System for contamination-free reaction processes.

The invention relates to a system for automatically carrying out reactions in reaction vessels as well as suitable reaction vessels and closures.

An essential component of health systems is the detection of diseases on the so-called medical diagnostics. In this case be with samples possibly diseased persons were removed, detection reactions the analyte-passback performed. Suitable analytes have substrates of enzymatic reactions, enzymes themselves, antigens and nucleic acids also proved in recent times. The analysis of the latter substances is often considerably more difficult, as the present concentrations are sometimes very low. A prerequisite for reliable results is a carryover free working, as the lowest concentrations of interfering substances have a substantial influence on the measurement result. Particularly in tests in which multiple analyzes are performed sequentially in a single device, there is a considerable carry-over risk.

The nucleic acid diagnostics, such as the polymerase chain reaction (US-A-4,683,195), the detection limit was determined by the provision of amplification is significantly reduced. For this is also a risk of falsification of the measurement increases by contamination, especially by formation of aerosols in the air. For this reason, the preparation of samples to be analyzed, and the amplification has also been proposed to carry out in separate rooms. Other solutions are overlaying with oil or wax as well as the application of an internal enzymatic decontamination system with lower sensitivity (US Patent 5,035,996). These solutions are expensive and for automatic execution of several reactions ill suited.

In the Polymerase Chain Reaction special tubes are used which allow by conical shaping a good heat transfer from an underlying heating. Moreover, most used for performing the thermal cycling devices use a cover heater (100 ° C) to reduce evaporation and prevent condensation.

Also described are plates containing multiple reaction vessels, which can be closed via a manually breakable joint cover. A disadvantage of this design is that at the opening of the plate splashes can move from one reaction vessel in an adjacent, and thus the risk of carry-over is increased.

In EP-A-0408280 also reaction vessels in an open 96-well microtitration plate are described. The risk of contamination is likewise very large.

All previously described solutions for vessels for the automatic processing of multiple reactions have the disadvantage that the reaction vessels are either not effectively be closed, or that the reaction vessels have caps that are not suitable for automatic continuous. These systems are therefore to use for routine use with high throughput only at considerable risk of contamination.

The invention relates to a system for automatically carrying out reactions in reaction vessels comprising:<ul><li>a plurality of reaction vessels,</li><li>several individual closures for this reaction vessels,</li><li>a device for automatically opening the closures and</li><li>an automatic pipetting device.</li></ul>

Similarly, to use the technology above sample and Reagenzvorratsgefäße.

Likewise provided by the invention are suitable tubes and closures for such systems.

The inventive system is suitable for automatically carrying out reactions. a performance is referred to as automatic in this context, be avoided in the manual steps after the introduction of the sample into the system until receipt of the measurement result. The processing steps occur in a predetermined manner, preferably separated by a computer program in the system from. The system is also to carry out several consecutive reactions and suitable even in different Reagenzvorratsgefäßen.

In principle, any kind of chemical reactions in the system according to the invention are performed. These include in particular those intended to be completed in the course of an analysis of components of a sample. The occurring reactions depend on the type of analyte to be determined. For example, immunologically active compounds are usually determined by immunological methods. The interaction between antigens and haptens one hand and antibodies directed against them on the other hand used. In the case of nucleic acid tests the relatively specific interaction between each other substantially complementary nucleic acids is used.

Analytical methods for nucleic acids can be essentially in the steps of sample preparation, nucleic acid processing and detection of nucleic acids treated divided. The inventive system is designed to perform the particular nucleic acid treatment responses. These reactions include in particular the amplification of nucleic acids. For this, there are a number of ways. While it isothermally run amplifications, as the method described in EP-A 0329822 are many other amplifications are performed by performing temperature change cycles (thermal cycles), as described for example in EP-A-0200362.

The inventive system includes a plurality of reaction vessels which are suitable for accommodating reaction fluids. The volume of the reaction liquors is preferably 5 ul to 5 ml, more preferably between 10 .mu.l and 200 .mu.l. The reaction vessels are made of a reaction-inert material solution z. B. plastics such as polystyrene or polypropylene. Provided that the implementation of thermal cycles is provided, these reaction vessels must be thermostable. The PCR tubes from Perkin Elmer commonly used are suitable for example as reaction vessels of the present invention.

An essential feature of the present invention is the use of individual closures for these reaction vessels. The individual closure of reaction vessels has not been taken into consideration, possibly supported by the fact that the designers of Tube composite plates were fixed on the use of composite lids particularly suitable. Moreover, the use of individual closures represents a technical challenge that is managed by the individual closures according to the invention. The closures are, like the vessels, preferably made from a preferably thermally stable material, such. As polystyrene, polypropylene, particularly preferably produced by a known injection molding technique.

Reactions which may be carried out in the reaction vessel, physical (for example, adsorption), but also biochemical species (eg, enzyme reactions, amplifications, etc.) can be.

Also essential to the system of the invention is a device for the automatic opening of the shutters. This device is designed technically to the single shutter used in each case.

For automatically carrying out reactions of the fluids will be filled in the reaction vessel. For this purpose, the use of an automatic pipetting device, preferably, exposed with disposable tips to be particularly advantageous. The components of the reactions to be carried out in any appropriate order can be dispensed. It is for example possible to make first a sample containing the analyte in the liquid Reagenzvorratsgefäß. This can, if desired, already happened outside of the inventive system and without the automatic pipetting device. In a subsequent step, which can be made, if desired, by means of the automatic pipetting device, the reagents required for the reaction are added to the sample liquid in the Reagenzvorratsgefäß, wherein the inventive method can also be applied.

In another embodiment, the necessary reagents in solid or liquid form are given in the reaction vessel, and the sample liquid is pipetted.

In the following, a system for automatically carrying out reactions in a composite panel 96 will wells described in Mikrotitrationsplattenraster, this being a particularly preferred embodiment of the arrangement of several reaction vessels. This system is especially well suited for carrying out the polymerase chain reaction by performing thermal cycles. 1 shows a composite panel for the instrument Perkin Elmer Cycler 9600 is indicated. Located A reaction vessel 1 is drawn as in one of the holes 3 of a retaining plate 2 for 96 Tubes. The reaction vessel is fixed by means of barb 102 in the retaining plate. Once the composite panel with the reaction vessels in the PCR cycler is inserted ranging peaks of the reaction vessels into the heating block 4 of the cycler. In this figure, a suitable for the invention lid 5 is shown schematically. For closing the reaction vessel, the cover 5 is pressed into the reaction vessel 1 up to a stop five hundred and first

Also subject of the invention is a lid for a reaction vessel having a projecting into the opening of the reaction vessel part, characterized in that said portion includes means for removal of the lid from the reaction vessel.

The lid according to the invention comprises at least two parts. One of the portions (reference numeral 550) is so designed that it protrudes into the opening of the associated reaction vessel when the reaction vessel is closed by the lid. Another portion 500 protrudes then out of the reaction vessel.

The portion 550 includes means 551 for removing the lid of the reaction vessel. In addition, this part comprises means for sealing the lid to the reaction vessel, eg. As sealing lips or sealing beads 552nd

The portion 500 preferably includes a stop 501 that prevents the lid in the closing of the reaction vessel disappears completely in this. Thereby an exact orientation of the cover surface 503 is assured. The part 500 of the cover preferably projects only very little about the opening of the reaction vessel out. The outstanding from the reaction vessel parts of the lid close substantially with a surface 503 from. This is advantageous if a cover heater for preventing condensation of water vapor from the reaction liquid to be used on the cover. This may then lie flat on the surfaces 503 of several reaction vessels.

As a means 551 for removing the cover all structural embodiments may be used, which cause the resistance between the device for automatically removing the lid and the lid is greater than the resistance between the cover and the inner wall of the reaction vessel. Technical design possibilities are offered for example by using conical surfaces or ridges for engaging a device in part 550th

In figure 2 a first embodiment for an inventive cover (in the state of a closed reaction vessel) and a suitable opening arrangement 7 is shown. The means for removing the lid are here designed as a conically shaped recess 551 which is opposite to the reaction vessel closed, however, is open to the environment, to accommodate the removal of mandrel 700 of the apparatus for removal of the lid can. Against the inner wall 101 of the reaction vessel in this case pushes a member that has an all-round sealing bead running 552nd A particularly advantageous constructive detail of the lid according to the invention is in front of the structural separation of the center 551 of the seal lip 552, so that the pressure exerted during insertion of the access pin in the part 550 to the intermediate pressure does not lead to a substantial increase of the pressure on the sealing lip. This strong increase in the fixed seat of the lid upon insertion of the access pin 700 is avoided. The structural separation takes place in Figure 2, by providing a gap 553 between the means 551 and the sealing lip 552nd In Figure 2, this space is open toward the interior of the reaction vessel. Since the surface of the cover 503 is relatively large in this case, the support surface of a cover heater and the heat transfer from the heater to the cover is is particularly high.

3 shows a particularly advantageous cover is shown. It differs essentially characterized by the lid shown in Figure 2, that the space is not designed 553 open towards the reaction vessel, but toward the environment. This embodiment has the advantage that the contact surface of the lid to the reaction solution and the supernatant gas space is small so that only a limited surface is available for a possible condensation of liquid on the cover are available.

In Figure 4, a lid is shown, in which no gap 553 is provided. Although this lid is for the system according to the invention are also useful, however, with the disadvantage that the insertion of the access pin increases the pressure on the Reagenzvorratsgefäßinnenwand and thus a higher force to remove the lid from the reaction vessel is required, as in the cases described above.

The device for the automatic opening of the closures is adapted to the shape of the means for removal of the lid in the lid. Particularly advantageous mandrels which have proven to have a Entnahmewulst 701, which has to be pushed over a corresponding resistance of the cover in the part of the 550th Upon retraction of the mandrel the bead acts as a resistance. Characterized in that the pressure of the lid on the reaction vessel inner wall is smaller than the resistance of the bead with the lid, the lid is removed from the reaction vessel.

Thus, the access pin 7 can be used repeatedly, the device preferably includes a component for stripping of the reaction vessel lid remote from the access pin. This may for example be a Abstreiferhülse 702nd

The device is designed for the processing of samples in reaction vessels on the arrangement of the reaction vessels. In case of using a composite panel, the device is therefore located at an XY transport unit with which the device can be driven via the respective reaction vessel. By movement of the access pin 700 to the sealed reaction vessels to (Z-direction), the tip of the access pin inserted in the cover. This movement will be generally perpendicular to the transport plane. Although the two motions are in principle executed by each computer-controlled robot, it is also possible to retrofit conventional automatic pipetting eg Tecan by replacing the rack normally used for pipetting by a rack with a device. 7 It is also possible to attach the means for automatically opening the lid, in addition to the pipette at a pipetting rod of a tooth. The pipetting apparatus is programmed accordingly.

For parallel processing of multiple reaction vessels, z. B. a row a microtiter plate, a plurality of devices 7 can be attached to a transport unit, so that a plurality of individual lids can be opened simultaneously. However, this embodiment does not appear preferable in view of the prevention of contamination.

The present invention, in particular for vending machines lid is not only suitable for reaction vessels, but for any kind of vessels, in a system for performing several reactions. These include not only the vessels in which a reaction between several reactants expires, and which are called here the reaction vessels, and vessels in which the individual reactants to the desired reaction are kept. In the case of the analysis of liquid samples can be made between sample vessels and Reagenzvorratsgefäßen here. In a sample container, a liquid to be examined for the presence or amount of one or more analytes, held. The sample fluid is transferred to sampling (for example, recovery of a patient who is to be examined blood) and possibly work-up (extraction of serum or plasma) in a sample vessel and, if desired, with a machine veschlossen common lid according to the invention. Then, the sample container is an automatic analysis machine introduced individually or together with other sample vessels in the system, for example,. For flow of a method according to the invention, before a transfer of sample liquid from the sample container to be carried out in a reaction vessel, the lid from the sample container and removed the desired amount of sample fluid. An advantage of the present system, in which the sample containers are closed by a common automatic lid, is the possibility of easy re-closure of the sample vessel by means of the same or a new lid. For the preferred case of the re-closure with a new lid from the lid of the old access pin is transferred through the Abstreiferhülse in a waste container and a new lid mounted from a supply vessel for the removal of the cover on the mandrel. The new lid is then placed on the sample container and the access pin removed, the lid remains on the sample vessel. For a further determination of another analytes from the same sample, the cover is removed again and again removed a certain amount of sample liquid. If the cover includes a septum, a sampling from the sample vessel can also be performed without removing the lid with a lancing pipette.

During the execution of reactions it may be necessary to transfer additional reagents in the reaction vessel. These are held in Reagenzvorratsgefäßen, which can also be inventively closed and opened. Under a Reagenzvorratsgefäß therefore is defined as a storage vessel for the reagents, and these reagents are used in carrying out reactions in reaction vessels. Since the reagents usually in large quantities, for example for carrying out several reactions, need to be kept, this Reagenzvorratsgefäße often have the form of bottles with volumes between 20 and 500 ml. Also such Reagenzvorratsgefäße can be closed by an automatic current cover according to the invention , This will be done by the manufacturer, as a rule. The customer sets the Reagenzvorratsgefäß into an appropriate position of the system. Once in a reaction vessel, a reaction to be performed, which requires the use of in the pre-determined Reagenzvorratsgefäß preserved reagent, the Standard machine lid is removed from the Reagenzvorratsgefäß and removed the required amount of reagent and transferred to the reaction vessel. Then the Reagenzvorratsgefäß can be resealed by the removed or a new cover (see sample vessel).

In the case of large-volume Reagenzvorratsgefäßen the Reagenzvorratsgefäße previously used can be used in principle, when the screw cap customarily used are modified by use of the machine according to the invention common lid. These preferably m the upper side of the screw cap a small opening is provided, which is then sealed with a lid according to the invention. This system has the advantage that the Reagenzvorratsgefäß can be filled by a relatively large, closed by the screw cap opening, however, the removal of reagent fluid through the relatively small, according to the invention closed opening can be made.

Also subject of the invention is a method for automatically carrying out chemical reactions in reaction vessels, characterized by the steps of:<ul><li>Providing of reaction mixtures in reagent and / or reaction vessels</li><li>Closure of the reaction vessels by individual closures and</li><li>automatically opening the individual closures by means of a device for removing the lid of the vessels.</li></ul>

The process is similar in principle from previously known as the manual method, but using a conventional machine and the lid in a system. Of course, the automatic standard cover can be opened and closed manually. The central feature is the provision of reaction vessels containing a reaction mixture and which are closed by a common cover machines. This is especially true for reactions which extend in the thermal cycles. After completion of the reaction of the lid by means of a device for the removal of the lid is detached from the reaction vessel. This is done by inserting the device in the provided in the cover means and pulling out of the device together with the lid. The cover can then be stripped by retracting the access pin in a Abstreiferhülse the access pin. Thereafter, the reaction mixture can be removed from the reaction vessel, or further processed in it and, if desired, even closed again by means of a (new) lid.

Advantage of the cover of the invention is the availability of a cover heater due to the particularly simple possibility of providing a flat surface. By shifting the center to open the cover in the space below the vessel material is saved and a compact design allows. Another advantage is the ability to keep the gas space above the reaction solution by projecting into the reaction vessel parts of the lid as small as possible. This advantage has the result that the height of the walls of the reaction vessel need not be reduced, which would be disadvantageous in view of injection options for pipetting. The Automatengängigkeit of the lid according to the invention facilitates the Vollautomatisierbarkeit of systems for detecting analytes in fluids.

The invention can be applied in particular for the analysis of components of body fluids according to principle of immunoassays or nucleic acid diagnostics. Because of the above-mentioned advantages, the application in the detection of nucleic acids is particularly preferred. Detection method for nucleic acids can include, as processing steps for the amplification of nucleic acids in a liquid, for example on the type of polymerase chain reaction (EP-B-0200362) or of another system. Also the carrying out of hybridization reactions connected thereto with detection probes may be carried out in the reaction vessels of the invention. Here, the multiple opening of the reaction vessel with the aid of a lid according to the invention is possible.

In Figure 1, a composite panel with reaction vessel 1, thermoblock 4 and inventive cover is shown.

In Figure 2 to 4 cover for carrying out the method of the invention are shown in conjunction with appropriate Entnähmevorrichtung and reaction vessel.

5 shows a system according to the invention for carrying out reactions is shown. Here, a composite panel with reaction vessels and lids is indicated by reference numeral 010th It can reagent reservoirs 011, 012 and 013 as well as several samples primary receptacles 014 may be provided 015 and 016, from which are taken the necessary for the reaction amounts of the pipette unit 017 and pipetted into the reaction vessels. 5 After closure of the reaction vessels, which can be, if desired, also be carried out with the removal device 7, the reaction and the lid is opened with the removal device, other reagents can be pipetted. If desired, a measurement of signals either in the reaction vessel itself, or by removal of the reaction mixture is then made in a measuring unit. In the figure, the rack 018 of the XYZ transport unit of a conventional automatic pipetting (eg. B Tecan) is indicated.

The aforementioned risk of contamination can also refer to reagents <u>open</u> standing on one device. Figure 6 shows an inventive embodiment of a screw, which can be processed in production with standard equipment and is then accessible system. The screw 8 has a screw thread 801 and a discharge opening 802 which can be opened or closed by means of a lid according to the invention.

Analogously, for the samples immediately after blood collection in a special test tube can be transferred to an embodiment with a septum by syringe. this is a modified inventive cover is shown in FIG. 7 It has a septum 555 which can be pierced by a conventional pipette needle. The advantage of this embodiment is the possibility of multiple sampling, without that the lid of the invention must be removed. The septum is preferably located in the protruding into the sample vessel part of the lid.

8 shows a locking unit for a removal device is shown. This is controlled via a pressure piece 2101 which is held together with the locking element in 2103 by a kickout 2106 to a pressure pin 2105 in a starting position. The locking element 2103 in this case runs in a locking sleeve 2102 and the push pin 2105 in a Abdrückhülse 2104th

This latching unit can be used both manually as well as within a system for removal of covers. Preferably, the detent element is mounted in an outer sleeve 2002 that is shown in FIG. 9 The locking unit by reference numeral 2100 in.

In Figure 10, a manual sampling device is shown. It contains as a part a device for removing the cover or for attaching the lid and as a second part a device for pipetting a liquid

In Figure 11, steps A to F for the removal of a cover are shown by a vessel. Step A shows the starting position of the withdrawal section and a sealed by a lid according to the invention vessel. In step B, the removal member is applied to the unit which contains the vessel, is placed. In Step C of the access pin is inserted into the part 550 of the lid. By retracting the access pin to a short distance, locked the access pin at the designated narrow one (level D). By further retraction of the access pin together with the Abstreifehülse the lid is removed from the vessel (level E). In step F the extraction unit is lifted from the assembly with the reaction vessel. In a not shown further step G of the cover is stripped by advancing the Abstreiferhülse about Entnahmewulst addition. After the extraction unit is back to position, or can be transferred back to the starting position (A level).

Also subject of the invention is an apparatus for processing a liquid which is located in a closed or closable vessel, comprising a device for pipetting the liquid and a device for removing or / and attaching a closure of the vessel. As a device for pipetting the liquid on the one hand, a commercially available pipette (z. B. company Eppendorf, Germany) are used. at this pipette, preferably mounted in the vicinity of the handle, a lid handler. This acts preferably by pressure of the overall apparatus on a sealed vessel when the lid is to be removed or by pressure on a non-sealed vessel when the vessel with the aid of a closure to be sealed. Be particularly effective cover of the invention have been found.

For the case of automatic processing of liquids, the lid handler on a X-, Y-, (Tecan) or a cylindrically arranged robot arm be attached (eg. B. from Zymark) Z-arm of a robot. It is also possible to use the lid handler in different styles, for. Example, in 8-fold or 12-fold adjacently positioned manner. The lid handler can in another embodiment also to an arm of a conventional Mikrotiterplattenwaschers (also this leads X, Y, Z motion) be attached. Further, the lid handler according to the invention can also with a thermal cycler for carrying out thermal cycling reactions, such as, for example, the polymerase chain reaction may be attached, so that hereby a coordinated removal of lids of containers, in which reagents are to be introduced possible. It is also possible to combine the lid handler invention with magnetic separators, heating blocks or water baths. All these devices are in the handling of liquids of use.

Another object of the invention is therefore a device for grasping a vessel cover comprising a component (700), which engages in the interior of the lid and firmly clamped in this, and a component (710) with which the gripped cap from the component, which is clamped in this, can be stripped. Preferably, this device includes a locking mechanism, eg. As in the manner of a ball point mechanism, simply by applying pressure to the device can leave a predetermined sequence of locking positions. Especially preferred is the case that the pressure on the outer casing of the apparatus is exerted.

example 1

Using the example of the flow chart of an amplification reaction, a possible protocol for the interaction of the laboratory robot is presented with the objects of the invention. The interaction of all elements allows automatic execution of the reaction process.

The upstream steps to recover the analyte-containing sample from the body's fluids (eg., Blood) done by generally customary processes and are therefore not described. At the beginning of the sequence of the automatic Amplifikationsdurchführung is therefore of an analyte in a sample liquid can be assumed (in this case, of purified nucleic acid from a blood sample) which is present in a reaction vessel according to the invention with a single fastener.

The system for contamination-free processing of reactions further includes:<ul><li>a laboratory robot Tecan (type RSP9652SLD) Arm 1, the automatic pipetting unit (017) Arm 2, the opening and removal device (7),</li><li>a thermocycler Perkin Elmer with a composite panel (2) with reaction vessels (1) incl. single fasteners (5), the composite panel consists of a retaining plate with recesses for receiving of the reaction vessels and arranged underneath the heating block (4), which also has corresponding recesses.</li></ul>

In the laboratory robot platform are the necessary reagent vessels, the sealed sample with the analyte for the amplification reaction, the measuring cells or storage receptacles, waste container and pipette tip receptacle with pipettes. All vessels are closed with locks and are opened before use.

The following flow chart assumes that all units, vessels, elements and substances are present and ready for a new start of an amplification reaction is present. As "standby" position of the arm is called a defined position on the laboratory robot platform that is located outside the vessel region and allowed further access of the other arm on the same vessel. For each of the two arms has its own "standby" position.

flowchart

step Actions

Start of the flowchart<ul><li>1 Arm 1 with pipette leads from the initial position to the pipette tip receiving and stocked the unit with a new pipette tip,</li><li>2 arm 2 with opening and removal device (7) moves from the initial position to the sample vessel with the analyte (014),</li><li>3 opening and removal device outlet (Arm 2), Device move to the home position, Placing the removal element to the lid surface, Introduction of the access pin (700) into the lid interior, Pressing in of the mandrel withdrawal by lifting of the removal device (7), Remove the lid from the vessel Arm 2 with the cover moves out of the region of the reaction vessel opening adjacent to the vessel in a "stand-by" position.</li><li>4 Arm 1 with pipette and pipette tip moves from the pipette tip receiving the sample vessel of the analyte,</li><li>5 <b>Sampling sequence (arm 1)</b> Lowering the pipette with the pipette tip, Sucking the sample liquid in the tip, Lifting the pipette with pipette tip plus sample liquid,</li><li>6 Arm 1 with pipette with pipette tip plus sample liquid travels next to the opening of Amplifikationsgefäßes in "standby" position</li><li>7 Arm 2 with lid leads to the sample vessel of Analytenöffnung </li><li>8th <b>Aufsetzungs- and Verschließungsablauf (Arm 2)</b> Arm 2 with lid moves over the vessel opening, Lowering of the lid on the vessel, Removing the cover from the access pin of the opening device, Vessel is sealed with a lid, Arm 2 moves to the "standby" position.</li><li>9 Arm 2 with opening and removal device moves from the "standby" position to the reaction vessel (1) (Amplifikationsgefäß) in a defined position on the composite panel</li><li>10 <b>Opening and removal device outlet (Arm 2)</b> Device (7) moves to the starting position, Placing the removal element to the cover surface of a reaction vessel (1), Introduction of the access pin into the cover interior, Pressing of the access pin, Remove the lid from the vessel, Lifting the opening and extraction unit with cover from the vessel, Arm 2 with lid moves from the area of ​​the vessel opening next to the vessel in "standby" position</li><li>11 Arm 1 with pipette with pipette tip plus sample fluid moves from the "standby" position to the reaction vessel (1) (Amplifikationsgfäß) opening</li><li>12 <b>Sample dispensing outlet (Arm 1)</b> Lowering the pipette with pipette tip plus sample Emptying the sample liquid from the tip, Lifting the pipette with the pipette tip, Arm 1 goes to Waste container Discharge of used lace, Arm 1 moves into position</li><li>13 Arm 2 with lid moves to Amplifikationsgefäßöffnung </li><li>14 <b>Aufsetzungs- and Verschließungsablauf (Arm 2)</b> Arm 2 with lid moves over the vessel opening, Lowering of the lid on the vessel, Removing the cover from the access pin of the opening device, Vessel is sealed with a lid, Arm 2 moves into "standby" position</li><li>15 Arm 1 with pipette moves from the initial position to the pipette tip receiving and stocked the unit with a new pipette tip</li><li>16 Arm 2 with opening and removal device moves from the "standby" position for reagent bottle on the platform</li><li>17 <b>Opening and removal device outlet (Arm 2)</b> Device (7) moves to the starting position, Placing the removal element to the lid surface, Introduction of the access pin on the lid surface, Introduction of the access pin into the cover interior, Pressing of the access pin, Cover the vessel removed, Opening and extraction unit with cover from the vessel stand, Arm 2 with lid moves from the area of ​​the vessel opening next to the vessel in "standby" position</li><li>18 Arm 1 with pipette and pipette tip moves from the pipette tip receiving the reagent vessel (011) on the platform</li><li>19 <b>Reagenzentnahmeablauf (Arm 1)</b> Lowering the pipette with the pipette tip (optionally septum puncture) Sucking the required amount of liquid in the tip, Lifting the pipette with pipette tip plus liquid,</li><li>20 Arm 1 with pipette with pipette tip plus liquid runs alongside the opening of Amplifikationsgefäßes in "standby" position </li><li>21 Arm 2 with lid moves to Reagenzgefäßöffnung</li><li>22 <b>Aufsetzungs- and Verschließungsablauf (Arm 2)</b> Arm 2 with lid moves over the vessel opening, Lowering of the lid on the vessel, Peeling the lid from the access pin of the opening device with the Abstreiferhülse (710) Vessel is sealed with a lid, Arm 2 moves into "standby" position</li><li>23 Arm 2 with opening and removal device moves from the "standby" position to Amplifikationsgefäß in a defined position on the composite panel,</li><li>24 <b>Opening and removal device outlet (Arm 2)</b> Device move to the home position, Placing the removal element to the lid surface, Introduction of the access pin into the cover interior, Pressing of the access pin, Cover the vessel removed, Opening and extraction unit with cover from the vessel stand, Arm 2 with lid moves from the area of ​​the vessel opening next to the vessel in "standby" position</li><li>25 Arm 2 with pipette with pipette tip plus liquid moves from the "standby" position to Amplifikationsgefäßöffnung</li><li>26 <b>Reagenzabgabeablauf (Arm 1)</b> Lowering the pipette with pipette tip plus sample Emptying the reagent liquid from the tip, Lifting the pipette with the pipette tip, Arm 1 goes to Waste container Discharge of used lace, Arm 1 moves into position</li><li>27 Arm 2 with lid moves to Amplifikationsgefäßöffnung </li><li>28 <b>Aufsetzungs- and Verschließungsablauf (Arm 2)</b> Arm 2 with lid moves over the vessel opening, Lowering of the lid on the vessel, Removing the cover from the access pin of the opening device with the Abstreiferhülse, Vessel is sealed with a lid, Arm 2 moves into "standby" position</li></ul> The steps 15 to 28 represent a cycle which has to be negotiated for each renewed addition of reagents in the Amplifikationsgefäß.

Will 3 Reagenzzugabezyklen provided as in the commercial kit from Roche (PCR buffer, Taq polymerase, primers) are therefore to go through another 28 steps.<ul><li>68 Arm 2 with pipette moves to the starting position</li><li>69 amplification solution in the vessel passes through a temperature program as in the aforementioned kit and thermal cycler recommended</li><li>70 end</li><li>71 Arm 2 with opening and removal device moves from the initial position to Amplifikationsgefäß in a defined position on the composite panel</li><li>72 <b>Opening and removal device outlet (Arm 2)</b> Device move to the home position, Placing the removal element to the lid surface, Introduction of the access pin into the cover interior, Pressing of the access pin, Cover the vessel removed, Opening and extraction unit with cover from the vessel stand, Arm 2 with lid moves from the area of ​​the vessel opening next to the vessel in "standby" position </li><li>73 Arm 2 with pipette with pipette tip moves from the initial position to Amplifikationsgefäßöffnung</li><li>74 <b>Amplification mixture (Arm 1)</b> Lowering the Pipiettiereinheit with pipette tip, Aspirating the desired quantity of liquid in the tip, Lifting the pipette with pipette tip plus liquid</li><li>75 Arm 1 with pipette with pipette tip plus liquid runs alongside the opening of Amplifikationsgefäßes in "standby" position</li><li>76 Arm 2 with lid moves to Amplifikationsöffnung</li><li>77 <b>Aufsetzungs- and Verschließungsablauf (Arm 2)</b> Arm 2 with lid moves over the vessel opening, Lowering of the lid on the vessel, Removing the cover from the access pin of the opening device, Vessel is sealed with a lid, Arm 2 moves into "standby" position</li><li>78 Arm 2 with opening and removal device moves from the "standby" position to the measuring cell or to a containment vessel in a defined position on the platform</li><li>79 <b>Opening and removal device outlet (Arm 2)</b> Device move to the home position, Placing the removal element to the cover surface of the measuring cell or the containment vessel, Introduction of the access pin into the cover interior, Pressing of the access pin, Cover the vessel removed, Opening and extraction unit stand with lid from the vessel, arm 2 with lid moves from the area of ​​the vessel opening next to the vessel in "standby" position</li><li>80 Arm 2 with pipette with pipette tip plus sample fluid moves from the "standby" position to the measuring cell or storage opening</li><li>81 <b>Sample dispensing outlet (Arm 1)</b> Lowering the pipette with pipette tip plus sample Emptying the sample liquid from the tip, Lifting the pipette with the pipette tip, Arm 1 goes to Waste container Discharge of used lace, Arm 1 moves into position</li><li>82 Arm 2 moves with lid for measuring cell or storage vessel opening</li><li>83 <b>Aufsetzungs- and Verschließungsablauf (Arm 2)</b> Arm 2 with lid moves over the vessel opening, Lowering of the lid on the vessel, Removing the cover from the access pin of the opening device, Vessel is sealed with a lid, Arm 2 moves to starting position</li></ul>

End of the process

The amplification mixture is now ready for the direct detection of the amplificates formed or to carry out further reactions.

example 2

Below is recalling FIG 8 - described 11 as a lid can be removed or attached by means of a lid handler. <ul><li>A) Open the lid The lid handler must be in the position with a pen mechanism latching position A.<ul><li>1) placing the element 2003 (outer shell) on the webs of the tube.</li><li>2.) applying a force downward causes the lowering of the element (2006) on the cover with simultaneous tension of the spring (2004). Now, a first pressure point is reached.</li><li>3.) Exercise greater force than in Step 2.) causes:<ul><li>a.) the mandrel (700) moves to a cap pin receptacle.</li><li>b.) the spring (2007) is compressed</li><li>c.) the point mechanism (2005) shortens the distance II and goes into locking position B, wherein the spring (2007) remains in a tense state.</li></ul> A second pressure point is reached.</li><li>4.) releasing the lid handler (withdrawal of the downward force) causes the removal of the cap from the tube. The force necessary to overcome the friction resulting from the discharge of the spring (2004). The force is transmitted to the cover and the webs of the tube over element (2003) and the mandrel (700).</li><li>5.) The lid handler with the cover can now be lifted off and removed. The mandrel (700) is withdrawn by the shortening of distance II now in the element (2002). The lid is now partially protected by contamination member (2003), but extends functionally 1-2 mm above element (2003) addition.</li></ul></li><li>B) Close the lid<ul><li>6) placing the lid handler directed with lid on the mouth of the tube and exert a force pushes down the plate to the mouth of the tube.</li><li>7) reinforcing the power of 6) causes the complete insertion of the lid and clamping via frictional forces. The force is transmitted through the elements 2005, 2009, 2008, 2010 to the mandrel (700). Simultaneously spring 2004 is stretched. A third pressure point is reached.</li><li>8) reinforcing the power of 7) pushes the mandrel (700) of element (2006). At the same time the spring (2004) tightens further, while the point mechanism (2005) snaps back to its original resting position A and extended distance II.</li><li>9) reducing the downward force (= release) pushes the lid handler fully upwards, the tensioned spring (2004), member (2003), the tube keeps low. The spring relaxes in this process and the lid handler is ready for another opening process.</li></ul></li></ul>

LIST OF REFERENCE NUMBERS

<dl id="dl0001"><dt>1</dt><dd>reaction vessel</dd><dt>101</dt><dd>Inner wall of the reaction vessel</dd><dt>102</dt><dd>barb</dd><dt>2</dt><dd>retaining plate</dd><dt>3</dt><dd>Holes in the retaining plate</dd><dt>4</dt><dd>thermoblock</dd><dt>5</dt><dd>cover</dd><dt>500</dt><dd>also projecting from the reaction vessel of the reaction vessel</dd><dt>501</dt><dd>stop</dd><dt>503</dt><dd>Surface of the lid</dd><dt>550</dt><dd>In the opening of the reaction vessel into protruding part</dd><dt>551</dt><dd>Means for removing the lid</dd><dt>552</dt><dd>sealing bead</dd><dt>553</dt><dd>clearance</dd><dt>554</dt><dd>directed onto the inner wall 101 of outer surface of the lid</dd><dt>555</dt><dd>septum</dd><dt>7</dt><dd>Opening device / retrieval device</dd><dt>700</dt><dd>access pin</dd><dt>701</dt><dd>Entnahmewulst</dd><dt>710</dt><dd>Abstreiferhülse</dd><dt>2100</dt><dd>removal device</dd><dt>2101</dt><dd>pressure piece </dd><dt>2102</dt><dd>locking sleeve</dd><dt>2103</dt><dd>locking element</dd><dt>2105</dt><dd>pushpin</dd><dt>2106</dt><dd>kickout</dd><dt>2001</dt><dd>admission</dd><dt>2002</dt><dd>shell</dd><dt>2003</dt><dd>backstop</dd><dt>2004</dt><dd>detent spring</dd><dt>2005</dt><dd>Phillips screw (ball point mechanism)</dd><dt>2006</dt><dd>Abstreiferhülse</dd><dt>2007</dt><dd>feather</dd><dt>2008</dt><dd>pusher</dd><dt>2009</dt><dd>pusher</dd><dt>2010</dt><dd>pusher</dd><dt>010</dt><dd>Composite panel with reaction vessels and lids</dd><dt>011, 012, 013</dt><dd>Reagenzvorratsgefäße</dd><dt>014, 015, 016</dt><dd>sample containers</dd><dt>017</dt><dd>pipette</dd><dt>018</dt><dd>rack</dd><dt>8th</dt><dd>screw</dd><dt>801</dt><dd>screw</dd><dt>802</dt><dd>Removal opening of the screw cap</dd><dt>9</dt><dd>pipette</dd></dl>