330 at the bottom of the yoke 1346 and the top cover of the photometer housing 1372 at the upper stop 1354 (not shown). It is preferably limited to 12 degrees by engagement with.

0322To engage the MTU inserted in the luminometer 1360, the lead thread 1340 rotates in the first direction and due to friction in the threads between the thread splicing 1344 and the lead thread 1340, the transfer 1342 is transferred to the upper stop 1354. Rotate upward with the lead screw 1340 until it hits the top cover (not shown) of the luminometer 1360. At that time, if the rotation of the lead screw 1340 is continued, the transfer 1342 moves backward to the position shown by the phantom in FIG. Lateral arms 1348, 1350 pass the tip of the MTU as the transfer 1342 moves backwards. Reverse rotation of the lead screw 1340 causes the transfer 1342 to first rotate downward with the lead thread 1340 until the bottom of the yoke 1346 hits the top edge of the wall 1330, where the lateral arms 1348 and 1350 of the yoke 1346 , Straddles the MTU160 located inside the illuminometer 1360.

0323The MTU transfer mechanism 1332 is then used to move the MTU 160 further forward to hold each of the individual reaction tubes 162 of the MTU 160 in front of the light sensor opening 1292. After measuring the last reaction tube 162 with an optical receiver in the photometer, the transfer 1342 moves the MTU160 to a position adjacent to the exit door, where the lead screw 1340 reverses direction, thereby as described above. In addition, the transfer 1342 is retracted to the initial position behind the MTU160. The rotation of the lead screw 1340 is reversed again to advance the transfer 1342 as described above. The exit door assembly 1200 opens and the longitudinal extension 1352 of the yoke 1346 engages the MTU operating structure 166 of the MTU 160 to push the MTU 160 out of the photometer exit door and into the deactivation preparation room 750.

0324(Deactivation station) At the amplicon deactivation station 750, a deactivation solution such as buffered bleach is added to the reaction tube 162 of MTU160 by a dedicated dropping line (not shown) to inactivate nucleic acids in the residual fluid of MTU160. Examples of nucleic acid deactivated solutions are disclosed, for example, in Dattagupta et al., US Pat. No. 5,612,200, and Nelson et al., US Pat. No. 2005-0202491 A1. The fluid contents of the reaction tube are sucked by a tubular member (not shown) connected to a dedicated suction line and collected in a dedicated liquid waste container in the lower chassis 1100. The tubular member preferably has a length of 4.7 inches and an inner diameter of 0.041 inches.

0325The MTU shuttle (not shown) moves the MTU 160 further (to the right in Figure 3) by sending each subsequent MTU 160 from the photometer 950 to the deactivation station 750. Before the MTU is sent to the deactivation station 750 by the photometer 950, the MTU shuttle must be retracted to its home position as detected by a strategically held optical slot switch. After receiving the MTU160 from the illuminometer, the shuttle moves the MTU160 to a deactivation station where a dedicated drop line connected to a dedicated injector dispenses the deactivating solution into each reaction tube 162 of the MTU160. If there is a previous MTU in the deactivation preparation room, push it forward by the distance traveled by the MTU shuttle. Sensors at the deactivation station verify the presence of both the MTU and the MTU shuttle, thereby preventing the occurrence of deactivation fluid injections into non-existent MTUs or double injections into the same MTU.

0326The suction station (not shown) contains five mechanically connected suction tubes, which are mounted for vertical movement on the suction tube rack and connected to the actuator to lift and lower the suction tubes. Has been done. The suction station is in the last position along the deactivation preparation room, before the MTU descends through a hole in the reference plate 82 and enters the waste bin 1108. With or without the MTU in the suction station, the suction tube circulates up and down once each time the MTU moves into the deactivation station. If the MTU is present, the suction tube sucks the fluid contents from the MTU. When the next MTU is moved into the deactivation station by the MTU shuttle, the previously sucked MTU is pushed out of the end of the deactivation preparation room and falls into the waste bin 1108.

0327Ideally, the analyzer 50 can perform about 500 preferred assays in 8 hours or about 1,000 preferred assays in 12 hours. Once the analyzer 50 is prepared and initialized, it usually does not require or rarely requires operator assistance or intervention. The analyzer can simultaneously perform multiple assay types in which different MTUs may or may not be treated the same, but in one assay each sample is treated the same. As a result, manual pipetting, incubation time regulation, temperature control, and other limitations associated with manually performing multiple assays are circumvented, thereby increasing reliability, efficiency, and throughput. Also, the operator's exposure to the sample is generally limited to sample input, which significantly reduces the risk of possible infection.

0328(Real-time amplification assay) Real-time amplification assays can be used, for example, to determine the presence and amount of nucleic acid of interest in samples derived from pathogens or viruses. By determining the amount of nucleic acid of interest in the sample, practitioners can estimate the amount or input of organisms or viruses in the sample. In one application, real-time amplification assays include hepatitis C virus (HCV) and human immunodeficiency. Used to screen blood or blood products for transfusion for blood-borne pathogens, such as Virus; HIV), or to monitor the efficacy of treatments in patients infected with the pathogen or virus. Good. The real-time amplification assay may be used for diagnostic purposes as well as for gene expression determination. In the preferred use of the invention described above, the presence of the desired organism or virus was obtained from the nucleic acid sequence of interest (ie, the organism or virus) derived from the desired organism or virus under certain conditions of use. Determined using a probe that exhibits specificity in the sample (contained in the nucleic acid of interest, or its amplification product). To demonstrate specificity, the probe so that under selective assay conditions, the probe hybridizes so that it is detectable with the target sequence or its complement rather than any non-target nucleic acid that may be present in the sample. It must have a nucleotide sequence that is substantially complementary to the target or its complement.

0329In addition to the "end point" amplification assay described above, where the amount of amplification product containing the target sequence or complement thereof is determined at a detection station such as an illuminometer 950 at the end of the amplification procedure, the present invention is the target sequence or A "real-time" amplification assay can also be performed in which the amount of amplification product containing complement is determined during the amplification procedure. In a real-time amplification assay, the concentration of the nucleic acid of interest can be determined by periodically determining the amount of amplification product in the sample containing the target sequence or its complement and calculating the rate at which the target sequence is amplified. it can. It is preferred that the instrument can be used selectively in the endpoint or real-time detection mode, or simultaneously in both modes.

0330In a real-time amplification assay, the probes interact to emit different signals depending on whether the probe is self-hybridizing or hybridizing to the target sequence or its complement, an interaction label. It is preferably a single molecule self-hybridizing probe having a pair. For example, Diamond et al., US Pat. No. 4,766,062 "Displacement Polynucleotide Assay Method and Polynucleotide Complex Reagent Therefor"; Tyagi et al., US Pat. No. 5,925,517 "Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays and Kits"; Tyagi et al., US Pat. No. 6,150,097. Issue "Nucleic Acid Detection Probes Having Non-FRET Fluorescence Quenching" and Kits and Assays See "Including Such Probes"; and Becker et al., US Pat. No. 6,361,945, "Molecular Torches." Use of other probes in the present invention, including the use of complementary two molecule probes, probes labeled with insert dyes, and insert dyes to distinguish single-stranded and double-stranded nucleic acids. May be considered. For example, Morrison, US Pat. No. 5,928,862 "Competitive Homogenous Assay"; Higuchi, US Pat. No. 5,994,056 "Homogenous Methods for Nucleic Acid Amplification and Detection": and Yokoyama et al., US Pat. No. 6,541,205 "Method for Assaying Nucleic". See "Acid". Examples of interaction labels include enzymes / substrates, enzymes / cofactors, luminescent / quenchers, luminescent / addition compounds, dye dimers and Forrester energy transfer pairs. Methods and materials for ligating the interaction label to the probe for optimal signal discrimination are described in the references above.

0331In a preferred real-time amplification assay, the interaction label comprises a fluorescent moiety and a quencher moiety such as, for example 4- (4-dimethylaminophenylazo) benzoic acid (DABCYL). The fluorescent portion emits (ie, fluoresces) the light energy of a particular emission wavelength when excited by the light energy of the appropriate excitation wavelength. When the fluorescent portion and the quencher portion are held close to each other, the light energy emitted from the fluorescent portion is absorbed by the quencher portion. However, when the probe hybridizes to the nucleic acid present in the sample, the fluorescent and quencher moieties are separated from each other and the light energy emitted by the fluorescent moiety can be detected. Fluorescent moieties that are excited and emit at different identifiable wavelengths can be combined with different probes. Different probes can be added to the sample, and the presence and amount of nucleic acid of interest associated with each probe alternately exposes the sample to light energies of different excitation wavelengths, from samples of different wavelengths corresponding to different fluorescent moieties. It can be determined by measuring the light emission of.

0332In an example of a multiple real-time amplification assay, the following may be added to the sample prior to initiating the amplification reaction: the quencher moiety and the HCV-derived nucleic acid tethered to its 5 ́ and 3 ́ ends. First fluorescent dye having specificity for the sequence (excitation wavelength λ_ex1And emission wavelength λ_em11st probe with); a second probe that is tethered to the quencher moiety and its 5 ́ and 3 ́ ends and has specificity for the nucleic acid sequence from HIV type 1 (HIV-1). Fluorophore (excitation wavelength λ_ex2And emission wavelength λ_em2A second probe with) and a third probe that is tethered to the quencher moiety and its 5 ́ and 3 ́ ends and has specificity for a West Nile virus (WNV) -derived nucleic acid sequence. Fluorophore (excitation wavelength λ_ex3And emission wavelength λ_em3With) and a third probe. After combining the probe in the sample with the amplification reagent, the wavelength λ_ex1, Λ_ex2, And λ_ex3The sample was periodically and alternately exposed to the excitation light of the wavelength λ._em1, Λ_em2, And λ_em3The emission light in can be measured to detect the presence (or absence) and amount of all three viruses in a single sample. The components of the amplification reagent vary depending on the assay performed, but generally in a suitable buffer, at least one amplification oligonucleotide such as a primer, promoter primer, and / or promoter oligonucleotide, nucleoside triphosphate, and magnesium. Contains cofactors such as ions.

0333If you use amplification procedures to increase the amount of target sequence or complement present in the sample before detection can occur, make sure that amplification has taken place, thereby avoiding false negatives. It is desirable to include a "control" for this. Such a control may be a known nucleic acid sequence unrelated to the desired sequence. A combination of a probe (ie, control probe) with a specificity to the control sequence and a unique fluorescent dye (ie, control dye) and a quencher is required to amplify the control sequence as well as the target sequence. Add to sample with one or more amplification reagents. After exposing the sample to appropriate amplification conditions, the sample is alternately exposed to light energies of different excitation wavelengths (including the excitation wavelength of the control dye) to detect emitted light. Detection of emitted light at a wavelength corresponding to the control dye confirms successful amplification (ie, the control sequence was indeed amplified) and therefore detection of emitted light corresponding to the probe of the target sequence. It is considered that any failure of is not due to amplification failure. Conversely, failure to detect emitted light from the control dye is considered to indicate amplification failure and therefore any results of the assay are questionable.

0334The real-time amplification assay is performed within a real-time incubator (RT incubator), which is a modified version of the AT incubator 602 described above. The RT incubator designated by reference number 608 in FIGS. 61 and 64-65 is essentially a rotating incubator such as incubators 600, 602, 604, and 606. The RT incubator is a reaction tube of the MTU160 carried into the RT incubator by measuring the fluorescence emitted by the dye in each reaction tube 162 of the MTU160 when the MTU160 is irradiated with the excitation light corresponding to each dye. Includes an instrument attached to it for detecting the amplification occurring within 162 in a real-time fashion. The RT incubator 608 may be integrated into the automated diagnostic analyzer 50 by modifying the AT incubator 602 to function as an AT incubator 602 for end point amplification assays or as an RT incubator 608 for real-time amplification assays. Alternatively, the RT incubator 608 may be secured on a structure (not shown) attached to the housing 60 if it is desirable to keep the AT incubator 602 away from the RT incubator 608. In this case, additional mechanisms such as transfer mechanisms 500 and 502 are required to transfer the MTU160 from the processing desk 200 of the analyzer 50 to the RT incubator 608 transported to the ancillary structure adjacent to the processing desk 200. I can fully understand that.

0335The instrument attached to the RT incubator 608 for real-time fluorescence detection is known as a photodetector module (a type of signal measuring device), as described herein.

0336The photodetector module, generally designated by reference number 1700, is shown in the side sectional view of FIG. FIG. 61 also shows a portion of the floor 613 of the RT incubator 608, with the photodetector module 1700 extending through an opening 615 formed in the floor 613. In FIG. 61, a portion of the cylindrical wall 610 is shown, but the insulating coating 612 is not shown for clarity. Also, the reference plate 82, to which the RT incubator 608 is mounted and most of the photodetector modules 1700 are located beneath it, is not shown in FIG. A portion of the MTU160 is shown to be held above the photodetector module 1700, which is held under the first reaction tube 162a of the photodetector module 1700. The substantially identical photodetector module 1700 is preferably held for each of the other reaction tubes 162b, 162c, 162d, and 162e at different locations in the RT incubator 608.

0337As shown in FIGS. 61-63, the photodetector module 1700 includes a housing 1710 attached to the printed circuit board 1790. The housing 1710 includes four sections: an excitation light housing 1714, an excitation lens housing 1712, an adapter pipe 1718, and an emission lens housing 1716. The excitation lens housing 1712, the excitation light housing 1714, and the emission lens housing 1716 are each preferably formed from machined 6061-T6 aluminum with a black anodized finish. The adapter pipe 1718 is preferably made of Delrin (R) resin. As can be seen from FIG. 61, the adapter pipe 1718 is in close proximity to the incubator floor 613 of the RT incubator 608. Therefore, in order to provide a certain level of thermal separation of the photodetector module 1700 from the RT incubator 608, the adapter pipe 1718 is formed from a material with low thermal conductivity, such as Delrin (R) resin. preferable. The adapter pipe 1718 also provides additional electrical separation between the housing 1710 and the circuit board 1790.

0338The excitation light housing 1714 houses the excitation light assembly 1730 (discussed in more detail below) and is attached to the printed circuit board 1790 at its lower end and to the end of the excitation lens housing 1712 at its upper end. .. The excitation light housing 1714 is attached to the excitation lens housing 1712 using mechanical fasteners such as screws (not shown). The assembly may also include a location pin 1721 (see FIG. 62) that extends between the excitation light housing 1714 and the excitation lens housing 1712 to facilitate accurate relative positioning of each housing during its assembly.

0339The excitation lens housing 1712 includes a first portion 1713 that is horizontally oriented in the explanatory view and a second portion 1715 that extends perpendicularly from the first portion 1713 and is vertically oriented in the drawing. The slanted surface 1717 preferably has an angle of 45 ° with respect to the vertical axis of the first portion 1713 and the second portion 1715.

0340The adapter pipe 1718 includes a base portion 1720 adapted to join the end of a second portion 1715 of the excitation lens housing 1712 in a light-tight manner. More specifically, the base portion 1720 of the adapter pipe 1718 preferably comprises a circular overhang 1724 (see FIG. 61), which is within the upper end of the second portion 1715 of the excitation lens housing 1712. Extends into the recesses of the size and shape of the joint formed in. The upper portion 1722 of the adapter pipe 1718 projects above the base portion 1720. The upper portion 1722 is preferably circular in shape and is adapted to project through an opening 615 formed in the floor 613. The upper portion 1722 preferably has a lateral dimension smaller than that of the base portion 1720, thereby forming a step portion 1726 between the upper portion 1722 and the base portion 1720, wherein the step portion 1726 is a light detection module. When 1700 is installed in RT incubator 608, it leans against the bottom of floor 613.

0341The adapter pipe 1718 is preferably secured to the excitation lens housing 1712 using mechanical fasteners such as screws (not shown) to facilitate accurate relative positioning of each part during its assembly. A locator pin (not shown) may extend between the adapter pipe 1718 and the second portion 1715 of the excitation lens housing 1712.

0342The emission lens housing 1716 is attached to the printed circuit board 1790 at its lower end at a position separated from the excitation light housing 1714. The lower end of the emission lens housing 1716 has a preferred 45 ° angle and is in the form of an oblique surface 1719 that coincides with the oblique surface 1717 of the excitation lens housing 1712. The excitation lens housing 1712 and the emission lens housing 1716 are preferably connected to each other by mechanical fasteners such as screws (not shown), between the housings to facilitate accurate positioning of the housing during its assembly. May include location pin 1723 extending to.

0343Gaskets (not shown) may be placed on the joint surfaces between any of the housings to limit light entry into the housings 1710. Such gasket materials may include, for example, foaming materials.

0344As shown in FIGS. 61 and 63, the internal optics of the photodetector module 1700 includes an excitation light assembly 1730, an excitation lens assembly 1740, and an emission lens assembly 1770.

0345The Excited Light Assembly 1730 includes a Light Emitting Diode (LED) 1732 connected to the printed circuit board 1790 in a conventional fashion. Different fluorochromes are excited at different wavelengths. In one multiple application of the invention, the preferred dyes are the rhodamine dyes tetramethyl-6-rhodamine (TAMRA) and tetrapropano-6-carboxyrhodamine (ROX), respectively, in combination with the DABCYL quencher, as well as fluorescence. Dye 6-carboxyfluorescein (FAM) and 2 ́,7 ́-dimethoxy-4 ́,5 ́-Dichloro-6-carboxyrhodamine (JOE) is included. The excitation spectrum of the preferred dye is shown in FIG. Since preferred dyes are excited at different wavelengths, the light detection module 1700 is tailored to emit excitation light at or near the desired excitation wavelength (ie, color) for the particular dye of interest. Is preferable. Therefore, the component selection of the optical system is often dominated by the particular dye of interest for the photodetector module. For example, for LED1732, the choice of a particular LED (manufacturer and model number) depends on the dye targeted by the photodetector module. For FAM dyes, the preferred LED is commercially available from Kingbright Corporation (Industry, CA) as model number L7113PBCH, and for TAMRA dyes, the preferred LED is commercially available from Kingbright as model number L7113VGC / H, ROX. For dyes, the preferred LED is Agilent with model number HLMP-EL16-VY000 It is commercially available from Technologies, Inc. (Palo Alto, CA), and for JOE dyes, the preferred LED is commercially available from Kingbright as model number L7113VGC / H.

0346The optical pipe 1733 is held on top of the LED 1732 and includes a base portion 1734 and an elongated portion 1735 protruding from the base portion 1734. Optical pipe 1733, also known as a mixing rod, is preferably cast or extruded clear acrylic. The light pipe 1733 captures the light emitted by the LED 1732 and sends it upwards away from the LED 1732, creating a spatially homogeneous light distribution at the end of the elongated portion 1735 of the light pipe 1733 opposite the LED 1732. .. The optical pipe acts as an optical transmitter and as a physical spacer that matches the height of the excitation light assembly 1730 with the height of the emission lens assembly 1770. Also, the spatially homogeneous excitation light improves fluorometric reading and facilitates radiation reproducibility. The optical pipe 1733 extends through a narrow opening 1711 formed inside the excitation light housing 1714.

0347The light from the optical pipe 1733 is directed towards the mirror 1736 located at the upper end of the optical pipe housing 1714. Preferred mirrors include mirrors commercially available from Edmund Optics Inc. (Burlington, NJ) as part number Y43-790 (reinforced aluminum) and part number Y43-791 (protection gold). The mirror 1736 is preferably oriented at an angle of about 45 °. The cover 1738 is preferably made of Delrin (R) resin or other suitable material and is attached to the upper end of the excitation light housing 1714 on the mirror 1736. A mirror 1736 is placed in an opening with a counterbore formed at the upper end of the optical pipe housing 1714, after which the housing is closed using a cover 1738.

0348Excitation lens assembly 1740 includes a first lens 1744 and a second lens 1746. A preferred lens for the first lens 1744 is a lens commercially available from Edmund Optics as part number Y32-913, and a suitable lens for the second lens 1746 is Edmund as part number Y45-348. Examples include lenses commercially available from Optics. Lenses 1746 and 1744 are preferably separated from each other by a spacer element 1743 made of 6061-T6 aluminum with a black anodic finish. The light reflected by the mirror 1736 is preferably directed by the lenses 1744 and 1746, which collimate the reflected light within a tolerance of ± 10 °. Light that has passed through the second lens 1746 then passes through the baffle aperture 1750. The baffle opening 1750 is a ring with an inner surface at an angle of 35 ° with respect to the vertical axis (ie, the optical axis) of the first portion 1713 of the housing 1712, preferably machined 6061-T6 with a black anodized finish. Made of aluminum. The purpose of the baffle 1750 is to block out stray light outside the ± 10 ° margin of error. The second lens 1746 is preferably separated from the aperture baffle 1750 by a spacer element 1748 made of 6061-T6 aluminum with a black anodic finish.

0349Following the aperture baffle 1750, the light passes through the excitation filter 1752, removing unwanted spectral components of the excitation light. Again, the particular filter used depends on the excitation spectrum of the dye of interest for the photodetector module 1700. Preferred filters are part number HQ480 / 43x for dye FAM, part number HQ525 / 50x for dye TAMRA, part number HQ590 / 20x for dye ROX, and part number HQ514 / 22x for dye JOE, Chroma Technology Corp. Commercially available from (Rockingham, Vermont). The elements of the excitation lens assembly 1750 are gripped in place within the excitation lens housing 1712 using a retaining ring 1742. Suitable fastening rings are commercially available from Thorlabs, Inc. (Newton, NJ) under stock number SM18RR.

0350Next to the excitation filter 1752, the light hits the dichroic beam splitter 1754. The dichroic beam splitter 1754 is oriented at 45 ° and redirects the excitation light through the excitation filter 1752 90 ° towards the lens 1756 located in the adapter pipe 1718 (see below). The specific beam splitter used depends on the dye targeted by the photodetector module 1700. Preferred beam splitters are as follows: For FAM dyes, a suitable beam splitter is commercially available from Chroma Technology as stock number 511LP, and for TAMRA dyes, a suitable beam splitter is stock number 560LP, Chroma Technology. For ROX dyes, a suitable beam splitter is marketed by Chroma Technology as part number 615LP, and for JOE dyes, a suitable beam splitter is as part number 535LP for Chroma. It is commercially available from Technology. The lens 1756 is a focused lens that directs light through the window 1758, which then hits the reaction tube 162 of the MTU 160. Suitable focusing lenses are commercially available from Edmund Optics under stock number Y32-913.

0351When excited by light of the correct bandwidth, the contents of the reaction tube 162 fluoresce, thereby emitting light, assuming the presence of the particular dye in question. The light emitted from the contents of the reaction tube 162 passes through the window 1758 and returns to the lens 1756, which collects and focuses the emitted light as much as possible. Lens 1756 also collimates the emitted light so that it is preferably within a tolerance of ± 10 °. The light that has passed through the lens 1756 then hits the dichroic beam splitter 1754, and if the wavelength is different from the excitation light, the emitted light passes through and cannot be redirected by the beam splitter 1754. After passing through the dichroic beam splitter 1754, the light enters the emission lens assembly 1770, where it first collides with the aperture baffle 1772. The baffle 1772 is preferably made of 6061-T6 aluminum with a black anodic finish and has an internal opening at an angle of 35 ° to its vertical axis. The aperture baffle 1772 blocks light outside the ± 10 ° margin of error.

0352After passing through the aperture baffle 1772, the light collides with the emission filter 1774, which removes unwanted spectral components present in the emission light. The preferred particular filter depends on the wavelength of light emitted by the particular dye of interest for the photodetector module. FIG. 67 shows the emission spectra of preferred dyes used according to the present invention. Preferred emission filters are: For FAM dyes, a suitable filter is commercially available from Chroma Technology as part number HQ533 / 24m; for TAMRA dyes, a suitable filter is Chroma as part number HQ588 / 35m. Commercially available from Technology; for ROX dyes, the appropriate filter is commercially available from Chroma Technology as part number HQ640 / 40m; for JOE dyes, the appropriate filter is commercially available from Chroma Technology as part number HQ560 / 30m. Has been done.

0353The light then passes through the focusing lens 1778. Suitable lenses are commercially available from Edmund Optics under stock number Y45-348. The emitted light is focused by the lens 1778 on a photodiode 1780 that produces a current signal in proportion to the intensity of the emitted light. Suitable photodiodes are commercially available from UDT Sensors, Inc. (Hawthorne, Calif.) As model number PIN-10DI. The lens 1778 and filter 1774 are preferably separated from each other by a spacer element 1776 made of 6061-T6 aluminum with a black anodic finish. Elements of the emission lens assembly 1770 other than the photodiode 1780 are gripped in place within the emission lens housing 1716 using a retaining ring 1777. Suitable fastening rings are commercially available from Thorlabs, Inc. (Newton, NJ) under stock number SM18RR. The photodiode 1780 is connected to the printed circuit board 1790.

0354Figures 68A-68F illustrate circuits suitable for the circuit board 1790, including the LED 1732 and photodiode 1780, including an amplifier circuit that produces a voltage proportional to the current generated by the photodiode 1780.

0355The electronic circuit 1790 includes the following components and subcircuits: power supply 1800 and power supply filter formed by capacitors C6, C10, C17, resistors R11, R25 (Fig. 68E), excitation source (LED) 1732 (Fig. 68F). ), Excitation drive source circuit including U1 and various components (Fig. 68F), receiver (photodioden) 1780 and various components (Fig. 68A), preamplifier circuit U6 (pins 5-7), U7, and various Components (Fig. 68A), offset compensation circuit U6 (pins 1-3) and various components (Fig. 68A), microprocessor circuit U5 and various components (Fig. 68B), analog switch circuit SW1 and various configurations. Elements (Fig. 68B), as well as low-pass filter differential circuits U2, U3 (Fig. 68C), U4 (Fig. 68D) and various components.

0356To reject the effect of fluctuating background constant light, circuit 1790 incorporates microprocessor U5, which controls LED1732 (on / off) to modulate LED1732 and control analog switch SW1. Create the clock used for (FAM / ROX set to 250Hz, TAM set to 350Hz). Harmonize by modulating LED1732 (excitation) and changing the state of analog switch SW1 at the same frequency (changing the gain of subsequent differential filter U2 from positive gain to negative gain and vice versa). A transmitter / receiver pair is created. Only those optical signals arriving at the same frequency as this clock will be amplified, and all constant light and optical signals modulated at different frequencies will be suppressed.

0357The preamplifier (transimpedance) circuit-including U6 (pins 5-7) and U7-receives current from the photodiode 1780 and converts it to an amplified voltage. The offset compensation circuit-including U6 (pins 1-3) -provides a bias current that compensates for the current emitted from the photodiode 1780 in response to any constant light (unmodulated) incident on the photodiode 1780. This is because, given the gain of this preamplifier (20 mV / nA), the output of the preamplifier, which is easily and frequently realized, is not saturated with constant light (which may be orders of magnitude larger than the desired modulated light). It is an object.

0358By measuring high gain and small signals, the preamplifier circuit can be highly sensitive to measurement errors and changes in temperature and humidity as a result of EMI / RFI interference. To minimize these effects, the circuit traces, as well as the components including the high impedance circuits, especially the photodiode 1780 and the connection points, are located as far as possible from the other circuits. The printed circuit board 1790 is also preferably configured to facilitate the complete removal of contaminants capable of collecting adjacent critical high impedance components, in particular components R33, R36, and C21. To minimize the amount of contaminants and residual flux remaining on the circuit board 1790 after assembly, first wash the substrate with a saponifying agent suitable for the solder / flux used for soldering and then rinse with deionized water. Following these preparatory steps, it is preferred to solder the photodiode 1780 to the circuit board 1790 with "uncleaned flux" core solder, and any residual flux remaining on the circuit board 1790 provides a protective barrier and is therefore preferably preferred. Is not removed. These steps should mitigate the effects of long-term drift and circuit sensitivity associated with changes in temperature and humidity. Also, the preamplifier portion of the circuit board 1790 is completely contained within the grounded housing (Faraday cage) to suppress any EMI / RFI interference.

0359With reference to FIG. 68A, amplifiers U7 and U6 (pins 5-7) form the first two stages in the amplification of the optical signal. The components C20, C22, C24, C26, C27, C28, R35, and R45 provide power bypass / filtering for the amplifier. C18, D2, R32, and R34 form a -2.5V power supply with a filter that biases the anode of the photodiode 1780. The feedback resistors R33 and R36 convert the current from the photodiode 1780 to a voltage, while the C21 provides filtering for signals with frequencies above 3.6 KHz. The voltage divider formed by R37 and R38 provides a voltage gain of 10 in the next preamplifier stage, while the capacitor C23 provides additional lowpass filtering.

0360Amplifier U6 (pins 1-3) creates a DC bias current that negates the current from the photodiode 1780 due to background constant light and other inherent DC offsets in the circuit. The circuit forms an integrator amplifier that produces a current that is fed back to the input of the first preamplifier circuit (formed by U7). This results in an output signal at U7 and U6, where pin 7 has a zero DC component, i.e. the center of the signal is 0V.

0361The microprocessor U5 (Fig. 68B) provides LED functionality (eg, turns off LED1732 or modulates LED1732 to the target operating frequency: 250Hz for FAM and ROX, 350Hz for TAM) and differential amplifier gain. Control. Depending on its input signals (pins 6 and 7), the microprocessor U5 controls the LED 1732 to an "off" or "modulated" state. The gains of the differential filter circuits U2, U3, and U4 can be adjusted within the range of plus or minus 12 according to the phase relationship between the control signal to the LED1732 and the analog switch SW1.

0362Referring to Figure 68F, components C1, R3, and VR1 form a reference voltage circuit, which, along with resistors R18 and R27, establish LED current (when LED1732 is on). .. The components R1, R2, and Q1 form an LED control circuit, in which when the FET switch Q1 is on, the voltage on the input pin of the amplifier U1 rises, causing the amplifier output to go down. , LED current switch Q2 is effectively turned off. When Q1 is not turned on, U1 controls the voltage at the gate of FET switch Q2 so that the current through LED1732 is controlled at the established setpoint. The LED modulation frequency and off / on control are controlled by inputting "LED_ON" from the microprocessor U5 (Fig. 68B) described above.

0363Referring to FIG. 68B, analog switch SW1 is used to "invert" the input voltage to the next derivative filter. The frequency at which the signal is inverted is set and controlled by the microprocessor U5. Depending on the settings of the inputs A0 and A1 of the analog switch SW1, one set of switches (inside the analog switch SW1) is turned on to pass the signal through the device by wire (connected to the outputs D1 and D2, respectively). Switches S1A and S2A are "on" or switches S1B and S2B are "on"). In this circuit, when LED1732 is on, the signal from the preamplifier U6 (pins 5-7) is directed to the positive input of the derivative filter U2 (pin 3), while grounding is the derivative filter U2. The input to analog switch SW1 is wired so that it applies to the negative input of (pin 5). The output signal (after filtering) of the differential filter (U4) is approximately 12 times its amplitude. When LED1732 is turned off (modulating), the output of the preamplifier circuit is negative and the amplitude is about the same as when LED1732 was on. The input to the derivative filter is wired here, but the negative signal from the preamplifier U6 (pin 7) is directed to the negative input of the derivative filter U2 (pin 5), while the ground is a positive input. It may be reversed as applied to U2 (pin 3). The output signal (after filtering) is still about the same amplitude as if the LED was on and the analog switch was in another position.

0364Derivative amplifiers / filters U2, U3, U4 provide minimum gain (12x) and provide multi-pole lowpass filtering (cutoff at 10Hz) of the signal while treating the signal with distinction. This filter is used to attenuate any signal from a preamplifier outside the 10Hz range (240-260Hz for FAM / ROX, 340-360Hz for TAM) before and after the operating frequency of the LED / analog switch. The attenuation of the electrical signal caused by the photodiode 1780 increases rapidly as the frequency deviates outside this range.

0365The final amplifier circuit (U4) functions as a differential amplifier with zero gain. Its function is to convert the voltage differential between the two signals from the differential filter into a positive voltage relative to the circuit ground.

0366Figures 64 and 65 both show a top view of the RT incubator 608 and illustrate the positioning of the photodetector module 1700 mounted on the bottom of the RT incubator 608. In the embodiments shown in FIGS. 64 and 65, the RT incubator comprises 15 photodetector modules 1700, with 5 photodetector modules 1700 (MTU160 reaction tubes 162a ~) for each of the three different dyes, FAM, TAMRA, and ROX. One for each of 162e). Therefore, there are three photodetector modules 1700, one for each of the dyes, for each of the five radii corresponding to each of the five reaction tubes 162a-162e of the MTU160. The photodetector module 1700 is held incremented by 24 ° around the RT incubator. Stray light from adjacent reaction tubes that are simultaneously excited during detection in one particular module 1700 can affect the emissions detected in module 1700. Further, the excitation light is scattered in the reaction tube 162 and can excite the adjacent reaction tube 162. This condition is known as crosstalk. The photodetector modules are preferably held so as to maximize the distance between the detection windows of the adjacent photodetector modules 1700 and thereby minimize the crosstalk between the adjacent photodetector modules 1700. Crosstalk can also be prevented by providing a photoisolated baffle (not shown) in the form of concentric walls held between adjacent reaction tubes 162 of the MTU160.

0367Another method for reducing crosstalk between emissions from adjacent reaction tubes, eg, reducing background signals due to stray light, is by using phase-locked detection techniques. The excitation light is often modulated by applying a signal of known frequency to the LED 1732. An excitation signal frequency of 350 Hz is used for TAMRA dyes and a 250 Hz excitation signal frequency is used for FAM and ROX dyes. Therefore, the resulting emitted light will display a frequency dominated by the frequency of the excitation light, and any emission signal with a frequency inconsistent with the frequency of the excitation light will be discarded as not originating from the excitation light. It's okay. A known phase detection circuit can be used to output a voltage proportional to the phase difference between the excitation and emission signals.

0368As shown in FIG. 65, the photodetector module 1700 is preferably classified according to the dye targeted by the module. That is, modules 1 to 5 target FAM dyes, modules 6 to 10 target TAMRA dyes, and modules 11 to 15 target ROX dyes. It has been found that crosstalk is actually greater between adjacent photodetector modules with different wavelength excitation signals than between adjacent photodetector modules with the same wavelength excitation signal. Therefore, as shown in FIG. 64, crosstalk is reduced by collecting detectors of similar wavelengths.

0369Also, the internal components of the RT Incubator 608 are preferably black in order to minimize stray light transmission and reflection.

0370In a preferred embodiment, the photodetector module 1700 mounted on the bottom floor 613 of the RT incubator 608 and in most cases under the reference plate 82 of the processing desk 200 is a stray electromagnetic interference that can affect the photodetector module. Surrounded by a shield (known as the Faraday Shield, not shown) that blocks the light.

0371Figures 64 and 65 show an RT incubator 608 with 15 photodetector modules 1700. Such an arrangement allows real-time scanning of the MTU160's five reaction tubes 162 and three dyes. For example, as mentioned above, if it is desirable to incorporate four dyes into the procedure to detect amplification products associated with three different viruses and internal controls, RT 20 photodetector modules in this embodiment. It will need to be incorporated into the incubator 608, but as can be fully understood from FIGS. 62 and 63, this would be nearly impossible given the spatial constraints and the size of the illustrated embodiment of the photodetector module 1700.

03724 photodetectors moved to RT incubator 608, one for each dye, to avoid requiring 20 photodetectors, i.e. 5 photodetectors for each of the 4 individual dyes A scanning real-time fluorometer mounted as possible may be used, whereby each photodetector module can be selectively held under each of the five reaction tubes 160a-162e of the MTU160. (The number of photodetector modules can be adjusted within the scanning real-time fluorometer based on the number of dyes detected.) Scanning fluorometer assemblies for such scanning real-time fluorometers are shown in Figures 69-72. Is generally specified by reference number 2000. In the scanning fluorometer assembly shown, four photodetector modules 1700 are mounted so that they can be moved radially with respect to the fixed scanning disk 2002. Each photodetector module 1700 is mounted on a photodetector module mounting bracket 2004 carried to a translation assembly 2006 to exert the effect of radial motion of the photodetector module 1700 on the scanning disk 2002. The translation assembly 2006 comprises a linear sliding bearing 2008 to which a mounting bracket 2004 is attached and a bearing track 2010 that is mounted on a scanning disc 2002 and the bearing 2008 can be slidably translated along the linear sliding bearing 2008. .. The slotted photosensor 2014 is fixed to the scanning disk 2002 at the radial outward end of the bearing track 2010, and the protrusion 2016 extending from the bearing 2008 is the sensor when the photodetector 1700 is farthest from the radial position. Extends into 2014, thereby providing a "home" signal. The scanning disk 2002 is generally formed with radial slots 2040 (see Figure 72).

0373FIG. 72 shows a bottom view of the scanning fluorometer assembly 2000. The cam disc 2030 is located coaxially and parallel to the scanning disc 2002 and is rotatable around the shaft 2024 (see Figure 71). The cam disk 2030 has four arched cam slots 2032 formed therein, one for each of the photodetector modules 1700. Pin 2050 extending from bearing 2008 of each translation assembly 2006 extends through radial slot 2040 to each one of cam slot 2032.

0374Motor 2020 is attached to the tip of the fixed scanning disc 2002. The output shaft 2022 of the motor is connected to the cam disk 2030 (eg, by a belt and pulley arrangement (not shown) or a meshing gear arrangement (not shown)). In a preferred embodiment, a pulley arrangement is used that achieves a speed of 50 ° (ie, 200 ° / sec) in 0.25 seconds at a ratio of 6: 1. The rotation of the output shaft 2022 connected to the cam disk 2030 causes the cam disk 2030 to rotate. As the cam disc 2030 rotates, the radius of the corresponding one bearing 2008 along its respective bearing track 2010 by the engagement of each pin 2050 with each one of the cam slots 2032 formed within the cam disc 2030. A directional translation occurs, which results in a radial translation of the corresponding module mounting brackets 2004 and the light detection module 1700. An encoder (not shown) on the motor can monitor the rotation of the motor, thereby monitoring the position of the cam disk 2030. Alternatively, other position-sensing devices, such as optical sensors, can be used to directly monitor the position of the cam disk 2030.

0375Each cam slot 2032 includes positioning points 2032a, 2032b, 2032c, 2032d, and 2032e. (To minimize clutter, positioning points 2032a, 2032b, 2032c, 2032d, and 2032e are labeled for only one of the cam slots 2032.) Positioning points 2032a-e are during rotation of the cam disk 2030. Position the photodetector module 1700. If the pin 2050 associated with a particular photodetector module 1700 is at position 2032a, the photodetector module 1700 is the farthest radial from the center of disk 2002 to scan farthest from the MTU160 reaction tube 162a. It is in the position of. When the cam disk 2030 rotates and the pin 2050 associated with the photodetector module 1700 moves to point 2032b, the corresponding photodetector module is radially radial by the distance corresponding to the distance of the MTU160 to the next reaction tube 162b. It will move inward. The photodetector module 1700 can then scan the reaction tube 162b at that position. As the cam disk 2030 rotates further, the pin 2050 associated with the module 1700 moves to position 2030c, so that the module 1700 moves inward in the radial direction by a distance corresponding to the distance of the MTU160 to the reaction tube 162c. To do. Module 1700 can then scan the reaction tube 162c at that position. As the cam disk 2030 rotates further, the pin 2050 associated with the module 1700 translates inward in the radial direction by a distance corresponding to the distance to the reaction tube 162d. Module 1700 can then scan the reaction tube 162d at that position. As the cam disk 2030 rotates further, the pin 2050 associated with the module 1700 translates inward in the radial direction by a distance corresponding to the distance to the reaction tube 162e. Module 1700 can then scan the reaction tube 162e at that position. To. Thus, a single mobile photodetector module 1700 can detect emissions from each of the reaction tubes 162a-e of each MTU160.

0376Each photodetector module 1700 in the scanning fluorometer assembly 2000 extends to the incubator housing of the RT incubator 608. As the detector 1700 moves radially, an elongated radial opening (not shown) is formed in the floor 613 of the RT incubator 608, through which each photodetector scans the MTU160 in the RT incubator. To do. In one embodiment, each radial opening holds a shutter mechanism (not shown). The shutter mechanism has a movable opening, and the adapter pipe 1718 of each photodetector module 1700 extends through the opening. When the light detection module 1700 translates in the radial direction, the opening of the shutter mechanism translates with it, while the rest of the radial opening remains closed, thereby causing heat loss from the radial opening and Limit stray light.

0377As an alternative to the light detection module described above, the light detection module is a multi-wavelength fluorometer, eg, a fluorescence microscope with a filter changer, or a fluorescence microscope with multiple bandwidth filters and multiple bandwidth beam splitters. Good.

0378The present inventors have determined that the magnetic particles used for target capture in a preferred embodiment of the present invention can affect the real-time detection of amplification products. Two specific interference effects have been identified. First, magnetic particles can inhibit amplification by adsorption of oligonucleotides (eg, amplified oligonucleotides and probes) and enzyme reagents (eg, nucleic acid polymerases). Also, the presence of magnetic particles (in fixed or suspension) results in the dissipation of fluorescence emission, thereby the amount of excitation light reaching the detection dye and the light emitted from the reaction tube 162 of the MTU160. The amount of can be blocked or partially blocked. This is known as the dark cloud effect.

0379To minimize this effect, one embodiment of the RT Incubator 608 is provided with a magnetic partition 1500, as shown in FIGS. 73, 74, 75, and 75A. In a preferred embodiment of the invention, the RT incubator 608 grips 15 MTU160s at a time, each spaced 24 ° increments around the turntable. Assuming that a turntable in position 30, such as turntable 1656, is used, this means that only every other MTU station 1663 grips the MTU160 in the RT incubator 608. Therefore, as shown in the figure, the magnetic divider 1500 is held over every other MTU station 1663 on turntable 1656 (above) so that only 15 of the 30 stations can accommodate the MTU160. Can be kept like this. In an alternative embodiment, the magnet holder may be configured to fit between each of the 30 stations or to be held adjacent to every other station, thereby an RT incubator. Every other MTU160 content in the 608 can be processed according to an alternative assay procedure. Such a magnet holder can be formed from an iron sheet metal to which the magnet adheres. The griddle material will also have the advantage of significantly reducing the magnetic field on the opposite side of the magnet.

0380As shown primarily in FIGS. 74 and 75A, where only a single magnetic partition 1500 is shown for simplicity, the magnetic partition includes a magnet holder 1502 with a magnet block 1504 and an attachment arm 1510. A rectangular recessed area 1506 is formed within the magnet block 1504 and an opening 1508 is formed within the magnet block 1504 to receive magnets 1520 of similar size and shape. In the illustrated embodiment, the opening 1508 is circular and the magnet 1520 is disc-shaped. Currently preferred magnets are nickel-plated neodymium iron boron discs (Force Field) with dimensions of 1/2 inch (diameter) x 1/8 inch (thickness), with a maximum residual magnetic flux density of 12,100 and a maximum energy product of MGOe. (Fort Collins, Colorado); Item No. 0022). The magnet 1520 is placed in the associated opening 1508 and is gripped into the block 1504 using a clasp plate 1522, which is the openings 1524 and 1526 formed in the clasp plate 1522 and the magnet block 1504, respectively. It may be secured to the magnetic holder 1502 using mechanical fasteners such as screws or bolts (not shown) that pass through.

0381The attachment arm 1510 extends from the magnet block 1504 and includes a fastener hole 1512 formed within the lower plate 1662 and partition 1660 of the turntable 1656, along with the corresponding fastener hole 1661. The magnetic divider 1500 may be secured to turntable 1656 with suitable mechanical fasteners such as screws or bolts (not shown) extending through fastener holes 1512 and 1661.

0382The magnetic partition 1500 may also include an inner arm 1528 (see FIG. 75). The inner arm 1528 stabilizes the magnetic partition 1500 and provides additional attachment points for attaching the magnetic partition 1500 to the turntable 1656.

0383As shown in Figures 75 and 75A, when the MTU160 ́ is placed in the MTU slot of the turntable 1656, each reaction tube 162 ́ is adjacent to one of the magnets 1520 carried into the magnetic partition 1500. It is being held. The magnet 1520 attracts at least a portion of the magnetic particles towards the wall of the reaction tube 162 ́ adjacent to the magnet 1520, thereby in suspension in the residue of the contents of the reaction tube 162 ́ or in the reaction tube. The concentration of magnetic particles fixed to the bottom of 162 ́ remains substantially reduced.

0384As mentioned above, the preferred material for MTU160 is polypropylene. However, polypropylene has been found to fluoresce under certain conditions. Therefore, alternative MTU materials such as acrylics, polystyrenes, and cyclic olefins are considered. It also concentrates the sample at the bottom of each MTU160 reaction tube 162 ́, as illustrated by the MTU160 ́ reaction tube 162 ́ in Figures 75 and 75A thereby facilitating more consistent excitation and release from the sample. To allow the use of smaller reagent volumes, for example, use an MTU with a reaction tube with a conical end instead of the round end of the reaction tube 162 of the MTU 160 shown in FIG. 74, for example. Is being considered.

0385The processing steps of the real-time and endpoint amplification assays performed in accordance with the present invention are described in the flowchart shown in FIG. 76 (FIG. 76A shows the steps of the complete real-time TMA amplification assay and the steps of the endpoint TMA amplification assay by amplification, FIG. 76B shows the steps of the endpoint TMA amplification assay after exposing the contents of reaction tube 162 to amplification conditions.) The above steps only represent a typical TMA procedure. Those skilled in the art may modify or omit the steps below, or may add or substitute other steps according to other currently known or undeveloped real-time and endpoint amplification assay procedures. You will recognize that it is good. Reagent formulations for performing many amplification procedures are known in the art and can be used in the present invention or easily adapted for use. For example, Kachan et al., U.S. Pat. No. 5,399,491; Becker et al., U.S. Patent Application No. 2006-0046265 A1; Linnen et al., U.S. Patent Application No. 2004-0259108. A1 Gazette "Compositions and Methods for Detecting West Nile Virus"; Weisburg et al., US Patent Application No. 2004-0235138 A1 Gazette "Compositions, Methods and Kits for Determining the Presence of Trichomonas Vaginalis in a Test Sample"; See US Patent Application No. 10 / 825,757, "Compositions and Methods for Determining the Presence of SARS Coronavirus in a Sample," which has common ownership with the book.

0386The processing step of a typical real-time and end point TMA amplification assay begins in step 1902, in which the MTU 160 is located in the sample transmission station 250 under the sample preparation opening 252 provided in the jig plate 130. Moved to pipette position. In step 1904, the sample pipette assembly 450 dispenses 400 μL of Target Capture Reagent (TCR) into each reaction tube 162 of the MTU 160. Target capture reagents include capture probes, detergent-containing cytolytic agents such as lithium lauryl sulfate that lyse cells and inhibit the activity of ribonucleases present in the sample material, and approximately 40 μg of Sera-Mag (R) MG. -CM Carboxylate Modified (Seradyn, Inc. (Indiana Police, India) Part No. 24152105-050250), Covalently Bonded Poly (dT)₁₄Includes 1 micron superparamagnetic particles with. The capture probe is a poly (dT) bound to magnetic particles.₁₄Poly (dA) for binding to₃₀Includes a 5 ́ target binding region with a tail and a 3 ́ region. The target binding region of the capture probe is designed to bind to a region of the nucleic acid of interest that is different from the region targeted by the primer and detection probe.

0387In step 1906, pipette assembly 450 dispenses 500 μL of sample into each of the reaction tubes. In step 1908, the right transfer mechanism 500 moves the MTU 160 to the right rotary stirrer 550, preferably mixing the sample and TCR at 10 Hz for 30 seconds. It should be noted that the time defined in FIG. 76 and its description are desirable times and the actual real time may differ from this defined desirable time.

0388In step 1910, the right transfer mechanism 500 moves the MTU 160 from the right rotary stirrer 550 to one of the temperature gradient stations 700 located below the jig plate 130. The MTU160 is preferably in the temperature gradient station 700 for 312 seconds at a temperature of 65 ° C. In step 1912, the right transfer mechanism 500 moves the MTU 160 from the gradient station 700 to the TC incubator 600 and places it at 62 ° C for 20 minutes for hybridization of the capture probe to the nucleic acid of interest that could be extracted from the sample. (At this temperature, immobilized poly (dT)₁₄No perceptible hybridization of the capture probe to the oligonucleotide occurs. ) In step 1914, the left transfer mechanism 502 moves the MTU 160 from the TC incubator to one of the temperature gradient stations 700 located to the left of the processing desk 200 and grips it at room temperature for 174 seconds. In step 1916, the left transfer mechanism 502 moves the MTU 160 from the gradient station 700 to the AMP incubator 604 and binds the immobilized oligonucleotide associated with the magnetic particles to the capture probe at 43 ° C for 838 seconds. Put.

0389In step 1918, the left transfer mechanism 502 moves the MTU 160 from the AMP incubator 604 to the left rotary stirrer 552. The left rotary stirrer 552 includes a dispenser for dispensing oil to the MTU160, among other substances. In the left rotary stirrer 552, 200 μL of silicone oil, a surface treatment agent, is added to each reaction tube 162 of the MTU 160 and the MTU is mixed at 12 Hz for 30 seconds. In step 1920, the left transfer mechanism 502 moves the MTU 160 from the left rotary stirrer 552 to one of the magnetic separation stations 800 for the magnetic separation cleaning procedure described above.

0390The advantage of adding a surface treatment agent such as silicone oil to the sample solution in step 1918 is that it reduces the amount of material that adheres to the inner surface of the reaction tube 162 during the rinsing and suction steps of the magnetic separation cleaning procedure, thereby reducing the amount of material that adheres to the inner surface of the reaction tube 162. , To facilitate a more effective magnetic separation cleaning procedure. The MTU160 is preferably made of a hydrophobic material such as polypropylene, but even a small amount of material such as a cleaning solution may form on the inner surface of the MTU reaction tube 162 during the suction step of the magnetic separation cleaning procedure. If not sufficiently removed from the reaction tube 162 during the magnetic separation and cleaning procedure, this residue, which may contain nucleic acid amplification inhibitors, can affect the results of the assay. In an alternative approach, the surface treatment agent may be added to the reaction tube 162 and removed prior to the addition of the TCR and sample, or the surface treatment agent may be added to the reaction tube with the TCR and sample, optionally with a cleaning solution. It may be added to the reaction tube after being aspirated from and removed before adding the amplification reagent and the enzyme reagent to the reaction tube. The purpose is to provide a coating of a surface treatment agent on the inner surface of the reaction tube 162. Inhibitors of the amplification reaction are known in the art and will vary depending on the sample source used and the amplification procedure. Possible amplification inhibitors include: hemoglobin in blood samples; hemoglobin, nitrates, crystals and / or β-human chorionic gonadotropin in urine samples; nuclease; protease; sodium dodecyl sulfate (Sodium Dodecyl). Anionic detergents such as Sulfate (SDS) and Lithium Lauryl Sulfate (LLS); and divalent cations such as magnesium, which is a cofactor used in nucleic acid-based amplification reactions as described above. EDTA, an anticoagulant and fixative for some specimens. For example, Mahony et al., J.Clin.Microbiol., 36 (11): 3122-2126 (1998); Al-Soud, J.Clin.Microbiol., 39 (2): 485-493 (2001); and , Kacian et al., US Pat. No. 5,846,701, "Method for Suppressing Inhibition of Enzyme-Mediated Reactions By Ionic Detergents Using High Concentration of Non-Ionic Detergent."

0391In step 1922, the left transfer mechanism 502 returns the MTU 160 from the magnetic separation station 800 to the left rotary stirrer 552, with 200 μL in each reaction tube 162 of the MTU 160 to prevent evaporation and splashing of the fluid contents during post-operation. Add silicone oil. In step 1924, the reagent pipette assembly 470 dispenses 75 μL of amplification reagent into each reaction tube 162 of the MTU 160 placed in the left rotary stirrer 552. For a typical TMA reaction, the amplification reagents are an antisense promoter primer with a 3 ́ target binding region and a 5 ́ promoter sequence recognized by RNA polymerase, and a sense primer that binds to the extension product formed by the promoter primer. , Nucleoside triphosphates (ie, dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP) and sufficient cofactors to carry out the TMA reaction. For real-time TMA amplification assays, the amplification reagents are chain substitutions and their 5 ́ ends and 3 by interacting label pairs (eg, interacting fluorescent moieties and conventional means. ́A molecular torch probe with a quencher moiety tethered to the end) and to the amplification product when amplification is occurring, preferably other than any asymmetric nucleic acid that may be present in the reaction tube 162. On the other hand, it also contains a target-specific region that can hybridize detectably. Kacian et al., U.S. Pat. No. 5,399,491; Becker et al., U.S. Patent Application No. 2006-0046265 A1 "Single-Primer Nucleic Acid Amplification" (Disclosures an alternative TMA-based amplification assay in which sense promoter oligonucleotides are used); and see Becker et al., US Pat. No. 6,361,945. Then mix MTU160 at 16Hz for 15 seconds.

0392In step 1926, the left transfer mechanism 502 moves the MTU 160 from the left rotary stirrer 552 to one of the temperature gradient stations 700 located to the left of the processing desk 200. The MTU160 is then incubated at 65 ° C for 132 seconds. In step 1928, the left transfer mechanism 502 transfers the MTU160 from the temperature gradient station 700 to the TC incubator 600 and incubates at 62 ° C for 10 minutes to bind the promoter primer to the nucleic acid of interest. The preferred promoter primer in this particular TMA example has a promoter sequence recognized by T7 RNA polymerase. In step 1930, the left transfer mechanism 502 moves the MTU160 from the TC incubator 600 to the AMP incubator 604 and incubates the contents of the MTU160 at 43 ° C for 10 minutes to stabilize the MTU contents.

0393In step 1932, reagent pipette assembly 470 adds 25 μL of enzyme reagent kept at 20 ° C. from reagent cooling bay 900 to each reaction tube 162 of MTU160 located within AMP incubator 604. (By maintaining the temperature of the contents of each reaction tube 162 at a temperature slightly higher than the amplification temperature, the heat-sensitive enzyme can be maintained at a cold temperature before starting the amplification.) The enzyme reagent of this example. Contains TMA, a reverse transcriptase and T7 RNA polymerase for performing transcription-based amplification procedures. In step 1934, the linear stirrer 634 in the AMP incubator 604 mixes the MTU160 with the enzyme reagent added at 10 Hz for 15 seconds, and the temperature of the contents of each reaction tube 162 drops to about 42 ° C. In step 1936, the left transfer mechanism 502 moves the MTU 160 from the AMP incubator 604 to the RT incubator 608. MTU160 is maintained in RT incubator 608 at 42 ° C for 60 minutes to allow amplification of target sequences, and in the case of real-time amplification, a specified frequency to detect hybridization of the probe to amplification products during the amplification process. Is read. The MTU is processed in a continuous fashion by the instrument 50 (generally, the new MTU starts the assay process every 165 seconds), so the MTU is continuous (generally every 165 seconds). ) To RT incubator 608, and removed from it. In step 1938, after the final read is made, the right transfer mechanism 500 moves the MTU160 from the RT incubator 608 to the illuminometer 1360. At step 1940, the MTU160 is deactivated from the photometer 1360.Go to Biroom 750. Upon entering the inactivation preparation chamber 750, 2 mL of bleach-based agent is provided in each of the reaction tubes 162 to inactivate the nucleic acids present in the reaction tubes (ie, turn the nucleic acids into non-amplifiable). .. See, for example, Dattagupta et al., U.S. Pat. No. 5,612,200, and Nelson et al., U.S. Patent Application No. 2005-022491 A1.

0394Following step 1936, the MTU160 with contents processed according to a typical endpoint TMA amplification assay proceeds as shown in Figure 76B. In step 1942 of this process, the left transfer mechanism 502 transfers the MTU 160 from the RT incubator 608 to the temperature gradient station 700 on the left side of the processing desk 200 and heats it at 64 ° C for 362 seconds. Alternatively, the MTU160 is moved from the RT incubator 608 to the designated area of the HYB incubator 606 for temperature gradient. In step 1944, the left transfer mechanism 502 moves the MTU 160 from the temperature gradient station 700 to the HYB incubator 606, where 100 μL of probe reagent is added to each reaction tube 162. The probe reagent provides a sufficient amount of probe to detectably bind to the amplification product of the nucleic acid of interest, and preferably to any non-target nucleic acid that may be present in the reaction tube 162. contains. For a typical endpoint TMA embodiment, the probe is synthesized to include a non-nucleotide linker used to label the probe with a chemiluminescent acridinium ester. See U.S. Pat. Nos. 5,185,439 and 6,031,091 by Arnold et al. In step 1946, the MTU 160 is held in a HYB incubator 606 adjacent to the tilted disk linear stirrer 634, which is used to mix the contents of the MTU at 14 Hz for 15 seconds. In step 1948, the contents of MTU160 are incubated at 64 ° C for 1762 seconds.

0395For detection, in step 1950, 250 μL of selective reagent is first provided to the contents of each reaction tube 162 of MTU160. As mentioned above, the selected reagents in the HPA assay contain alkaline reagents that hydrolyze the acridinium ester label specifically associated with the non-hybridized probe, while the acrylic associated with the hybridized probe. The dinium ester label is not hydrolyzed under these conditions and can chemiluminescent in a detectable manner under the conditions described below, so that the user can use the binding and free probes in solution. Can be distinguished. See U.S. Pat. No. 5,639,604, Arnold et al. After adding the selective reagent to the reaction tube 162, the MTU 160 is held adjacent to the tilted disk linear stirrer 634 and the contents of the reaction tube are mixed at 13 Hz for 30 seconds. In step 1954, the contents of reaction tube 162 are incubated at 64 ° C for 606 seconds to facilitate the selection process.

0396In step 1956, the left transfer mechanism 502 transfers the MTU 160 from the HYB incubator 606 to the AMP incubator 604 and cools the contents of the reaction tube 162 at 43 ° C for 172 seconds. Lowering the temperature of the contents of the reaction tube 162 below 50 ° C generally interferes with the activity of the selective reagents, and therefore each MTU160 so that the final signal values from the various reaction tubes are comparable. It is important to cool the contents of the reaction tube at substantially the same rate. In step 1958, the left transfer mechanism 502 moves the MTU 160 from the AMP incubator 604 to the RT incubator 608, and then the right transfer mechanism 500 moves the MTU 160 from the RT incubator 608 to the stop station 210 on the right side of the processing desk 200. The contents of the reaction tube 162 are further cooled at room temperature for 560 seconds. In step 1960, the MTU 160 is transferred to the temperature gradient station 700 on the right side of the processing desk 200 and the contents of the reaction tube 162 are cooled at 21 ° C for 366 seconds. In HPA multiplex assays with multiple chemiluminescent labels, it is preferred to keep the temperature of the contents of the reaction tube 162 below 29 ° C so that the light emission characteristics of the label can be identified. See, for example, Nelson et al., US Pat. No. 5,756,709, "Compositions for the Simultaneous Detection and Quantitation of Multiple Specific Nucleic Acid Sequences."

0397In step 1962, the right transfer mechanism 500 moves the MTU 160 from the gradient station 700 to the photometer 1360, where each reaction tube 162 receives 200 μL of Detect I reagent and then about 2 seconds later, 200 μL of Detect II reagent. .. The rate at which the Detect I and Detect II reagents are injected into the reaction tube 162 should be strong enough to mix the contents of the reaction tube without agitation so that the flow of reagents is not interrupted by air bubbles or voids. , Prepare a drop line (not shown). The preferred maximum rate for injecting the Detect I and Detect II reagents into the reaction tube 162 is 1000 μL / sec. As mentioned above, the Detect I and Detect II reagents are combined to form a basic hydrogen peroxide solution that enhances the chemiluminescence of those acridinium ester labels that were not hydrolyzed during the selection process. These reagents are marketed as the GEN-PROBE (R) Detection Reagent Kit (Gen-Probe; product number 1791).

0398In step 1964, the MTU 160 passes from the illuminometer 1360 to the deactivation preparation room 750. In the inactivation preparation chamber 750, 2 mL of a bleach-based agent is provided in each of the reaction tubes 162 to inactivate the nucleic acids present in the reaction tubes. See, for example, Dattagupta et al., U.S. Pat. No. 5,612,200, and Nelson et al., U.S. Patent Application No. 2005-022491 A1.

0399Fluorescence measurements are preferably made at a rate of one measurement for each of the three or four spectral bands (ie, for each target dye) per reaction tube 162 every 30 seconds. The turntable 1656 must rotate once every 30 seconds. In a preferred embodiment, there are 15 MTU160s transported to turntable 1656 of RT Incubator 608, each spaced 24 ° apart. Each read must be completed in less than 2 seconds in order for each of the 15 MTU stations 1663 to be able to read during rotation. The reading is completed in less than 2 seconds, and the time to place the new MTU160 in the RT incubator 608 when removing the completed MTU from the RT608, while maintaining the desired reading speed of one measurement for each dye every 30 seconds. Ideally, each rotation should be completed in less than 30 seconds to make.

0400When data is collected by measuring fluorescence emission from each reaction tube 162 at specified intervals for a specified period of time, the data is processed to determine the concentration of a particular sample (eg, nucleic acid of interest) in the sample. To do. The measured data, or measurement signal, is referred to as the Relative Fluorescent Unit (RFU) and is based on the amount of emitted fluorescence focused on the photodiode 1780 by the printed substrate 1790 of the photodetector unit 1700. It is a generated signal. Each data point measured at a time interval is RFU (t). A plot of RFU (t) representing various datasets known as the "growth curve" is shown in Figure 78. In general, each RFU (t) plot is generally S-shaped, with an initial flat area at or near the lowest level (known as the "static level" or "reference period"), which is sudden and relatively steep. It is characterized by a sloping section (known as the "growth phase") followed by a substantially flat section (known as the "stop mitotic phase") at or near the highest level.

0401As used herein, "growth curve" refers to the characteristic pattern of appearance of synthetic products, such as amplicon, in a reaction as a function of time or number of cycles. The growth curve is conveniently represented as a two-dimensional plot of some index (y-axis) of product quantity such as time (x-axis) vs. fluorescence measurement RFU. The growth curve has an S-shape, but not all. The "reference period" of the growth curve is that the amount of product (such as amplicon) increases at a substantially constant rate, which is the increasing characteristic of the growth curve (which may have a log-linear profile). The initial stage of the curve that is less than velocity. The reference period of the growth curve generally has a very shallow slope that often approximates zero. The "growth period" of a growth curve is the portion of the curve in which measurable products substantially increase over time. The transition from the reference phase to the growth phase in a typical nucleic acid amplification reaction is characterized by an increase in the appearance of amplicon at a certain rate over time. The transition of the growth curve from the growth phase to the mitotic phase begins at the inflection point where the rate of amplicon appearance begins to decrease. "Stopping phase" refers to the final stage of the curve. In the mitotic phase, the measurable rate of product formation is substantially lower than the rate of amplicon production during the long logarithmic line formation and may be close to zero.

0402The process for calculating the sample concentration is shown using the flowchart of FIG. 77. The data RFU (t) from the photodetector module 1700 is entered as shown in box 2100. At step 2102, the data RFU (t) goes through a color separation procedure. As can be fully understood from FIG. 67, there is considerable overlap in the emission spectra of different dyes, especially spectrally adjacent dyes. Thus, the RFU (t) data obtained from a particular reaction tube 162 corresponds to the desired specimen (ie, with the desired specimen) as well as release data from one or more different dyes corresponding to different targets. It may also include release data (from the dye coupled to the binding probe). Standard mathematical techniques such as deconvolution of different signals obtained by different dye-specific photodetector modules 1700 can be used to separate that portion of the RFU (t) signal that is not from the desired sample. it can. Deconvolution is a known technique that assumes that the signal measured by each photodetector module can be represented as a mathematical function of emission from each of the dyes present in the sample. For example, assuming the measurement results are from four photodetector modules, RFU₁(t) = k₁RFU_A(t) + k₂RFU_B(t) + k₃RFU_C(t) + k₄RFU_D(t) RFU₂(t) = k₅RFU_A(t) + k₆RFU_B(t) + k₇RFU_C(t) + k₈RFU_D(t) RFU₃(t) = k₉RFU_A(t) + k₁₀RFU_B(t) + k₁₁RFU_C(t) + k₁₂RFU_D(t) RFU₄(t) = k₁₃RFU_A(t) + k₁₄RFU_B(t) + k₁₅RFU_C(t) + k₁₆RFU_D(t) here, RFU₁(t) = Signal measured by photodetector module # 1 RFU₂(t) = Signal measured by photodetector module # 2 RFU₃(t) = Signal measured by photodetector module # 3 RFU₄(t) = Signal measured by photodetector module # 4 RFU_A(t), RFU_B(t), RFU_C(t), RFU_D(t) = Part of the emission signal by each dye A, B, C, D k₁~ k₁₆= Constant Is.

0403The functions corresponding to the signals of all the photodetectors are placed in a matrix, and for each dye, matrix inversion is performed to derive the mathematical representation of the signal by the dye as a function of the signals from each of the photodetectors. Will be done. RFU_A(t) = f₁(RFU₁(t), RFU₂(t), RFU₃(t), RFU₄(t)) RFU_B(t) = f₂(RFU₁(t), RFU₂(t), RFU₃(t), RFU₄(t)) RFU_C(t) = f₃(RFU₁(t), RFU₂(t), RFU₃(t), RFU₄(t)) RFU_D(t) = f₄(RFU₁(t), RFU₂(t), RFU₃(t), RFU₄(t)).

0404From the color separation 2102, the data RFU (t) proceeds to the threshold time determination starting at 2104. The threshold time, that is, the T time (also known as the time of appearance) is the time when the normalized data RFU (t) reaches a predetermined threshold as described later. As described in more detail below, a calibration curve can be used to correlate the T time determined for a particular sample with the sample concentration, thereby indicating the sample concentration of that sample. In general, the higher the concentration of the desired sample, the faster the T time.

0405The first step in the T-time determination procedure is background correction and data normalization, as shown in Box 2106. Background correction is performed to reduce the background "noise" portion of the signal data RFU (t) from, for example, stray electromagnetic signals from other modules of the instrument 50. That is, the background noise includes the relevant portion of the RFU (t) signal from a source other than the desired sample. Background correction is adjusted data RFU^*Executed by subtracting the background value "BG" from the data RFU (t) to obtain (t). That is, RFU^*(t) = RFU (t) -BG.

0406Background BG can be determined in many ways.

0407According to one method for determining background noise, the first step is to determine the time interval between data points. The time interval is 60, which is the cycle time (ie, the time between successive data measurements) multiplied by the data points (ie, 0th data point, 1st data point, 2nd data point, ... nth data point). Determined by dividing by seconds. For example, assuming that the cycle time is 30 seconds, the time interval of the 15th data point is (15 × 30 seconds) / 60 seconds = 7.5.

0408The next step is to find the midpoint of the signal data by adding the minimum and maximum signal data points and dividing by two. That is, (RFU_max+ RFU_min) / 2. Start from the time corresponding to the median and calculate in the opposite direction to calculate the slope of each data point pair: (RFU (t) -RFU (t-1)) / Δt (t t-1).

0409Then, by finding the first slope value less than the static slope value, it is determined where to flatten the slope of RFU (t) (ie, the value before the RFU (t) curve starts the upward slope. To do). A typical static slope value, also known as the "delta value", is 0.0001. After finding this slope, find the next cycle in which the non-negative or negative slope exceeds, for example, the negative delta value (ie -0.0001), and set this value to H._indexAnd. Then, starting from the first data point, average the entire range of RFU (t) values and H_indexProceed to the RFU value corresponding to. The average of this data uses a static back trim value of 0.15 (ie, excluding 7.5% from the lower limit of the RFU value within the specified range and 7.5% from the upper limit of the RFU value within the specified range). Data ranges can be calculated using Excel's TRIMMEAN function. Let this average value be the background, BG.

0410Alternatively, the background can be determined according to the procedure described above, using a delta value other than 0.0001.

0411A further alternative method for determining background is to eliminate the delta value criterion and instead take the TRIMMEAN average of the RFU data from cycle 1 to the specified end point, such as the first cycle 5.5 minutes ago. In this alternative, the static back trim value can be adjusted to, for example, 0.40 (ie, 20% from the lower limit of the RFU value within the specified range and 20% from the upper limit of the RFU value within the specified range, back. Excluded from ground calculation).

0412A further alternative method for determining background is to perform curve fitting on all or part of the RFU data to derive an estimate of the underlying value to be reduced. Any curve fitting technique suitable for fitting curves to RFU data may be used.

0413A typical curve fitting method uses a part of the equation for curve fitting of a general S-shaped curve related to nucleic acid amplification, which was derived by Weusten et al. See Weusten et al., Nucleic Acids Research, 30 (6e26): 1-7 (2002). In the background subtraction method, it is only necessary to confirm the reference level. Therefore, it is also at least necessary to fit the curve to the first part of the RFU data, which includes the baseline, usually towards the start of the curve.

0414Curve fitting can be performed on RFU (t) data from cycle 1 to the cycle immediately preceding 75% of the maximum RFU. The following polynomial (3), which is part of the equation derived by Weusten et al. As described above, is used to generate the best fit model for RFU. RFU (t) = Y0 + ala2 [e^{a2 (t-a3)}/ (1 + e^{a2 (t-a3)})] ln (1 + e^{a2 (t-a3)}) (3) The initial approximations for the variables Y0, a1, a2, and a3, as described below, are inputs to the curve fitting equations, for example Microsoft EXCEL to produce the final equation and the final values for Y0, a1, a2, and a3. Using the SOLVER function of, an iterative solution method is performed to fit the equation to the RFU data. Y0 = baseline; the initial value may be RFU (1). a1 = RFU (t) for the steeply sloping portion of the data (growth phase); 0.05 can be a good initial estimate for a1. a2 = RFU (t) for the steeply sloping portion of the data (growth phase); 1.0 can be a good initial estimate for a2. a3 = on transition between baseline and tilt feature; RFU_maxThe time or cycle to reach the value immediately preceding 25% of is a good initial estimate of a3.

0415When the final values of Y0, a1, a2, and a3 are derived, Y0 is treated as the background and subtracted from the RFU (t) data for which curve fitting was performed.

0416Curve fitting equations other than the above may be used. For example, a commercially available TABLECURVE software package (SYSTAT Software Inc. (Richmond, CA)) can be used to identify and select equations that describe typical real-time nucleic acid amplification curves. Such a typical resulting equation used in mathematical modeling is given by equation (4). RFU (t) = Y0 + b (1-exp (-(td)^*ln (1-2 ^^{(-1 / e)}) -c) / d)) ^^e (Four) Yet another typical equation that results is given by equation (5). RFU (t) = Y0 + b / (1 + exp (-(td)^*ln (2 ^^{(1 / e)}-1) -c) / d))) ^^e (Five) In either case, as mentioned above, the equation can be solved using, for example, the Microsoft EXCEL SOLVER function to produce the final equation and the final value of Y0 and other parameters, the solution being RFU ( t) Produces Y0, which is the background that is subtracted from the data.

0417To normalize the data, divide each background-adjusted data point by the same background-adjusted maximum data point, i.e.

0418<chemistry num="1"><img id="000002" he="22" wi="154" file="JP5773533B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>That is, RFU_n(t) goes from -1 to 1.

0419In step 2108, the range of data is RFU_{n (max)}From RFU_{n (min)}Is calculated by subtracting. If the calculated range does not meet or exceed the specified minimum range (eg, 0.05), then the T time is not calculated because the data is suspected to be unreliable. The minimum range is empirically determined and may differ between one fluorescence measuring instrument and the next. Ideally, the specified minimum range should be selected to ensure that the variation of the data values from the minimum to the maximum exceeds the noise of the system. At step 2110, the curve fitting procedure is applied to the normalized background adjusted data. Any of the known curve fitting methodologies may be used, but in a preferred embodiment, the linear least squares method is used. Curve fitting by squares; "LLS") is used. Curve fitting is performed only on a portion of the data between a given lower and upper bounds. The ultimate goal after finding the curve that fits the data is to find the time corresponding to the point at which the curve intersects a given threshold. In a preferred embodiment, the threshold for normalized data is 0.11. The upper and lower bounds are empirically determined as the range of curves applied to the various control datasets shows the least variation over the time associated with a threshold. In a preferred embodiment, the lower limit is 0.04 and the upper limit is 0.36. The curve is suitable for data that extends from the first data point below the lower limit to the first data point above the upper limit.

0420At step 2110, determine if the slope of the fit is statistically important. For example, if the p-value of the first-order coefficient is less than 0.05, the fit is considered important and processing continues. If not, the process ends. Alternatively, R²The validity of the data can be judged by the value.

0421The slope m and intercept b of the linear curve y = mx + b are determined according to the fitted curve. According to that information, the T time can be determined in step 2104 as follows.

0422<chemistry num="2"><img id="000003" he="20" wi="67" file="JP5773533B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>The method of determining the T time using the fitted curve is graphically illustrated in Figure 79.

0423With reference to FIG. 77, in step 2116 it is determined whether internal control / calibrator adjustment is desirable. In general, the test procedure appears to include at least one reaction vessel as a control with a known concentration of nucleic acid (other than the desired nucleic acid), or a control nucleic acid sequence may be added to each sample. Known concentrations may simply be used as controls to confirm that the reaction took place in the reaction vessel. That is, it is confirmed that the reaction was successful when the known concentration was amplified as expected, and it can be concluded that the poor results for the target sample were due to the absence of the target in the sample. On the other hand, failure to amplify known concentrations as expected indicates reaction failure and any consequences for the target are ignored.

0424Known concentrations can be used to calibrate the concentration of the target. The T-time corresponding to a set of standards containing internal controls and target sequences is determined for a substantially valid number of datasets. This data is used to construct a calibration plot in which the concentration of the test sample is interpolated as described below.

0425One way to construct a calibration plot is to place the known concentration of the sample of interest on the x-axis and the difference in T-time between the target and control on the y-axis. Subsequently, the concentration of the test sample is interpolated by calibration curve fitting. Another way to construct a calibration plot is to place the known concentration of the sample of interest on the x-axis and the ratio [target T time / internal control T time] on the y-axis. Subsequently, the concentration of the test sample is interpolated by calibration curve fitting. An example of this is disclosed in Haaland et al., US Pat. No. 6,066,458, "Methods, Apparatus and Computer Program Products for Determining Quantities of Nucleic Acid Sequences in Samples Using Standard Curves and Amplification Ratio Estimates." A further alternative method of constructing a calibration plot is Carrick et al., U.S. Provisional Application No. 60 / 737,334, which shares common ownership herein, "Parametric. Use a parametric calibration method such as the method described in "Calibration Method".

0426The dataset sometimes shows a dip immediately after the initial static baseline (ie, the initial flat part of the RFU (t) curve, see Figure 78) and just before the data begins to tilt upwards. To identify and correct such data, the following algorithm is used before determining the T-time of the data. H_indexStarting from, each RFU (t) value is checked to determine if it is less than the background value BG. If it is small, subtract RFU (t) from BG (the result should be a positive number). This is the Cor Value (repulsion coefficient value). CorValue is added to the value obtained by subtracting the background, so that RFU (t) becomes the baseline. Calculate forward for each RFU (t) value and run this analysis until the last CorValue is less than the previous CorValue. Add the largest CorValue to each of the remaining background-reduced RFU (t) values. Here, as described above, the corrected dataset can be normalized and the T time can be determined.

0427When using the curve fitting method to derive the background level, it may not be necessary to perform dip correction as described above.

0428It is also desirable to perform outlier detection on the dataset to identify data points that show anomalous values compared to the remaining data points and discard them if necessary. Any of the known outlier detection methodologies may be used.

0429The second part of the sample concentration determination is the quantification procedure 2120. Determine the T time for a sample of known concentration under known conditions. This data can be used to derive the relationship between sample concentration (commonly referred to as a Log copy) and T-time. After determining the T time for a particular sample, the derived relationship (Log copy = f (T time)) can be used to determine the sample concentration for that sample.

0430More specifically, in steps 2122 and 2124, calibration / control datasets for control specimens of known concentrations are validated, for example, by outlier analysis and / or any other known data validation methodology. Be verified. If the data is found to be valid, the calibration will continue, otherwise the calibration will end.

0431The T time is determined for the control dataset and a T time vs. Log copy is plotted for all samples in a particular state (eg, samples treated with reagents in a particular batch lot). In step 2126, curve fitting, such as linear least squares fitting, is performed on a portion of the T-time vs. Log copy plot to determine the optimal line slope m and intercept b for the data. If the number of T-time vs. Log copy data points (known as "calibrators") available is at least a given minimum number of calibrators (as determined in step 2128), then the lowest calibrator, if necessary. In step 2130, remove as follows.

0432After finding the optimum line for the calibrator data points, the same test is performed for quadratic and cubic curve fitting. If these fits are significantly better than the linear linear fit, discard the calibrator data points farthest from the linear curve fit, find the primary, secondary, and tertiary fits and compare them again with the rest of the calibrators. This process is repeated-assuming the number of calibrators is at least the minimum acceptable number of calibrators-until the secondary and tertiary fits are significantly worse than the linear linear fits.

0433Once the linear T-time vs. Log copy equation is derived, in step 2132 the desired sample concentration for that sample is determined (as a Log copy) by substituting the T-time of the sample into the equation. In this way, assay results are obtained 2134.

0434A possible enhancement of the RT Incubator 608 is a self-checking photodetector module. In such modules, a known standard excitation signal is emitted by the LED1732 (or, as an alternative, a separate dedicated LED), and the excitation light is the excitation signal, the emission signal, and the signal output of the printed substrate 1790. Is directed to the photodiode 1780 (and / or a separate dedicated comparator photodiode) to ensure that everything is correct.

0435All references referred to herein are incorporated herein by reference. However, no document is recognized as the prior art of the subject of the claims.

0436Although the present invention has been described in connection with what is currently considered to be the most practical and preferred embodiments, the invention is not limited to the disclosed embodiments, and the spirit of the appended claims. It should be understood that it is intended to extend to the various modifications and equivalents included in the scope.

0437In addition, those in the appended claims that do not include the wording of the "means for performing a particular function" form permitted under Article 112, paragraph 6 of the United States Patent Act are those of the United States Patent Act 112 It is not intended to be construed as being limited to the structures, materials, or actions described herein and its equivalents under Article 6.