Methods and systems for nucleic acid amplification

Method and system for nucleic acid amplification

[0001] Cross Reference

[0002] This application claims the priority of the Patent Cooperation Treaty Application No. PCT/CN2013/090425 filed on December 25, 2013, which is incorporated herein by reference in its entirety for all purposes.

Background technique

[0003] Nucleic acid amplification methods allow selective amplification and identification of nucleic acids of interest from complex mixtures such as biological samples. In order to detect nucleic acid in a biological sample, the biological sample is usually processed to separate the nucleic acid from other components of the biological sample and other substances that may interfere with the nucleic acid and/or amplification. After isolating the nucleic acid of interest from the biological sample, the nucleic acid of interest can be amplified by, for example, an amplification method known in the art, such as a method based on thermal cycling (eg, polymerase chain reaction (PCR)). increase. After amplifying the nucleic acid of interest, the amplified product can be detected, and the result can be interpreted by the end user. However, extracting nucleic acids from biological samples before nucleic acid amplification may be time-consuming, resulting in a decrease in the overall time efficiency of the process.

[0004] Point-of-care (POC) testing has the potential to improve the detection and treatment of infectious diseases in remote areas where laboratory results are delayed and follow-up of patients may be more complicated under resource-constrained conditions with poor laboratory infrastructure. potential. POC testing can also make existing health care facilities more capable of providing sample-to-answer results to patients during a single visit. However, the inefficiency of the POC method and device limits what can be achieved. For example, preparing nucleic acids (e.g., nucleic acids of pathogens) from complex sample types (e.g., biological samples) requires skilled technicians to manually perform multiple processing steps and subsequent detections in a dedicated laboratory space, so it often takes several hours or even several hours. The result report can only be issued after days.

[0005] Therefore, there is a need for fast and accurate methods and devices for analyzing nucleic acids from complex sample types. Such methods and devices can be used, for example, to achieve rapid sample-feedback detection and treatment of diseases that can be detected via their nucleic acids.

Summary of the invention

[0006] The present invention provides methods and systems for the efficient amplification of nucleic acids such as RNA and DNA molecules. The amplified nucleic acid product can be detected quickly and with high sensitivity.

[0007] In one aspect, the present invention provides a method of amplifying target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. In one embodiment, the method includes: (a) providing a reaction vessel containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagent Contains (1) reverse transcriptase, (ii) DNA polymerase, and (iii) a primer set for the target RNA; and (b) subjecting the reaction mixture in the reaction vessel to multiple cycles of primer extension reaction to generate an indication of the presence of the target RNA Each cycle includes: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, and then (ii) incubating the reaction mixture at an extension temperature of less than or equal to 60 seconds Seconds of extension duration, thereby amplifying the target RNA. In another embodiment, the method includes: (a) receiving a biological sample that has been obtained from a subject; (b) providing a biological sample containing the biological sample and the method for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification; Increase the necessary reagents in the reaction vessel to obtain the reaction mixture, which contains (1) reverse transcriptase and (ii) a primer set for the target RNA; (c) subject the reaction mixture to multiple cycles of primer extension reactions to produce an indicator The presence of a detectable amount of the target RNA in the biological sample

(D) detecting the amount of amplified DNA product of (c); and, outputting information about the amount of amplified DNA product to the recipient, where the amount of time for completing (a)-(e) is less than or Equal to about 30 minutes. In some embodiments, the amount of time is less than or equal to 20 minutes, less than or equal to 10 minutes, or less than or equal to 5 minutes.

[0008] In some embodiments, the reagent further comprises a reporter that generates a detectable signal indicative of the presence of an amplified DNA product. In some embodiments, the intensity of the detectable signal is proportional to the amount of amplified DNA product or target RNA. In some embodiments, the reporter is a dye. In some embodiments, the primer set includes one or more primers. In some embodiments, the primer set includes a first primer for generating a strand complementary to the target RNA. In some embodiments, the primer set includes a second primer for generating a strand complementary to a DNA product that is complementary to at least a portion of the target RNA. In some embodiments, the target RNA is viral RNA. In some embodiments, the viral RNA is pathogenic to the subject. In some embodiments, the viral RNA is selected from HIV I, HIV II, Ebola virus, Dengue virus, Orthomyxovirus, Hepevirus, and/or Type A, Type B, Type C (e.g., RNA-HCV with A), hepatitis D and E viruses.

[0009] In some embodiments, the reaction vessel includes a body and a lid. In some embodiments, the cover is removable. In some embodiments, the reaction vessel takes the form of a pipette tip. In some embodiments, the reaction vessel is part of an array of reaction vessels. In some embodiments, the reaction vessel portion of the reaction vessel array can be individually addressed by the liquid handling device. In some embodiments, the reaction vessel contains two or more thermal zones. In some embodiments, the reaction vessel is hermetically sealed, optionally hermetically sealed.

[0010] In some embodiments, the denaturation temperature is about 90°C to 100°C, or about 92°C to 95°C. In some embodiments, the extension temperature is about 35°C to 72°C, or about 45°C to 65°C. In some embodiments, the duration of denaturation is less than or equal to 30 seconds. In some embodiments, the extension duration is less than or equal to 30 seconds.

[0011] In some embodiments, the target RNA is not subjected to concentration before providing a reaction vessel containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification. In some embodiments, when a reaction vessel containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification is provided, the biological sample is not subjected to RNA extraction. In some embodiments, the method further includes adding lysis to the reaction vessel before or during the provision of the reaction vessel containing the biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification. Step of the agent. In some embodiments, the lysing agent comprises a buffer. In some embodiments, the target RNA is released from the biological sample during one or more cycles of the primer extension reaction.

[0012] In some embodiments, the biological sample is a biological fluid from a subject. In some embodiments, the biological sample is selected from exhaled breath, blood, urine, feces, saliva, cerebrospinal fluid, and sweat.

[0013] In some embodiments, DNA amplification is performed via polymerase chain reaction. In some embodiments, the polymerase chain reaction is a nested polymerase chain reaction. In some embodiments, DNA amplification is linear amplification. In some embodiments, the amplification produces a detectable amount of DNA product with a cycle threshold (Ct) of less than 50, less than 40, less than 30, less than 20, less than 10, or less than 5, indicating the presence of the target RNA in the biological sample. In some embodiments, the amplification produces a detectable amount of DNA product that indicates the presence of the target RNA in the biological sample in a period of 30 minutes or less, 20 minutes or less, or 10 minutes or less. In some embodiments, the amplification is not emulsion-based.

[0014] In some embodiments, the recipient is the treating physician, pharmaceutical company, or subject. In some embodiments, the reaction mixture is subjected to multiple cycles of a primer extension reaction to produce a detectable amount of amplified DNA product indicative of the presence of the target RNA in the biological sample for 30 cycles or less, 20 cycles or less , Or 10 cycles or more

less. In some embodiments, the detection is optical detection, electrostatic detection, or electrochemical detection. In some embodiments, the method includes providing a reaction vessel containing a biological sample and reagents necessary for performing reverse transcription amplification and deoxyribonucleic acid (DNA) amplification.

[0015] In some embodiments, the information is output as a report. In some embodiments, the report is an electronic report. In some embodiments, this information is output to an electronic display.

[0016] In another aspect, the present invention provides a method of amplifying a target nucleic acid present in a biological sample obtained from a subject. The method includes: (a) providing a reaction vessel containing a biological sample and reagents necessary for nucleic acid amplification to obtain a reaction mixture, the reagent containing (i) deoxyribonucleic acid (DNA) polymerase and optionally reverse transcription Enzymes, and (ii) a primer set for the target nucleic acid; and (b) subjecting the reaction mixture in the reaction vessel to multiple series of primer extension reactions to generate amplification products indicating the presence of the target nucleic acid in the biological sample, each The series includes two or more of the following cycles: (i) incubating the reaction mixture under denaturation conditions characterized by denaturation temperature and duration of denaturation, followed by (ii) incubating the reaction mixture under denaturation conditions characterized by extension temperature and extension duration The reaction mixture is incubated under conditions, wherein a single series is different from at least one other single series of the plurality of series in terms of denaturing conditions and/or extension conditions.

[0017] In some embodiments, the target nucleic acid is a ribonucleic acid. In some embodiments, the reagents are necessary for reverse transcription amplification in parallel with DNA amplification. In some embodiments, the amplification product is an amplified deoxyribonucleic acid product. In some embodiments, the biological sample is not purified in (a). In some embodiments, the method further comprises subjecting the target nucleic acid to one or more denaturing conditions before (b). In some embodiments, the one or more denaturation conditions are selected from a denaturation temperature profile and a denaturant.

[0018] In some embodiments, the biological sample is diluted. This may help minimize suppression. In some embodiments, the biological sample is concentrated. This may help increase or otherwise improve sensitivity.

[0019] In some embodiments, the method further comprises subjecting the target nucleic acid to one or more denaturing conditions between the first series and the second series of the plurality of series of primer extension reactions. In some embodiments, at least any one, at least any two, and at least any three of the ramping rate between denaturation temperature and extension temperature, denaturation temperature, denaturation duration, extension temperature, and extension duration For one or at least any four, each single series is different. In some embodiments, each individual series differs in terms of the ramp rate between the denaturation temperature and the extension temperature, the denaturation temperature, the denaturation duration, the extension temperature, and the extension duration.

[0020] In some embodiments, the plurality of series of primer extension reactions include a first series and a second series, the first series includes more than 10 cycles, and each cycle of the first series includes (i) at about 92C Incubate the reaction mixture at -95C for no more than 30 seconds, and then (ii) incubate the reaction mixture at about 35C-65C for no more than 1 minute. The second series includes more than 10 cycles, and each cycle of the second series includes: (i) Incubate the reaction mixture at about 92C-95C for no more than 30 seconds, and then (ii) incubate the reaction mixture at about 40C-60C for no more than 1 minute.

[0021] In some embodiments, compared to a single series of primer extension reactions under similar denaturation and extension conditions, the multiple series of primer extension reactions produce indicators in a biological sample with a lower cycle threshold There is a detectable amount of amplification product of the target nucleic acid. In some embodiments, the method further includes, before (b), placing the biological sample at 90°. . The pre-heating at the pre-heating temperature to 100C does not exceed the pre-heating time of 10 minutes, 2 minutes or 1 minute. In some embodiments, the preheating temperature is 92C to 95C. In some embodiments, the pre-heating duration does not exceed about 30 seconds.

[0022] In another aspect, the present invention provides a system for amplifying target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. In one embodiment, the system includes: (a) an input module, which receives a user request to amplify target RNA in a biological sample; (b) an amplification module, which responds to the user request: receives a reaction in a reaction vessel

A mixture, the reaction mixture containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification, the reagents including (1) reverse transcriptase, (ii) DNA polymerase, and (iii) A primer set for the target RNA; and, subjecting the reaction mixture in the reaction vessel to multiple cycles of primer extension reactions to generate amplified DNA products indicating the presence of the target RNA. Each cycle includes: (i) denaturing the reaction mixture Incubate at temperature for a denaturation duration of less than or equal to 60 seconds, and then (ii) incubate the reaction mixture at an extension temperature for an extension duration of less than or equal to 60 seconds, thereby amplifying the target RNA; and (c) operationally An output module coupled to the amplification module, wherein the output module outputs information about the target RNA or DNA product to the recipient.

[0023] In another embodiment, the system includes: (a) an input module that receives a user request to amplify target RNA in a biological sample; (b) an amplification module that responds to the user request: (i ) Receive a reaction mixture in a reaction vessel, the reaction mixture comprising a biological sample that has been obtained from a subject and reagents necessary for reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising: (1) Reverse transcriptase, and (2) a primer set for the target RNA; and (ii) subject the reaction mixture to multiple cycles of primer extension reactions to produce a detectable amount indicative of the presence of the target RNA in the biological sample Amplify the DNA product; (iii) detect the amount of amplified DNA product of (iii); and (iv) output information about the amount of amplified DNA product to the recipient, where it is used to complete (i)-(iv) The amount of time is less than or equal to about 30 minutes; and (c) an output module operatively coupled to the amplification module, wherein the output module transmits information to the recipient. In some embodiments, the output module is an electronic display. In some embodiments, the electronic display includes a user interface. In some embodiments, the output module is a communication interface operably coupled to a computer network.

[0024] In another aspect, the present invention provides a system for amplifying a target nucleic acid present in a biological sample obtained from a subject. The system includes: (a) an input module, which receives a user request for amplification of target RNA in a biological sample; (b) an amplification module, which responds to the user request: receives a reaction mixture in a reaction vessel, the reaction mixture containing Biological samples and reagents necessary for nucleic acid amplification, the reagents comprising (i) DNA polymerase and optional reverse transcriptase, and (ii) a primer set for the target nucleic acid; and, making the reaction mixture in the reaction vessel Go through multiple series of primer extension reactions to generate amplification products that indicate the presence of target nucleic acid in the biological sample. Each series includes two or more cycles as follows: (i) When the denaturation temperature and the denaturation duration are Incubate the reaction mixture under characteristic denaturing conditions, and then (ii) incubate the reaction mixture under extension conditions characterized by extension temperature and extension duration, where a single series differs from all in terms of denaturation and/or extension conditions. At least one other single series of the plurality of series; and (c) an output module operatively coupled to the amplification module, wherein the output module outputs information about the nucleic acid or amplification product to the recipient.

[0025] In another aspect, the present invention provides a computer-readable medium containing machine-executable code. When the machine-executable code is executed by one or more computer processors, the implementation of augmentation exists directly from the recipient. The method that the subject obtains the target ribonucleic acid (RNA) in the biological sample. In one embodiment, the method includes: (a) providing a reaction vessel containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagent Containing (i) reverse transcriptase, (ii) DNA polymerase, and (iii) a primer set for target RNA; and (b) subjecting the reaction mixture in the reaction vessel to multiple cycles of primer extension reaction to generate an indication of presence The amplified DNA product of the target RNA, each cycle includes: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, and then (ii) incubating the reaction mixture at an extension temperature for less An extension duration of 60 seconds or more, thereby amplifying the target RNA.

[0026] In another embodiment, the method includes: (a) receiving a biological sample that has been obtained from a subject; (b) providing a biological sample containing the biological sample and for performing reverse transcription amplification and optionally deoxyribonucleic acid ( DNA) a reaction container with reagents necessary for amplification to obtain a reaction mixture, which contains (1) reverse transcriptase, and (ii) a primer set for the target RNA; (c) makes the reverse transcriptase

The mixture should undergo multiple cycles of primer extension reactions to produce a detectable amount of amplified DNA product indicating the presence of target RNA in the biological sample; (d) detecting the amount of DNA product of (c); and (e) Information on the amount of DNA product is output to the recipient, where the amount of time for completing (a)-(e) is less than or equal to about 30 minutes.

[0027] In another aspect, the present invention provides a computer-readable medium containing machine-executable code, the machine-executable code when executed by one or more computer processors, the implementation of amplification exists in the subject The method of obtaining target nucleic acid in biological sample by the author. In one embodiment, the method includes: (a) providing a reaction vessel containing a biological sample and reagents necessary for nucleic acid amplification to obtain a reaction mixture, the reagent containing (i) a DNA polymerase and optionally a reversal Transcriptase, and (ii) a primer set for the target nucleic acid; and (b) subject the reaction mixture in the reaction vessel to multiple series of primer extension reactions to generate amplification products from the target nucleic acid, each series including two or More cycles as follows: (i) incubate the reaction mixture under denaturation conditions characterized by denaturation temperature and duration of denaturation, and then (ii) incubate the reaction under extension conditions characterized by extension temperature and extension duration Mixtures, wherein the single series is different from at least one other single series of the plurality of series in terms of denaturation conditions and/or extension conditions.

[0028] Another aspect of the present invention provides a system for amplifying a target nucleic acid in a biological sample obtained from a subject. The system may include an electronic display screen that includes a user interface displaying graphical elements that can be accessed by a user to execute an amplification scheme for amplifying target nucleic acids in a biological sample. The system may also include a computer processor coupled to the electronic display screen and programmed to execute the augmentation scheme when the user selects a graphical element. The amplification scheme may include subjecting a reaction mixture containing a biological sample and reagents necessary for nucleic acid amplification to a plurality of series of primer extension reactions to generate an amplification product indicative of the presence of the target nucleic acid in the biological sample. Each series of primer extension reactions may include two or more cycles as follows: incubate the reaction mixture under denaturation conditions characterized by denaturation temperature and duration of denaturation, and then incubate the reaction mixture at a temperature characterized by extension temperature and duration of denaturation. The reaction mixture is incubated under extension conditions. In terms of denaturation conditions and/or extension conditions, a single series may be different from at least one other single series of the plurality of series.

[0029] In some embodiments, the amplification protocol may further include selecting a primer set for the target nucleic acid. In some embodiments, the reagent may include a deoxyribonucleic acid (DNA) polymerase, an optional reverse transcriptase, and a primer set for the target nucleic acid. In some embodiments, the user interface may display multiple graphical elements. Each graphic element can be associated with a given amplification scheme among multiple amplification schemes. In some embodiments, each graphic element can be associated with a disease. A given amplification protocol among the plurality of amplification protocols may be directed toward determining the presence of a disease in the subject. In some embodiments, the disease may be associated with viruses such as, for example, RNA viruses or DNA viruses. In some embodiments, the virus may be selected from the group consisting of human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza virus, hepatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Hepatitis G virus, Epstein-Barr virus, Mononucleosis virus, Cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus and varicella virus. In some embodiments, the influenza virus may be selected from H1N1 virus, H3N2 virus, H7N9 virus, and H5N1 virus. In some embodiments, the gland The virus can be adenovirus type 55 (ADV55) or adenovirus type 7 (ADV7). In some embodiments, the hepatitis C virus may be RNA-hepatitis C virus with A (RNA-HCV). In some embodiments, the disease may be associated with pathogenic bacteria (e.g., Mycobacterium tuberculosis) or pathogenic protozoa (e.g., Plasmodium).

[0030] In some embodiments, the target nucleic acid may be associated with a disease. In some embodiments, the amplification protocol may be directed to determining the presence of disease based on the presence of amplification products. In some embodiments, the disease can be associated with viruses such as

For example, RNA viruses or DNA viruses are related. In some embodiments, the virus may be selected from the group consisting of human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza virus, hepatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Hepatitis G virus, Epstein-Barr virus, Mononucleosis virus, Cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus and varicella virus. In some embodiments, the influenza virus may be selected from H1N1 virus, H3N2 virus, H7N9 virus, and H5N1 virus. In some embodiments, the adenovirus may be adenovirus type 55 (ADV55) or adenovirus type 7 (ADV7). In some embodiments, the hepatitis C virus may be RNA-hepatitis C virus with A (RNA-HCV). In some embodiments, the disease may be associated with pathogenic bacteria (e.g., Mycobacterium tuberculosis) or pathogenic protozoa (e.g., Plasmodium).

[0031] From the following detailed description, other aspects and advantages of the present invention will become apparent to those skilled in the art, in which only illustrative embodiments of the present invention are shown and described. It will be realized that the present disclosure is capable of other and different embodiments, and its several details are capable of modification in many obvious respects, all without departing from the present disclosure. Accordingly, the drawings and descriptions will naturally be regarded as illustrative rather than restrictive.

[0032] Incorporation by reference

[0033] All publications, patents, and patent applications mentioned in this document are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference.

Description of the drawings

[0034] The novel features of the present invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed descriptions and drawings (also referred to as "figures" in this text) illustrating illustrative embodiments in which the principles of the present invention are utilized. In the attached picture:

[0035] FIG. 1 is a schematic diagram depicting an exemplary system.

[0036] FIGS. 2A and 2B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 1.

[0037] FIGS. 3A and 3B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 1.

4A and 4B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 2.

[0039] FIG. 5 is a graph depicting the results of the exemplary nucleic acid amplification reaction described in Example 3.

6A and 6B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 4.

[0041] FIGS. 7A and 7B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 4.

[0042] FIGS. 8A and 8B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 4.

9A and 9B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 4.

10A and 10B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 4.

[0045] FIG. 11 is a graph depicting the results of the exemplary nucleic acid amplification reaction described in Example 5.

[0046] FIG. 12 is a graph depicting the results of the exemplary nucleic acid amplification reaction described in Example 5.

[0047] FIG. 13 is a graph depicting the results of the exemplary nucleic acid amplification reaction described in Example 7.

14 is a graph depicting the results of the exemplary nucleic acid amplification reaction described in Example 9.

15A and 15B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 10.

[0050] FIGS. 16A and 16B are graphs depicting the results of the exemplary nucleic acid amplification reaction described in Example 10.

[0051] FIG. 17 is a graph depicting the results of the nucleic acid amplification reaction described in Example 11.

18 is a graph depicting the results of the nucleic acid amplification reaction described in Example 12.

[0053] FIGS. 19A and 19B are graphs depicting the results of the nucleic acid amplification reaction described in Example 13.

20 is a graph depicting the results of the nucleic acid amplification reaction described in Example 14.

21 is a graph depicting the results of the nucleic acid amplification reaction described in Example 15.

[0056] FIGS. 22A and 22B are graphs depicting the results of the nucleic acid amplification reaction described in Example 17.

[0057] FIGS. 23A, 23B, and 23C are graphs depicting the results of the nucleic acid amplification reaction described in Example 18.

[0058] FIGS. 24A and 24B are graphs depicting the results of the nucleic acid amplification reaction described in Example 19.

[0059] FIGS. 25A and 25B are graphs depicting the results of the nucleic acid amplification reaction described in Example 19.

[0060] FIGS. 26A and 26B are graphs depicting the results of the nucleic acid amplification reaction described in Example 20.

[0061] FIG. 27 is a graph depicting the results of the nucleic acid amplification reaction described in Example 21.

[0062] FIG. 28A is a schematic diagram of an exemplary electronic display with an exemplary user interface.

[0063] FIG. 28B is a schematic diagram of an exemplary electronic display with an exemplary user interface.

Detailed Description of the Invention

[0065] Although various embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art can think of many changes, changes and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0066] As used in the present and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0067] As used herein, the terms "amplifying" and "amplification" are used interchangeably, and generally refer to the production of one or more copies or "amplification products" of a nucleic acid. The term "DNA amplification" generally refers to the production of one or more copies of a DNA molecule or "amplified DNA product". The term "reverse transcription amplification" generally refers to the production of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template by the action of reverse transcriptase.

[0068] As used herein, the term "cycle threshold" or "Ct" generally refers to a cycle during thermal cycling in which the increase in detectable signal due to amplification products reaches a statistically significant higher than The level of the background signal.

[0069] As used herein, the terms "denaturing" and "denaturation" are used interchangeably, and generally refer to the complete or partial unwinding of the helical structure of a double-stranded nucleic acid, and in some cases Unwinding of the secondary structure of single-stranded nucleic acids. Denaturation can include the inactivation of pathogen cell walls or virus coats, as well as the inactivation of inhibitor proteins. Conditions under which denaturation can occur include "denaturation temperature" and "denaturation duration" "denaturation temperature" generally refers to the temperature at which denaturation is allowed to occur "denaturation duration" generally refers to the amount of time allocated for denaturation to occur.

[0070] As used herein, the term "elongation" generally refers to the incorporation of nucleotides into nucleic acids in a template-guided manner. Extension can occur by means of an enzyme such as, for example, polymerase or reverse transcriptase. Conditions under which extension can occur include "extension temperature" and "extension duration" "extension temperature" generally refers to the temperature at which extension is allowed to occur "extension duration" generally refers to the amount of time allotted for extension to occur.

[0071] As used herein, the term "nucleic acid" generally refers to a polymerized form of nucleotides (deoxyribonucleotides (dNTP) or ribonucleotides (rNTP)) or analogs thereof of any length. Nucleic acids can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of nucleic acids include coding or non-coding regions of DNA, RNA, genes or gene fragments, one or more loci determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant nucleic acid, protein

Support nucleic acid, plasmid, vector, isolated DNA of arbitrary sequence, isolated RNA of arbitrary sequence, nucleic acid probes and primers. The nucleic acid may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be performed before or after the nucleic acid assembly. The nucleotide sequence of a nucleic acid can be interrupted by non-nucleotide components. The nucleic acid can be further modified after polymerization, for example by coupling or binding to a reporter.

[0072] As used herein, the term "primer extension reaction" generally refers to the denaturation of a double-stranded nucleic acid, in which a primer is combined with one or both strands of the denatured nucleic acid, followed by extension of the primer.

[0073] As used herein, the term "reaction mixture" generally refers to a composition containing reagents necessary to complete nucleic acid amplification (eg, DNA amplification, RNA amplification), and non-limiting examples of such reagents include A primer set specific to target RNA or target DNA, DNA produced by reverse transcription of RNA, DNA polymerase, reverse transcriptase (for example, for reverse transcription of RNA), suitable buffers (including zwitterionic buffers) ), cofactors (e.g., divalent and monovalent cations), dNTPs and other enzymes (e.g., Uracidine-DNA glycosylase (UNG), etc.). In some cases, the reaction mixture may also include one or more reporter agents.

[0074] A "reporter" as used herein generally refers to a composition that produces a detectable signal, the presence or absence of which can be used to detect the presence or absence of an amplified product.

[0075] As used herein, the term "target nucleic acid" generally refers to a nucleic acid molecule having a certain nucleotide sequence in the starting population of nucleic acid molecules, its presence, amount, and/or sequence, or one or more of them. Changes in items need to be measured. The target nucleic acid can be any type of nucleic acid, including DNA, RNA and their analogs. "Target ribonucleic acid (RNA)" as used herein generally refers to a target nucleic acid as RNA. "Target deoxyribonucleic acid (DNA)" as used herein generally refers to a target nucleic acid as DNA.

[0076] As used herein, the term "subject" generally refers to an entity or medium that has testable or detectable genetic information. The subject can be a human or an individual. The subject may be a vertebrate, such as a mammal. Non-limiting examples of mammals include mice, apes, humans, domestic animals, sport animals, and pets. Other examples of subjects include food, plants, soil, and water.

[0077] In one aspect, the present invention provides a method of amplifying target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The method includes: (a) providing a reaction vessel containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagent comprising (i) reverse transcription An enzyme, (ii) a DNA polymerase, and (iii) a primer set for the target RNA; and (b) subjecting the reaction mixture in the reaction vessel to multiple cycles of primer extension reactions to generate amplified DNA indicating the presence of the target RNA The product, each cycle includes: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, and then (ii) incubating the reaction mixture at an elongation temperature for an extension duration of less than or equal to 60 seconds Time to amplify the target RNA.

[0078] In another aspect, the present invention provides a method of amplifying target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The method includes: (a) receiving a biological sample that has been obtained from a subject; (b) providing a reaction containing the biological sample and reagents necessary for reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification Container to obtain a reaction mixture, the reagent comprising (i) reverse transcriptase and (ii) a primer set for the target RNA; (c) subjecting the reaction mixture to multiple cycles of primer extension reactions to produce an indication of the presence in the biological sample A detectable amount of the amplified DNA product of the target RNA; (d) detecting the amount of the amplified DNA product of (c); and (e) outputting information about the amount of amplified DNA product to the recipient, where it is used to complete The amount of time (a)-(e) is less than or equal to about 30 minutes.

[0079] In one aspect, the present invention provides a method of amplifying a target nucleic acid present in a biological sample obtained from a subject. The method includes: (a) providing a reaction volume containing a biological sample and reagents necessary for nucleic acid amplification

Reactor to obtain a reaction mixture, the reagent comprising (i) deoxyribonucleic acid (DNA) polymerase and optionally reverse transcriptase, and (ii) a primer set for the target nucleic acid; and (b) making the reaction in the reaction vessel The mixture undergoes multiple series of primer extension reactions to generate amplification products that indicate the presence of target nucleic acids in the biological sample, and each series includes two or more cycles as follows: (i) When the denaturation temperature and the denaturation duration are The reaction mixture is incubated under characteristic denaturing conditions, followed by (ii) incubating the reaction mixture under extension conditions characterized by extension temperature and extension duration, wherein a single series is different from the multiple in terms of denaturation and/or extension conditions. At least one other single series in the series.

[0080] In any of the various aspects, nucleic acid from a biological sample obtained from the subject is amplified. In some cases, the biological sample is obtained directly from the subject. A biological sample obtained directly from a subject generally refers to a biological sample that, after it is obtained from the subject, has not undergone further processing other than any means used to collect the biological sample from the subject for further processing. For example, blood is obtained directly from the subject by the following steps: entering the circulatory system of the subject, removing the blood from the subject (for example, through a needle), and allowing the removed blood to enter the reservoir. The reservoir may contain reagents (eg, anticoagulants) so that the blood sample can be used for further analysis. In another example, a swab can be used to access epithelial cells on the oropharyngeal surface of the subject. After obtaining the biological sample from the subject, the swab containing the biological sample may be contacted with a fluid (eg, buffer) to collect the biological fluid from the swab.

[0081] In some embodiments, the biological sample has not been purified when it is provided in the reaction vessel. In some embodiments, when the biological sample is provided into the reaction vessel, the nucleic acid of the biological sample has not been extracted. For example, when the biological sample is provided into the reaction container, the RNA or DNA in the biological sample may not be extracted from the biological sample. Furthermore, in some embodiments, the target nucleic acid (eg, target RNA or target DNA) present in the biological sample may not be concentrated before the biological sample is provided into the reaction vessel.

[0082] Any suitable biological sample containing nucleic acid can be obtained from the subject. The biological sample may be a solid substance (for example, biological tissue), or may be a fluid (for example, biological fluid). Generally, biological fluids may include any fluids associated with living organisms. Non-limiting examples of biological samples include blood (or components of blood-for example, white blood cells, red blood cells, platelets) obtained from any anatomical location of the subject (e.g., tissue, circulatory system, bone marrow), Cells, skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, tissue fluid derived from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, obtained from any anatomical location, Placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, cavity fluid, sputum, pus, microbiota (micropiota), meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine Fluid, gastric juice and digestive juice, tears, eye fluid, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, nails, skin cells, plasma, nasal swabs or nasopharyngeal lotion, spinal fluid, Cord blood, emphatic fluid and/or other excreta or body tissues.

[0083] The biological sample can be obtained from the subject by any means known in the art. Non-limiting examples of means for obtaining biological samples directly from a subject include: entering the circulatory system (for example, intravenously or intraarterially via a syringe or other needle), collecting secreted biological samples (for example, feces, urine) , Sputum, saliva, etc.), surgery (e.g., biopsy), wiping (e.g., oral swab, oropharyngeal swab), pipetting and breathing. In addition, the biological sample can be obtained from any anatomical site where the biological sample is desired in the subject.

[0084] In any of the various aspects, the target nucleic acid is amplified to generate an amplified product. The target nucleic acid may be target RNA or target DNA. Where the target nucleic acid is a target RNA, the target RNA can be any type of RNA, including the types of RNA described elsewhere herein. In some embodiments, the target RNA is viral RNA. In some embodiments, the viral RNA may be pathogenic to the subject. Non-limiting examples of pathogenic viral RNA include human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza virus (e.g., H1N1, H3N2,

H7N9 or H5N1), hepatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus (for example, RNAHCV virus with A), hepatitis D virus, hepatitis E virus, hepatitis G virus, EB virus, Mononucleosis virus, cytomegalovirus, SARS virus, West Nile virus, polio virus and measles virus.

[0085] In the case where the target nucleic acid is target DNA, the target DNA can be any type of DNA, including the types of DNA described elsewhere herein. In some embodiments, the target DNA is viral DNA. In some embodiments, the viral DNA may be pathogenic to the subject. Non-limiting examples of DNA viruses include herpes simplex virus, variola virus, adenovirus (eg, type 55 adenovirus, type 7 adenovirus), and varicella virus (eg, fowlpox). In some cases, the target DNA may be bacterial DNA. Bacterial DNA can be derived from bacteria that are pathogenic to the subject, for example, Mycobacterium tuberculosis, a bacterium known to cause tuberculosis. In some cases, the target DNA may be DNA from a pathogenic protozoan (eg, one or more protozoa of the Plasmodium type that can cause malaria).

[0086] In any of the various aspects of the present invention, a biological sample obtained from a subject is provided with reagents necessary for nucleic acid amplification in a reaction vessel to obtain a reaction mixture. Any suitable reaction vessel can be used. In some embodiments, the reaction vessel includes a body, which may include an inner surface, an outer surface, an open end, and an opposite closed end. In some embodiments, the reaction vessel may include a lid. The lid may be configured to contact the main body at its open end so that the open end of the reaction vessel is closed when contact is made. In some cases, the lid is permanently associated with the reaction vessel so that it remains attached to the reaction vessel in the open and closed configuration. In some cases, the lid is removable so that when the reaction vessel is opened, the lid is separated from the reaction vessel. In some embodiments, the reaction vessel can be sealed, optionally hermetically sealed.

[0087] The reaction vessel can have different sizes, shapes, weights, and configurations. In some examples, the reaction vessel may be a circular or elliptical tube. In some embodiments, the reaction vessel may be rectangular, square, diamond, circular, oval, or triangular. The reaction vessel may be of regular shape or irregular shape. In some embodiments, the closed end of the reaction vessel may have a tapered, rounded, or flat surface. Non-limiting examples of reaction vessel types include tubes, holes, capillaries, cartridges, dishes, centrifuge tubes, or pipette tips. The reaction vessel can be constructed of any suitable material, and non-limiting examples of such materials include glass, metal, plastic, and combinations thereof.

[0088] In some embodiments, the reaction vessel is part of an array of reaction vessels. The reaction vessel array is especially useful for automated methods and/or for processing multiple samples simultaneously. For example, the reaction vessel may be a well of a microplate composed of many wells. In another example, the reaction vessel may be contained in a hole of a thermal block of a thermal cycler, wherein the thermal cycle block includes a plurality of holes each capable of receiving a sample container. The array of reaction vessels may include any suitable number of reaction vessels. For example, the array may include at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 35, 48, 96, 144, 384 or more reaction vessels. The reaction container portion of the reaction container array can also be individually addressed by the fluid processing device, so that the fluid processing device can correctly identify the reaction container and dispense appropriate fluid materials into the reaction container. The fluid handling device can be used to automate the addition of fluid materials to the reaction vessel.

[0089] In some embodiments, the reaction vessel may contain multiple hot zones. The hot zone in the reaction vessel can be realized by exposing different areas of the reaction vessel to different temperature cycling conditions. For example, the reaction vessel may include an upper hot zone and a lower hot zone. The upper hot zone can receive biological samples and reagents necessary to obtain a reaction mixture for nucleic acid amplification. The reaction mixture can then undergo a first thermal cycling scheme. After a desired number of cycles, for example, the reaction mixture may slowly but continuously leak from the upper hot zone to the lower hot zone. In the lower hot zone, the reaction mixture then undergoes a desired number of cycles of a second thermal cycle scheme that is different from the scheme of the upper hot zone. These strategies may be particularly useful when using nested PCR to amplify DNA. In some embodiments, the hot zone can be reacted with the aid of the thermosensitive layered material in the reaction vessel.

Produced in the container. In such cases, the heating of the thermosensitive layering material can be used to release the reaction mixture from one hot zone to the next. In some embodiments, the reaction vessel includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more hot zones.

[0090] In some embodiments, a reaction vessel containing a hot zone can be used to process biological samples before nucleic acid amplification. For example, the lysing agent can be added to the first hot zone of the reaction vessel before adding the biological sample and the reagents necessary for nucleic acid amplification. When the biological sample and the reagent are added to the reaction container containing the lysing agent, a reaction mixture capable of lysing the species (for example, cells or virus particles) in the biological sample is obtained. Alternatively, the lysing agent can be added to the first hot zone of the reaction mixture at the same time as the biological sample and reagent. Subjecting the first hot zone to temperature conditions suitable for the action of the lysing agent can be used to lyse the cells and virus particles in the biological sample in the first hot zone so that the nucleic acid in the biological sample is released into the reaction mixture. After lysis, the reaction mixture can then be allowed to enter the second hot zone of the reaction vessel for amplification of the released nucleic acid using the amplification method described herein.

[0091] Where a lysing agent is required, any suitable lysing agent known in the art can be used, including commercially available lysing agents. Non-limiting examples of lysing agents include Tris-HCl, EDTA, detergents (e.g., Triton X-100, SDS), lysozyme, glucolase, protease E, viral endolysin, exolysin ), zymolose, lyticase, proteinase K, endolysin and exolysin from phage, endolysin from phage PM2, from Bacillus subtilis (B. subtilis) Endolysin of bacteriophage PBSX, endolysin from Lactobacillus prophage Lj928, Lj965, phage 15Phiadh, endolysin from Streptococcus pneumoniae phage Cp-I, bifunctional peptidoglycan from Streptococcus agalactiae phage B30 Lysin, endolysin and exolysin from prophage bacteria, endolysin from Listeria phage, holin-endolysin, cell 20 lysis gene, holWMY Staphylococcus wokerii ( Staphylococcus wameri) M phage varphiWMY, Staphylococcus wameri M phage Iy5WMY of varphiWMY, and combinations thereof. In some cases, the buffer may include a lysis agent (e.g., a lysis buffer). An example of a lysis buffer is sodium hydroxide (NaOH).

[0092] Any type of nucleic acid amplification reaction known in the art can be used to amplify a target nucleic acid and generate an amplification product. In addition, the amplification of nucleic acids can be linear, exponential, or a combination thereof. Amplification can be emulsion-based or can be non-emulsion-based. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification, and multiple displacement amplification (MDA ). In some embodiments, the amplification product may be DNA. In the case of amplification of target RNA, DNA can be obtained by reverse transcription of RNA and subsequent DNA amplification can be used to generate amplified DNA products. The amplified DNA product can indicate the presence of target RNA in the biological sample. In the case of amplifying DNA, any DNA amplification method known in the art can be used. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR), variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR) (dial-out PCR), helicase-dependent PCR, nested PCR, hot-start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR PCR, overlap-extension PCR, thermal asymmetric interlaced PCR (thermal asymmetric interlaced PCR), step-down PCR) and ligase chain reaction (LCR). In some cases, DNA amplification is linear. In some cases, DNA amplification is exponential. In some cases, DNA amplification is achieved by nested PCR, which can improve the sensitivity of detecting amplified DNA products.

[0093] In various aspects, the nucleic acid amplification reactions described herein can be performed in parallel. Generally, parallel amplification reactions are amplification reactions that occur simultaneously in the same reaction vessel. Parallel nucleic acid amplification reactions can be performed as follows: for example, reagents necessary for each nucleic acid amplification reaction are included in a reaction vessel to obtain a reaction mixture, and the reaction mixture is subjected to conditions necessary for each nucleic acid amplification reaction. For example, reverse transcription amplification and DNA amplification can be performed in parallel as follows

Row: Provide the necessary reagents for these two amplification methods in the reaction vessel to form and obtain a reaction mixture, and subject the reaction mixture to conditions suitable for performing the two amplification reactions. DNA produced by reverse transcription of RNA can be amplified in parallel to produce amplified DNA products. Any suitable number of nucleic acid amplification reactions can be performed in parallel. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or More nucleic acid amplification reactions.

[0094] Advantages of performing nucleic acid amplification reactions in parallel may include rapid switching between coupled nucleic acid amplification reactions. For example, the target nucleic acid (for example, target RNA, target DNA) can be extracted or released from the biological sample during the heating phase of parallel nucleic acid amplification. In the case of target RNA, for example, a biological sample containing the target RNA can be heated and the target RNA can be released from the biological sample. The released target RNA can immediately begin reverse transcription (through reverse transcription amplification) to produce complementary DNA. The complementary DNA can then be amplified immediately, usually on the order of a few seconds. The short time interval between the release of the target RNA from the biological sample and the reverse transcription of the target RNA into complementary DNA can help minimize the effects of inhibitors in the biological sample that may hinder reverse transcription and/or DNA amplification.

[0095] In any of these various aspects, a primer set for the target nucleic acid can be used to perform a nucleic acid amplification reaction. The primer set usually contains one or more primers. For example, the primer set may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more primers. In some cases, the primer set may include primers for different amplification products or different nucleic acid amplification reactions. For example, the primer set may include a first primer and a second primer that is complementary to a nucleic acid strand product. The first primer is necessary to generate a first strand of a nucleic acid product that is complementary to at least a portion of the target nucleic acid, and the second primer is to generate a nucleic acid product that is complementary to at least a portion of the target nucleic acid. At least a portion of the first strand of the product is complementary to the second strand of the nucleic acid product.

[0096] For example, the primer set can be directed to the target RNA. The primer set may include a first primer that can be used to generate a first strand of a nucleic acid product complementary to at least a portion of the target RNA. In the case of a reverse transcription reaction, the first strand of the nucleic acid product may be DNA. The primer set may also include a second primer that can be used to generate a second strand of a nucleic acid product that is complementary to at least a portion of the first strand of the nucleic acid product. In the case of a reverse transcription reaction performed in parallel with DNA amplification, the second strand of the nucleic acid product may be a strand of a nucleic acid (eg, DNA) product complementary to the DNA strand generated from the RNA template.

[0097] If necessary, any suitable number of primer sets can be used. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more primer sets can be used. When multiple primer sets are used, one or more primer sets may each correspond to a specific nucleic acid amplification reaction or amplification product.

[0098] In some embodiments, a DNA polymerase is used. Any suitable DNA polymerase can be used, including commercially available DNA polymerases. DNA polymerase generally refers to an enzyme that can incorporate nucleotides into DNA strands in a template-bound manner. Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso Polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase Enzymes, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragments, and their variants and modifications Products and derivatives. For certain hot-start polymerases, a denaturation step of 2 minutes to 10 minutes at 94C-95C may be required, which may change the heat distribution according to different polymerases.

[0099] In some embodiments, reverse transcriptase is used. Any suitable reverse transcriptase can be used. Reverse transcriptase generally refers to an enzyme that can incorporate nucleotides into the DNA strand when combined with an RNA template. Non-limiting examples of reverse transcriptase include HIV1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and their variants, modified products, and derivatives.

[0100] In various aspects, primer extension reactions are used to generate amplification products. The primer extension reaction usually includes the following

Cycling: Incubation of the reaction mixture at the denaturation temperature for a period of denaturation duration, and incubation of the reaction mixture at the extension temperature for an extended duration of time.

[0101] The denaturation temperature may vary depending on, for example, the specific biological sample being analyzed, the specific source of the target nucleic acid in the biological sample (eg, viral particles, bacteria), the reagents used, and/or the desired reaction conditions. For example, the denaturation temperature may be about 80°C to about 110°C. In some examples, the denaturation temperature may be about 90°C to about 100°C. In some examples, the denaturation temperature may be about 90°C to about 97°C. In some examples, the denaturation temperature may be about 92°C to about 95°C. In still other examples, the denaturation temperature may be about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89° C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C.

[0102] The duration of denaturation may vary depending on, for example, the specific biological sample being analyzed, the specific source of target nucleic acid in the biological sample (eg, viral particles, bacteria), the reagents used, and/or the desired reaction conditions. For example, the duration of denaturation may be less than or equal to 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds. Seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the duration of denaturation may not exceed 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.

[0103] The extension temperature may vary according to, for example, the specific biological sample being analyzed, the specific source of the target nucleic acid in the biological sample (eg, viral particles, bacteria), the reagents used, and/or the desired reaction conditions. For example, the extension temperature may be about 30°C to about 80°C. In some examples, the extension temperature may be about 35°C to about 72°C. In some examples, the extension temperature may be about 45°C to about 65°C. In some examples, the extension temperature may be about 35°C to about 65°C. In some examples, the extension temperature may be about 40°C to about 60°C. In some examples, the extension temperature may be about 50°C to about 60°C. In still other examples, the extension temperature may be about 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44° C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69° C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C.

[0104] The extension duration may vary depending on, for example, the specific biological sample being analyzed, the specific source of the target nucleic acid in the biological sample (eg, viral particles, bacteria), the reagents used, and/or the desired reaction conditions. For example, the extended duration may be less than or equal to 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds Seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the extension duration may not exceed 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.

[0105] In any of the multiple aspects, multiple cycles of primer extension reactions can be performed. Any suitable number of cycles can be performed. For example, the number of cycles performed may be less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 cycles. The number of cycles performed may depend on, for example, the number of cycles (e.g., cycle threshold (Ct) necessary to obtain a detectable amplification product (e.g., a detectable amount of amplified DNA product indicating the presence of target RNA in a biological sample)). )). For example, the number of cycles necessary to obtain a detectable amplification product (eg, a DNA product that indicates a detectable amount of target RNA in a biological sample) may be less than about or about 100 cycles, 75 cycles, 70 cycles. Cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 cycles, 10 cycles, or 5 cycles. Furthermore, in some embodiments, a detectable amount of amplification product (eg, a detectable amount of DNA product indicating the presence of target RNA in a biological sample) may be less than 100, 75, 70, 65, 60, 55, 50. , 45, 40, 35, 30, 25, 20, 15, 10 or 5 cycle threshold (Ct) is obtained.

[0106] The time required for the amplification to produce a detectable amount of the amplified product indicating the presence of the amplified target nucleic acid may depend on the biological sample from which the target nucleic acid is obtained, the specific nucleic acid amplification reaction to be performed, and the desired amplification Specific cycle of reaction

The number changes. For example, the amplification of the target nucleic acid can be in 120 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less. , 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less time period produces a detectable amount of amplification indicating the presence of the target nucleic acid Increase product.

[0107] In some embodiments, the amplification of the target RNA can be performed in 120 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less. Short, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less for a period of time to indicate the presence of the target A detectable amount of RNA amplified DNA product.

[0108] In some embodiments, the reaction mixture can be subjected to multiple series of primer extension reactions. A single series of the plurality of series may include a plurality of cycles of a specific primer extension reaction that is characterized by, for example, specific denaturation and extension conditions as described elsewhere herein. Generally, for example, in terms of denaturation conditions and/or extension conditions, each single series is different from at least one other single series of the plurality of series. For example, in terms of any one, two, three, or all four of the denaturation temperature, the denaturation duration, the extension temperature, and the extension duration, a single series may be different from another single series of the plurality of series. In addition, the plurality of series may include any number of individual series, for example, at least about or about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more individual series.

[0109] For example, multiple series of primer extension reactions may include a first series and a second series. The first series, for example, may include more than ten cycles of primer extension reactions, where each cycle of the first series includes (i) incubating the reaction mixture at about 92°C to about 95°C for no more than 30 seconds, and then (Ii) Incubate the reaction mixture at about 35°C to about 65°C for no more than about one minute. The second series, for example, may include more than ten cycles of primer extension reactions, where each cycle of the second series includes (i) incubating the reaction mixture at about 92°C to about 95°C for no more than 30 seconds, and then (Ii) Incubate the reaction mixture at about 40°C to about 60°C for no more than about 1 minute. In this specific example, the first and second series differ in their extension temperature conditions. However, this example is not intended to be limiting, as any combination of different extension and denaturation conditions can be used.

[0110] In some embodiments, ramp time (ie, the time it takes for a thermal cycler to transition from one temperature to another temperature) and/or ramp rate are important factors in amplification. For example, the temperature and time required for amplification to produce a detectable amount of amplified product indicative of the presence of the target nucleic acid may vary according to the ramp rate and/or ramp time. The ramp rate can affect the temperature and time used for amplification.

[0111] In some cases, the ramp time and/or ramp rate may be different between cycles. However, in some cases, the ramp time and/or ramp rate between cycles may be the same. The ramp time and/or ramp rate can be adjusted based on the sample being processed.

[0112] In some cases, for example, the ramp time between different temperatures can be determined according to the nature of the sample and the reaction conditions. The precise temperature and incubation time can also be determined according to the nature of the sample and the reaction conditions. In some embodiments, a single sample can be processed (eg, subjected to amplification conditions) multiple times using multiple thermal cycles, each thermal cycle being different in, for example, ramp time, temperature, and/or incubation time. The best or optimal thermal cycle can then be selected for that particular sample. This provides a robust and efficient method of tailoring thermal cycling for the specific sample or combination of samples being tested.

[0113] In some embodiments, the target nucleic acid can be subjected to denaturing conditions before the primer extension reaction is initiated. In the case of a plurality of series of primer extension reactions, the target nucleic acid may be subjected to denaturing conditions before performing the plurality of series, or may be subjected to denaturing conditions between the plurality of series. For example, the target nucleic acid can be subjected to denaturing conditions between the first series and the second series in the plurality of series. Non-limiting examples of such denaturation conditions include denaturation temperature distribution (e.g., one or more denaturation

Temperature) and denaturant.

[0114] The advantage of performing multiple series of primer extension reactions may be that, compared to a single series of primer extension reactions under similar denaturation and extension conditions, multiple series of methods produce indications at a lower cycle threshold. There is a detectable amount of amplification product of the target nucleic acid in the biological sample. Compared with a single series under similar denaturation and extension conditions, the use of multiple series of primer extension reactions can reduce these cycle thresholds by at least about or about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0115] In some embodiments, the biological sample may be preheated before performing the primer extension reaction. The temperature (e.g., preheating temperature) and duration (e.g., preheating duration) of preheating the biological sample may vary according to, for example, the specific biological sample being analyzed. In some examples, the biological sample may be preheated for no more than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds. In some examples, the biological sample may be preheated at a temperature of about 80°C to about 110°C. In some examples, the biological sample may be preheated at a temperature of about 90°C to about 100°C. In some examples, the biological sample may be preheated at a temperature of about 90°C to about 97°C. In some examples, the biological sample may be preheated at a temperature of about 92°C to about 95°C. In still other examples, the temperature may be at about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, Preheat biological samples at 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C.

[0116] In some embodiments, the reagents necessary for nucleic acid amplification (including those necessary for parallel nucleic acid amplification) may also include a reporter that generates a detectable signal, the presence or absence of the detectable signal The presence indicates whether the amplified product is present. The intensity of the detectable signal can be proportional to the amount of amplified product. In some cases, when the amplification product is generated by a different type of nucleic acid from the initially amplified target nucleic acid, the intensity of the detectable signal may be proportional to the amount of the initially amplified target nucleic acid. For example, in the case of amplifying target RNA by parallel reverse transcription and amplification of DNA obtained from reverse transcription, the reagents necessary for these two reactions may also include a reporter that can generate a detectable signal. The signal indicates the presence of amplified DNA product and/or amplified target RNA. The intensity of the detectable signal can be proportional to the amount of amplified DNA product and/or amplified original target RNA. The use of reporters also makes real-time amplification methods possible, including real-time PCR for DNA amplification.

[0117] The reporter can be covalently or non-covalently linked to the nucleic acid including the amplified product. Non-limiting examples of non-covalent means include ionic interactions, van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. In some embodiments, the reporter can be combined with the initial reactant, and the change in the level of the reporter can be used to detect the amplification product. In some embodiments, the reporter agent may only be detectable (or undetectable) while nucleic acid amplification is in progress. In some embodiments, optically active dyes (e.g., fluorescent dyes) can be used as reporters. Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium, acridine, proflavin, acridine orange, acridine yellow, fluorescent Coumarin (fluorcoumanin), ellipticine, daunorubicin, chloroquine, hemendomycin D, chromomycin, homidium, mithromycin, ruthenium polypyridyl, antrimethylene Anthramycin, phenanthridine and acridine, ethidium iodide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and ethidium homodimer Body-2, ethidium monoazide and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, Acridine Orange, 7-AAD, Actinomycin D, LDS751, Hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO- 3,YOYO-1, YOYO-3, TOTO-1,TOTO-3,JOJO-1, LOLO-1, BOBO-1, BOBO-3,PO-PRO-1,PO-PRO-3,BO-PRO- 1, BO-PRO-3 ,TO-PRO-1,TO-PRO-3,TO-PRO-5, JO-PRO-1,LO-PRO-1,YO-PRO-1,YO-PRO-3,

PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, 42, -43, -44, -45 (blue), SYTO-13, -16, -24 , -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 ( Orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate Esters (TRITC), rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red ( Texas Red), Phar-Red, Allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, Ethidium Homodimer I, Ethidium Homodimer II, Ethidium homodimer III, ethidium, umbelliferone, eosin, green fluorescent protein, erythrosine, coumarin, methyl coumarin, pyrene, malachite green, stilbene, fluorescent yellow, cascade Blue (cascade blue), dichlorotriazinamine fluorescein, dansyl chloride, fluorescent Optical lanthanide complexes (such as those containing europium and terbium), carboxytetrachlorofluorescein, 5 and/or 6-carboxyfluorescein (FAM), 5-(or 6-)iodoacetamide Fluorescein, 5-{[2 (and 3) -5-(acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5 and/or 6 Cyclodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1 ,3,6-trisulfonic acid trisodium salt, 3,6-disulfonic acid-4-amino-zeodicarboximide, phycobiliprotein, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.

[0118] In some embodiments, the reporter may be a sequence-specific oligonucleotide probe that is optically active when hybridized with the amplified product. Due to the specific binding of the probe to the sequence of the amplified product, the use of oligonucleotide probes can improve the specificity and sensitivity of detection. The probe may be linked to any optically active reporter (eg, dye) described herein, and may also include a quencher capable of blocking the optical activity of the associated dye. Non-limiting examples of probes that can be used as reporters include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

[0119] In some embodiments, the reporter may be an RNA oligonucleotide probe, which includes an optically active dye (eg, a fluorescent dye) and a quencher adjacent to the probe. The close proximity of the dye and the quencher can block the optical activity of the dye. The probe can bind to the target sequence to be amplified. Once the exonuclease activity of the DNA polymerase breaks the probe during amplification, the quencher is separated from the dye, and the free dye regains its optical activity, which can then be detected.

^[0120] In some embodiments, the reporter may be a molecular beacon. Molecular beacons include, for example, a quencher attached to one end of an oligonucleotide in a hairpin conformation. At the other end of the oligonucleotide is an optically active dye, for example, a fluorescent dye. In the hairpin configuration, the optically active dye and the quencher are close enough so that the quencher can block the optical activity of the dye. However, once hybridized to the amplified product, the oligonucleotide assumes a linear conformation and hybridizes to the target sequence on the amplified product. The linearization of the oligonucleotide leads to the separation of the optically active dye and the quencher, so that the optical activity is restored and can be detected. The sequence specificity of the molecular beacon to the target sequence on the amplified product can improve the specificity and sensitivity of detection.

[0121] In some embodiments, the reporter agent may be a radioactive species. Non-limiting examples of radioactive types include¹⁴C,¹²³I.¹²⁴I.¹²⁵I.¹³¹I, Tc99m,³⁵S or³H.

[0122] In some embodiments, the reporter may be an enzyme capable of generating a detectable signal. The detectable signal can be generated by the activity of the enzyme on its substrate, or on a specific substrate in the case where the enzyme has multiple substrates. Non-limiting examples of enzymes that can be used as reporters include alkaline phosphatase, horseradish peroxidase, I2-galactosidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and fluoresceinVegetarianase.

[0123] In various aspects, amplification products (eg, amplified DNA products, amplified RNA products) can be detected. The detection of the amplified product (including the amplified DNA) can be achieved by any suitable detection method known in the art. The test used

The specific type of method may depend on, for example, the specific amplification product, the type of reaction vessel used for amplification, other reagents in the reaction mixture, whether the reporter is included in the reaction mixture, and the reporter used when using the reporter The specific type of reporting agent. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, and the like. Optical detection methods include, but are not limited to, fluorescence measurement and ultraviolet-visible light absorption. Spectral detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques, such as gel electrophoresis. Electrochemical detection methods include, but are not limited to, electrochemical detection of amplified products after high-performance liquid chromatography separation of the amplified products.

[0124] In any of the multiple aspects, the time required to complete the elements of the method may vary according to the specific steps of the method. For example, the amount of time for completing the elements of the method may be about 5 minutes to about 120 minutes. In other examples, the amount of time for completing the elements of the method may be from about 5 minutes to about 60 minutes. In other examples, the amount of time for completing the elements of the method may be from about 5 minutes to about 30 minutes. In other examples, the amount of time used to complete the elements of the method may be less than or equal to 120 minutes, less than or equal to 90 minutes, less than or equal to 75 minutes, less than or equal to 60 minutes, less than or equal to 45 minutes, less than or equal to 40 minutes , Less than or equal to 35 minutes, less than or equal to 30 minutes, less than or equal to 25 minutes, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, or less than or equal to 5 minutes.

[0125] In some embodiments, information regarding the presence and/or amount of amplification products (eg, amplified DNA products) may be output to the recipient. The information about the amplified product can be output via any suitable method known in the art. In some embodiments, this information may be provided to the recipient orally. In some embodiments, this information may be provided in the report. The report can include any number of desired elements, non-limiting examples of such elements include raw data about the subject (e.g., gender, age, race, health, etc.), processed data (e.g., graphical display ( For example, graphs, charts, data tables, data summary), determined cycle threshold, calculated value of target polynucleotide starting amount) information, conclusions about the existence of target nucleic acid, diagnostic information, prognostic information, disease information, etc. , And combinations thereof. The report can be provided as a printed report (for example, a hard copy) or it can be provided as an electronic report. In some embodiments (including the case where an electronic report is provided), such information may be via a screen such as a monitor or TV, a screen operably connected to the unit for obtaining amplification products, a tablet computer screen, a mobile device screen, etc. Electronic display (for example, electronic display) output. Both printed reports and electronic reports can be stored in files or databases, respectively, so that they can be accessed for comparison with future reports.

[0126] In addition, any suitable communication medium (including, for example, a network connection, a wireless connection, or an Internet connection) may be used to send the report to a recipient at a local or remote location. In some embodiments, the report may be sent to the recipient's device, such as a personal computer, phone, tablet, or other device. The report can be viewed online, saved on the recipient's device, or printed. The report may also be transmitted by any other suitable means for transmitting information, non-limiting examples of such means include mailing a hard copy report for receiving and/or viewing by the recipient.

[0127] In addition, this information can be output to various different types of recipients. Non-limiting examples of such recipients include the subject from which the biological sample was obtained, the physician, the physician treating the subject, the clinical monitor used in the clinical trial, nurses, researchers, laboratory technicians, representatives of pharmaceutical companies , Healthcare companies, biotech companies, hospitals, human assistance organizations, healthcare administrators, electronic systems (e.g., one or more computers and/or one or more computer servers that store, for example, subjects' medical records ), public health workers, other medical personnel and other medical facilities.

[0128] In one aspect, the present invention provides a system that implements a method according to any of the methods disclosed herein. In another aspect, the present invention provides a system for amplifying target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The system includes: (a) an input module, which receives a user request for amplification of target RNA in a biological sample; an amplification module, which responds to the user request: receives a reaction mixture in a reaction vessel, the reaction mixture containing

Biological samples and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification, the reagents include (1) reverse transcriptase, (ii) DNA polymerase, and (ii) primers for target RNA Group; and, subjecting the reaction mixture in the reaction vessel to multiple cycles of primer extension reactions to generate amplified DNA products indicative of the presence of target RNA, each cycle including: (i) incubating the reaction mixture at a denaturation temperature for less than A denaturation duration of 60 seconds or less, followed by (5) incubating the reaction mixture at an extension temperature for an extension duration of less than or equal to 60 seconds, thereby amplifying the target RNA; and (c) operably coupled to the amplification The output module of the module, wherein the output module outputs information about the target RNA or DNA product to the recipient.

[0129] In another aspect, the present invention provides a system for amplifying target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The system includes: (a) an input module, which receives a user request to amplify target RNA in a biological sample; (b) an amplification module, which responds to the user request: (i) receives a reaction mixture in a reaction vessel, and The reaction mixture contains the biological sample that has been obtained from the subject and the reagents necessary for reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification. The reagents include (1) reverse transcriptase, and (2) against the target Primer set for RNA; and (ii) subjecting the reaction mixture to multiple cycles of primer extension reactions to produce a detectable amount of amplified DNA product indicating the presence of target RNA in the biological sample; (iii) detecting the amplification of (iii) Increasing the amount of DNA product; and (iv) outputting information about the amount of amplified DNA product to the recipient, wherein the amount of time for completing (i)-(iv) is less than or equal to about 30 minutes; and (c) An output module operatively coupled to the amplification module, wherein the output module transmits the information to the recipient.

[0130] In another aspect, the present invention provides a system for amplifying a target nucleic acid present in a biological sample obtained from a subject. The system includes: (a) an input module, which receives a user request for amplification of target RNA in a biological sample; (b) an amplification module, which responds to the user request: receives a reaction mixture in a reaction vessel, the reaction mixture containing Biological samples and reagents necessary for nucleic acid amplification, the reagents comprising (i) DNA polymerase and optional reverse transcriptase, and (ii) a primer set for the target nucleic acid; and, making the reaction mixture in the reaction vessel Go through multiple series of primer extension reactions to generate amplification products indicating the presence of the target nucleic acid in the biological sample. Each series includes two or more cycles as follows: (i) When the denaturation temperature and the denaturation duration are Incubate the reaction mixture under characteristic denaturing conditions, and then (ii) incubate the reaction mixture under extension conditions characterized by extension temperature and extension duration, wherein a single series is different from all in terms of denaturation and/or extension conditions. At least one other single series of the plurality of series; and (c) an output module operatively coupled to the amplification module, wherein the output module outputs information about the target RNA or DNA product to the recipient.

[0131] In another aspect, the present invention provides a system for amplifying a target nucleic acid in a biological sample obtained from a subject. The system may include an electronic display screen with a user interface displaying graphical elements that can be accessed by a user to execute an amplification protocol for amplifying target nucleic acids in a biological sample. The system may also include a computer processor (including any suitable device having a computer processor as described elsewhere herein) that is coupled to the electronic display screen and programmed to perform the expansion when the user selects the graphical element Increase program. The amplification scheme may include: subjecting a reaction mixture containing a biological sample and reagents necessary for nucleic acid amplification to multiple series of primer extension reactions to generate amplification products. The amplification product can be indicative of the presence of target nucleic acid in the biological sample. In addition, each series of primer extension reactions may include two or more cycles as follows: incubate the reaction mixture under denaturation conditions characterized by denaturation temperature and duration Incubate the reaction mixture under the extension conditions. In terms of denaturation conditions and/or extension conditions, a single series may be different from at least one other single series of the plurality of series.

[0132] In some embodiments, the target nucleic acid may be associated with a disease. For example, the disease can be related to RNA viruses or DNA viruses

turn off. Examples of viruses are provided elsewhere in this article. In some embodiments, the disease may be associated with pathogenic bacteria (eg, Mycobacterium tuberculosis) or pathogenic protozoa (eg, Plasmodium as in malaria) (including examples of such pathogens described elsewhere herein) . In some embodiments, the amplification protocol may be directed to determining the presence of the disease based on the presence of the amplification product.

[0133] In some cases, the user interface may be a graphical user interface. In addition, the user interface may include one or more graphical elements. Graphical elements may include images and/or text information, such as pictures, icons, and text. Graphical elements can have different sizes and orientations on the user interface. In addition, the electronic display screen can be any suitable electronic display, including the examples described elsewhere herein. Non-limiting examples of electronic display screens include monitors, mobile device screens, laptop computer screens, televisions, portable video game system screens, and calculator screens. In some embodiments, the electronic display screen may include a touch screen (for example, a capacitive or resistive touch screen) so that graphical elements displayed on the user interface of the electronic display screen can be selected via the user touching the electronic display screen.

[0134] In some embodiments, the amplification protocol may further include selecting a primer set for the target nucleic acid. In these cases, the primer set may be a primer set specially designed for amplifying one or more sequences of the target nucleic acid molecule. In some embodiments, the amplification scheme may further include selecting a reporter specific for one or more sequences of the target nucleic acid molecule (e.g., oligonucleotide probes containing optically active species or those described elsewhere herein. Other types of reporters). In addition, in some embodiments, the reagents may include any suitable reagents necessary for nucleic acid amplification as described elsewhere herein, such as deoxyribonucleic acid (DNA) polymerase, primer sets for target nucleic acids, and (any) Optional) reverse transcriptase.

[0135] In some implementations, the user interface may display multiple graphical elements. Each graphic element can be associated with a given amplification scheme among multiple amplification schemes. Each of the multiple amplification protocols may include a different combination of a series of primer extension reactions. However, in some cases, the user interface may display multiple graphical elements associated with the same augmentation protocol. An example of a user interface with multiple graphical elements each associated with a given amplification scheme is shown in Figure 28A. As shown in FIG. 28A, an exemplary electronic display screen 2800 associated with a computer processor includes a user interface 2801. The user interface 2801 includes the display of graphical elements 2802, 2803, and 2804. Each graphic element Graphic element can be associated with a specific augmentation scheme (for example, graphic element 2802 is "Prot. 1" and graphic element 2803 is "Prot. 2 (Prot. 2)" and the graphic element 2804 is "Prot. 4"). Once the user selects (for example, when the electronic display 2800 includes a touch screen with a user interface, the user touches) a specific graphic element, and the graphic element The associated specific augmentation scheme can be executed by the associated computer processor. For example, when the user selects the graphic element 2803, the associated computer processor executes the augmentation "scheme 2". Although in the exemplary user of FIG. 28A Only three graphical elements are shown in the interface 2801, but the user interface may have any suitable number of graphical elements. In addition, although each graphical element displayed in the user interface 2801 of FIG. 28A is only associated with one augmentation scheme, the user Each graphic element of the interface may be associated with one or more amplification schemes (for example, a series of amplification schemes), so that the associated computer processor executes a series of amplification schemes after the user interacts with the graphic element.

[0136] In some embodiments, each graphic element and/or may be associated with a disease, and a given amplification scheme in the plurality of amplification schemes may be directed toward determining the presence of the disease in the subject. Therefore, in such cases, the user can select graphic elements to run an amplification program (or a series of amplification programs) to analyze a specific disease. In some embodiments, the disease may be associated with a virus (eg, any RNA virus or DNA virus, including the examples of such viruses described elsewhere herein). Non-limiting examples of viruses include human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza virus (e.g., H1N1 virus, H3N2 virus) , H7N9 virus or

H5N1 virus), hepatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus (for example, RNA-hepatitis C virus with A (RNA-HCV)), hepatitis D virus, hepatitis E virus, Hepatitis G virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus (for example, type 55 Adenovirus (ADV 55), adenovirus type 7 (ADV 7)) and varicella virus. In some embodiments, the disease may be associated with pathogenic bacteria (eg, Mycobacterium tuberculosis) or pathogenic protozoa (eg, Plasmodium in malaria) (including examples of such pathogens described elsewhere herein).

[0137] An example of a user interface with multiple graphical elements each associated with a given amplification scheme is shown in FIG. 28B. As shown in FIG. 28B, an exemplary electronic display screen 2810 associated with a computer processor includes a user interface 2811. The user interface 2811 includes the display of graphical elements 2812, 2813, and 2814. Each graphic element can be associated with a specific disease (for example, graphic element 2812 is "Ebola", graphic element 2813 is "H1N1", and graphic element 2814 is "Hep C (hepatitis C)"). Associated with one or more amplification schemes that point to a specific disease. Once the user chooses (e.g., When the electronic display screen 2810 includes a touch screen with a user interface, the user touches a specific graphic element, and the specific amplification scheme associated with the disease associated with the graphic element can be executed by the associated computer processor. For example, when the user interacts with the graphic element 2812, the associated computer processor executes one or more amplification schemes associated with analyzing the Ebola virus. Although only three graphical elements are shown in the exemplary user interface 2811 of FIG. 28B, the user interface may have any suitable number of graphical elements each corresponding to a variety of diseases. In addition, although each graphic element displayed in the user interface 2811 of FIG. 28B is only associated with one disease, each graphic element of the user interface can be associated with one or more diseases, so that the associated computer processor is When the user selects the graphic element, a series of amplification schemes are executed (for example, various individual amplification schemes directed to a specific disease). For example, the graphic element may correspond to the Ebola virus and the H1N1 virus, such that the selection of the graphic element causes the associated computer processor to execute an amplification scheme for both the Ebola virus and the H1N1 virus.

[0138] In various aspects, the system includes an input module that receives a user request to amplify a target nucleic acid (eg, target RNA, target DNA) present in a biological sample obtained directly from a subject. Any suitable module capable of receiving such user requests can be used. The input module may include, for example, a device including one or more processors. Non-limiting examples of devices that include a processor (e.g., computer processor) include: desktop computers, laptop computers, tablet computers (e.g., Apple® iPad, Samsung® Galaxy Tab), cellular phones, smart phones (e.g., Apple® iPhone. Android® supported phones), personal digital assistants (PDAs), video game consoles, TVs, music playback devices (for example, Apple® iPod), video playback devices, pagers, and calculators. The processor may be associated with one or more controllers, computing units, and/or other units of the computer system, or may be embedded in firmware when needed. If implemented in software, the routine (or program) can be stored in any computer readable memory such as RAM, ROM, flash memory, magnetic disk, laser disk or other storage media. Likewise, the software can be delivered to the device via any known delivery method, including, for example, via a communication channel, such as a telephone line, the Internet, a local intranet, a wireless connection, etc., or via a portable medium, such as a computer readable Disks, flash drives, etc. Each step can be implemented as various blocks, operations, tools, modules, or technologies, and the latter can be implemented in hardware, firmware, software, or any combination thereof. When implemented in hardware, some or all of these blocks, operations, technologies, etc. can be used in, for example, custom integrated circuits (QC), application-specific integrated circuits (ASIC), field programmable logic arrays (FPGA), programmable logic arrays ( PLA) etc.

[0139] In some embodiments, the input module is configured to receive a user request to perform target nucleic acid amplification. The input module may be directly (for example, through an input device such as a keyboard, mouse, or touch screen operated by the user) or indirectly (for example,

Receive user requests via wired or wireless connections, including via the Internet. The input module can provide the user's request to the amplification module via the output electronics. In some embodiments, the input module may include a user interface (UI), such as a graphical user interface (GUI), which is configured to enable a user to provide a request to amplify a target nucleic acid. The GUI may include text, graphics, and/or audio components. The GUI may be provided on an electronic display, which includes a display of a device containing a computer processor. These displays may include resistive or capacitive touch screens.

[0140] Non-limiting examples of users include subjects from which biological samples are obtained, medical personnel, clinicians (e.g., doctors, nurses, laboratory technicians), laboratory personnel (e.g., hospital laboratory technicians, research scientists, Pharmaceutical scientists), clinical monitors of clinical trials, or other users in the healthcare industry.

[0141] In various aspects, the system includes an amplification module for performing a nucleic acid amplification reaction on the target nucleic acid or a portion thereof in response to a user request received by the input module. The amplification module may be capable of performing any of the methods described herein, and may include a fluid handling device, one or more thermal cyclers, and a device for receiving one or more reaction vessels (for example, holes of a thermal block for thermal cycling) Devices, detectors capable of detecting amplification products (for example, optical detectors, spectral detectors, electrochemical detectors), and for outputting to the recipient the presence and/or amount of amplification products (amplified DNA products) Information (e.g., raw data, processed data, or any other type of information described herein). In some cases, the amplification module may include a device with a computer processor as described elsewhere herein, and may also be able to analyze the raw data obtained from the test with the aid of suitable software. Furthermore, in some embodiments, the amplification module may include input electronics necessary to receive instructions from the input module and may include output electronics necessary to communicate with the output module.

[0142] In some embodiments, one or more of the steps of providing materials to the reaction vessel, amplifying nucleic acids, detecting amplified products, and outputting information can be automated by the amplification module. In some embodiments, automated operations may include the use of one or more fluid handlers and associated software. Several commercially available fluid handling systems can be used to run the automated operations of these processes. Non-limiting examples of such fluid handlers include fluid handlers from Perkin-Elmer, Caliper Life Sciences>Tecan>Eppendorf>Apricot Design, and Velocity 11.

[0143] In some embodiments, the amplification module may include a real-time detection instrument. Non-limiting examples of these instruments include real-time PCR thermal cyclers, ABI PRISM® 7000 sequence detection system, ABI PRISM® 7700 sequence detection system, Applied Biosystems 7300 real-time PCR system, Applied Biosystems 7500 real-time PCR system, App 1 ied Biosystems 7900HT Fast real-time PCR system (all from App 1 ied Biosystems); LightCycler'M system (Roche Diagnostics GmbH); Mx3000P, m real-time PCR system, Mx3005P, m real-time PCR system and Mx4000® Multiplex Quantitative PCR System (Mx4000® Multiplex Quantitative PCR System) ) (Stratagene, La Jolla, Calif.); and Smart Cycler System (Cepheid, distributed by Fisher Scientific). In some embodiments, the amplification module may include another automated instrument, for example, COBAS@Amp 1 iPrep/COBAS® TaqMan® system (Roche Molecular Systems) ^TIGRIS DTS system (Hologic Gen-Probe, San Diego, CA), PANTHER system (Hologic Gen-Probe, San Diego, CA) >BD MAX'" system (Becton Dickinson ), GeneXpert system (Cepheid), Filmairay® (BioFire Diagnostics) system, iCubate system, IDBox system (Luminex)> EncompassMDxTM (Rheonix) system, Liat'" Aanlyzer (IQuum) system, Biocart is's Molecular Diagnostic Platform system, Enigma ® ML system (Enigma Diagnostics), T2Dx® system

(T2Biosystems), Verigeiie® system (NanoSphere), Great Basin's Diagnostic System, UnyveroTM system (Curetis), PanNAT system (Micronics) or Spartan'M RX system (Spartan Bioscience).

[0144] In various aspects, the system includes an output module operably connected to the amplification module. In some embodiments, the output module may include a device having a processor for the input module as described above. The output module may include an input device as described herein, and/or may include input electronics for communicating with the amplification module. In some embodiments, the output module may be an electronic display, and in some cases, the electronic display includes a UI. In some embodiments, the output module is a communication interface operatively coupled to a computer network such as the Internet. In some embodiments, the output module can use any suitable communication medium (including a computer network, a wireless network, a local intranet, or the Internet) to transmit information to a recipient at a local or remote location. In some embodiments, the output module can analyze the data received from the amplification module. In some cases, the output module includes a report generator capable of generating a report and transmitting the report to the recipient, where the report contains any information regarding the amount and/or presence of amplification products as described elsewhere herein. In some embodiments, the output module may automatically transmit information in response to information received from the amplification module, for example in the form of raw data or data analysis performed by software included in the amplification module. Alternatively, the output module can transmit information after receiving user instructions. The information transmitted by the output module can be electronically Check it out or print it out by the printer.

[0145] One or more of the input module, the amplification module, and the output module may be included in the same device, or may include one or more of the same components. For example, the amplification module may also include an input module, an output module, or both. In other examples, the device including the processor may be included in the input module or the output module. The user can use the device to request amplification of the target nucleic acid, and can also be used as a tool for transmitting information about the amplified product to the recipient. In some cases, the processor-containing device can be included in all three modules, so that the processor-containing device can also be used to control the instrument included in the amplification module or any other module (eg, thermal cycler, detection Device, fluid processing device), provide instructions to the instrument, and receive information returned from the instrument.

[0146] An exemplary system for amplifying a target nucleic acid according to the methods described herein is shown in FIG. 1. The system includes a computer 101 that can serve both as part of the input module and as part of the output module. The user puts the reaction container 102 containing the reaction mixture to be subjected to nucleic acid amplification into the amplification module 104. The amplification module includes a thermal cycler 105 and a detector 106. The input module 107 includes a computer 101 and a related input device 103 (for example, a keyboard, a mouse, etc.). The input device 103 can accept a user's request for amplification of the target nucleic acid in the reaction mixture. The input module 107 communicates the user's request to the amplification module 104, and nucleic acid amplification is started in the thermal cycler 105. As the amplification proceeds, the detector 106 of the amplification module detects the amplified product. Information about the amplification product (for example, raw data obtained by the detector) is transmitted from the detector 106 back to the computer 101, and the computer 101 also serves as a component of the output module 108. The computer 101 receives the information from the amplification module 104, performs any additional operations on the information, and then generates a report containing the processed information. Once the report is generated, the computer 101 then transmits the report via the computer network interface 110 via a computer network (eg, intranet, Internet), in hard copy via a printer 111, or via an electronic display 112 operably connected to the computer 101 Deliver to its ultimate recipient 109. In some cases, the electronic display 112

[0147] In one aspect, the present invention provides a computer-readable medium containing machine-executable code, which when executed by one or more processors, implements a method according to any of the methods disclosed herein. In another aspect, the present invention provides a computer-readable medium containing machine-executable code, which, when executed by one or more computer processors, implements amplification that exists in a biological sample directly obtained from a subject. Target ribonucleic acid (RNA) method, the method includes: (a)

A reaction vessel containing a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagents including (i) reverse transcriptase, (ii) DNA polymerization Enzymes, and (iii) a primer set for the target RNA; and (b) subject the reaction mixture in the reaction vessel to multiple cycles of primer extension reactions to generate amplified DNA products indicative of the presence of the target RNA, each cycle including ( i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, and then (ii) incubating the reaction mixture at an extension temperature for an extension duration of less than or equal to 60 seconds, thereby amplifying the target RNA.

[0148] In another aspect, the present invention provides a computer-readable medium containing machine-executable code, which, when executed by one or more computer processors, implements amplification that exists in biological data directly obtained from a subject. A method for target ribonucleic acid (RNA) in a sample, the method comprising: (a) receiving a biological sample that has been obtained from a subject; (b) providing a biological sample containing the biological sample and for performing reverse transcription amplification and optionally deoxyribose A reaction vessel containing reagents necessary for nucleic acid (DNA) amplification to obtain a reaction mixture, which contains (1) reverse transcriptase and (ii) a primer set for target RNA; (c) primer extension that allows the reaction mixture to undergo multiple cycles React to produce a detectable amount of amplified DNA product indicating the presence of the target RNA in the biological sample; (d) detect the amount of DNA product of (c); and (e) output information about the amount of DNA product to the receiver Or, where the amount of time to complete (a)-(e) is less than or equal to about 30 minutes.

[0149] In one aspect, the present invention provides a computer-readable medium containing machine executable code, which, when executed by one or more computer processors, implements amplification in a biological sample obtained from a subject The target ribonucleic acid (RNA) method, the method includes: (a) providing a reaction vessel containing a biological sample and reagents necessary for nucleic acid amplification to obtain a reaction mixture, the reagents including (1) DNA polymerase and optionally Reverse transcriptase, and (ii) a primer set for the target nucleic acid; and (b) subject the reaction mixture in the reaction vessel to multiple series of primer extension reactions to generate amplification products from the target nucleic acid, each series including two or More cycles as follows: (i) incubating the reaction mixture under denaturation conditions characterized by denaturation temperature and duration of denaturation, and then (ii) incubating the reaction mixture under extension conditions characterized by extension temperature and extension duration, Wherein in terms of denaturation conditions and/or extension conditions, the single series is different from at least one other single series in the plurality of series.

[0150] The computer-readable medium may take many forms, including, but not limited to, a tangible (or non-transitory) storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, etc., which can be used to implement calculation steps, processing steps, etc., for example. Volatile storage media include dynamic memory, such as the main memory of a computer. Tangible transmission media include coaxial cables; copper wire and optical fiber, including the wires that contain the bus in a computer system. The carrier wave transmission medium may take the form of electric or electromagnetic signals or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communication. Therefore, common forms of computer readable media include, for example: floppy disks, flexible disks, hard disks, tapes, any other magnetic media, CD-ROM, DVD or DVD-ROM, any other optical media, punched paper tape, Any other physical storage media with hole patterns, RAM, PROM and EPROM, FLASH-EPROM, any other memory chips or cassettes, carrier waves that transmit data or instructions, cables or links that transmit these carriers, or computers Any other medium from which program code and/or data can be read. Many of these computer-readable media forms can participate in carrying one or more sequences of one or more instructions to a processor for execution.

Example

[0151] Example 1: Amplification and detection of nucleic acids in virus stock samples and biological samples

[0152] Amplification and detection experiments were performed to compare the results obtained from virus standard samples and biological samples. The biological sample containing the RNA viral pathogen and the standard sample of the viral pathogen are subjected to amplification conditions, thereby amplifying the RNA of the pathogen. Correct

A set of experiments were performed for each of the H3N2 and H1N1 (2007) influenza viruses. Each biological sample was obtained directly from the subject via an oropharyngeal swab. Each virus standard sample was obtained as a serial dilution of the stock solution containing the virus. The concentration of H3N2 and H1N1 (2007) is 10⁶IU/mL. For H5N1 and H1N1 (2007), the dilutions of 1/2, 1/20, 1/200, 1/2000, and 1/2000 were amplified. In each experimental group, a negative control (for example, a sample that does not contain viral RNA) is also amplified.

[0153] Put 5 microliters of each sample in 25 uL with the reagents necessary for reverse transcription of viral RNA and the reagents necessary to complete the amplification of complementary DNA obtained from reverse transcription (for example, parallel nucleic acid amplification) Combine in the reaction tube. The reagents necessary for reverse transcription and DNA amplification are provided as a commercially available premix (for example, Qiagen One-Step RT-PCR or OneStep RT-qPCR kit), which contains reverse transcriptase (for example, Sensiscript and Omniscript transcriptase), DNA polymerase (for example, HotStarTaq DNA polymerase), and dNTP. In addition, the reaction tube also contains a TaqMan probe that contains FAM dye for detecting the amplified DNA product. In order to generate amplified DNA products, each reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions, which included 5 minutes at 95°C, followed by 20 minutes at 45°C, and then at 95°C Next 2 minutes, followed by 40 cycles of 5 seconds at 95°C and 30 seconds at 55°C. During the incubation period, the amplification product is detected.

[0154] The amplification results of H3N2 are graphically shown in Figure 2 (Figure 2A corresponds to each virus standard sample, and Figure 2B corresponds to biological samples), and the amplification results of H1N1 (2007) are graphically shown in Figure 3 ( Figure 3A corresponds to each virus standard sample, and Figure 3B corresponds to a biological sample). The recorded fluorescence of the FAM dye is plotted against cycle number.

[0155] As shown in FIG. 2A, each H3N2 virus standard sample showed a detectable signal relative to the negative control, and the Ct value ranged from 18 to 32. As shown in Figure 2B, each virus H3N2 biological sample showed a detectable signal relative to the negative control, and the Ct value ranged from 29-35.

[0156] As shown in FIG. 3A and except for the 1/20000 dilution, each H1N1 (2007) virus standard sample showed a detectable signal relative to the negative control, and the Ct value range was 24-35. As shown in Figure 3B, each H1N1 (2007) biological sample showed a detectable signal relative to the negative control, and the Ct value ranged from 28-35.

[0157] Overall, the data shown in Figures 2 and 3 show that the tested virus can be detected through the amplified DNA product at concentrations as low as 50 IU/mL and in the 4-log concentration range. , And has good sensitivity, the cycle threshold does not exceed about 40. In addition, the data also shows that the detection of viral RNA obtained from a biological sample obtained from a subject can also be detected in a similar manner.

[0158] Example 2: Amplification and detection of viral nucleic acid in different buffer systems

[0159] Perform amplification and detection experiments to compare the results obtained using different buffer systems for amplification. A set of experiments was performed for two different buffer systems (S1 and S2). S1 buffer contains zwitterionic buffer and BSA, while S2 buffer contains zwitterionic buffer and sodium hydroxide. A set of H5N1 influenza virus standard samples obtained as a serial dilution of the stock solution containing the virus was used to complete the experiment for each buffer. The concentration of H5N1 is 10⁶IU/mL. Amplify the 1/2, 1/20, 1/200, 1/2000, 1/20000, 1/200000 dilutions and negative control.

[0160] Put 5 microliters of each sample in 25 uL containing the reagents necessary for reverse transcription of viral RNA and the reagents necessary to complete the amplification of complementary DNA obtained from reverse transcription (for example, parallel nucleic acid amplification) Combine in the reaction tube. The necessary reagents for reverse transcription and DNA amplification include reverse transcriptase, DNA polymerase, dNTP and appropriate S1 or S2 buffer. In addition, the reaction tube also contains a TaqMan probe, which contains a FAM dye for detecting the amplified DNA product. To generate amplified DNA products, each reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions. The protocol included 5 minutes at 95°C, followed by 20 minutes at 45°C, and then at 95°C. °C for 2 minutes, followed by 95 °C for 5

Seconds and 40 cycles of 30 seconds at 55C. During the incubation period, the amplification product is detected.

[0161] The amplification result of the buffer system S1 is graphically shown in FIG. 4A, and the amplification result of the buffer system S2 is graphically shown in FIG. 4B. The recorded fluorescence of the FAM dye is plotted against cycle number.

[0162] As shown in FIG. 4A, each virus standard sample amplified in the buffer system S1 shows a detectable signal relative to the negative control, and the Ct value ranges from 25 to 36. As shown in Fig. 4B, each virus standard sample amplified in buffer S2 showed a detectable signal relative to the negative control, and the Ct value range was 25-35.

[0163] Overall, the data shown in Figure 4 shows that at concentrations as low as 50 IU/mL and in the 5-log concentration range, the tested virus can be detected through the amplified DNA product, and has Good sensitivity, the cycle threshold does not exceed about 40. In addition, the data also shows that using different buffer systems can achieve similar amplification results.

[0164] Example 3: Amplification and detection of hepatitis B virus (HBV) in plasma samples

[0165] An amplification experiment was performed to determine the robustness of the amplification method for detecting target nucleic acid in a biological sample. Amplification reactions were performed on diluted human plasma samples containing hepatitis B virus (HBV) of different concentrations (for example, 50 infectious units/ml (IU/mL), 200 IU/mL, 2000 IU/mL, 20000 IU/mL). HBV is a DNA virus that replicates via RNA intermediates. HBV is detectable via direct PCR of DNA viruses. In addition to multiple samples of the negative control (for example, plasma that does not contain HBV), multiple samples at various concentrations (n = 2-4) were also tested.

[0166] Put each sample of 2.5UL with the reagents necessary for reverse transcription of RNA and the reagents necessary to complete the amplification of complementary DNA obtained from reverse transcription (for example, parallel nucleic acid amplification) in a 50UL reaction tube To obtain the reaction mixture. The reagents necessary for reverse transcription and DNA amplification are provided as a commercially available premix (for example, Qiagen One-Step RT-PCR or One-Step RT-qPCR kit), which contains reverse transcriptase (for example, Sensiscript and Omniscript transcriptase), DNA polymerase (for example, HotStarTaq DNA polymerase), and dNTP. In addition, the mixture also contains a TaqMan probe that contains FAM dye for detecting the amplified DNA product. The reaction mixture also contains a zwitterionic buffer and Uracidine-DNA glycosylase (UNG) to prevent the inhibitory effect of amplification inhibitors found in plasma. Incubate each reaction mixture in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions, which includes 1 minute at 94 °C, then 10 minutes at 50 °C, then 2 minutes at 94 °C, and then proceed At 94. . 50 cycles of 5 seconds and 35 seconds at 58°C. During the incubation period, the amplification product is detected.

[0167] The amplification results are graphically shown in FIG. 5, and the determined Ct values are listed in Table 1. In Figure 5, the recorded relative fluorescence unit (RFU) of the FAM dye is plotted against the number of cycles. As shown in Figure 5 and Table 1, HBV can be detected at each concentration tested, and the cycle threshold ranges from 28.99 to 39.39. Generally, a higher concentration of samples corresponds to a lower cycle threshold.

[0168] In general, the data shown in Figure 5 and Table 1 show that at concentrations as low as 50IU/mL (the lowest value tested), HBV can be detected through amplified DNA products with good The sensitivity of the cycle threshold does not exceed about 40. Although the highest concentration tested (20000IU/mL) is 400 times more concentrated than the lowest concentration tested (50IU/mL), for the lower concentration, the cycle threshold is only about 25% higher, which indicates that the amplification scheme as a whole It is robust.

[0169] Table 1: Ct results of the experiment in Example 3

<td>[0170]</td><td>Sample serial number</td><td>IU/mL</td><td>Ct</td>

<td></td><td>1</td><td>2000</td><td>33.09</td>

<td></td><td>2</td><td>50</td><td>39.39</td>

<td></td><td>3</td><td>2.00E+04</td><td>29</td>

<td>[0171]</td><td>4</td><td>2000</td><td>32.97</td>

<td>5</td><td>200</td><td>35.51</td>

<td>6</td><td>2000</td><td>33.07</td>

<td>7</td><td>2.00E+04</td><td>30.03</td>

<td>8</td><td>200</td><td>35.78</td>

<td>9</td><td>50</td><td>37.91</td>

<td>10</td><td>2.00E+04</td><td>29.37</td>

<td>11</td><td>200</td><td>35.73</td>

<td>12</td><td>2.00E+04</td><td>28.99</td>

[0172] Example 4: Pre-heating the biological sample and a series of amplification reactions before amplifying the nucleic acid in the biological sample [0173] Perform amplification experiments to determine the effect of pre-heating the biological sample on detection sensitivity, and also determine The effect of using multiple series of amplification reactions on detection sensitivity.

[0174] Prepare 20 25UL reaction mixtures, each containing 1UL pathogenic species, and complete appropriate nucleic acid amplification reactions (for example, reverse transcription and DNA amplification of RNA species, and DNA amplification of DNA species) Necessary reagents and TaqMan probe containing FAM dye. Four reaction mixtures contained H1N1 (2007) (ie, RNA virus), four reaction mixtures contained H3N2 (ie, RNA virus), four reaction mixtures contained H1N1 (2009), and four reaction mixtures contained tuberculosis (TB) (Ie bacterial samples), and the four reaction mixtures contained Aleutian disease virus (ADV) (ie DNA virus) 0H1N1 (2007), H1N1 (2009), H3N2 and ADV pathogenic species obtained from subjects Oropharyngeal swab. TB was obtained from bacterial stocks.

[0175] Various combinations of pre-heating and amplification protocols were used and summarized in Table 2. For the first reaction mixture of various pathogenic species, the pathogenic species was preheated at 95C for 10 minutes before being added to the reaction mixture. After the pathogenic species were added to the reaction mixture, the reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions, which included a temperature of 95°C for 2 minutes, followed by a temperature of 95°C. . 40 cycles for 5 seconds and 30 seconds at 55 °C. During the incubation period, the amplification product is detected. These reaction mixtures are called PH-1 mixtures.

[0176] For the second reaction mixture of each pathogenic species, the pathogenic species was preheated at 50 C for 30 minutes before being added to the reaction mixture. After the pathogenic species were added to the reaction mixture, the reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions, which included 2 minutes at 95C, followed by 5 seconds at 95C and 55C 40 cycles of 30 seconds. During the incubation period, the amplification product is detected. These reaction mixtures are called PH2 mixtures.

[0177] For the third reaction mixture of each pathogenic species, the pathogenic species is not preheated before being added to the reaction mixture. Incubate these reaction mixtures in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions, which includes 1 minute at 95C, then 10 minutes at 55C, then 2 minutes at 95C, followed by 5 seconds at 95C and 40 cycles of 30 seconds at 55C. During the incubation period, the amplification product is detected. These reaction mixtures are called PTC-1 mixtures.

[0178] For the fourth reaction mixture of each pathogenic species, the pathogenic species is not preheated before being added to the reaction mixture. These reaction mixtures are subjected to a protocol that includes multiple series of amplification reactions, each series including multiple cycles of denaturation and extension conditions. Incubate the reaction mixture in a real-time PCR thermal cycler according to this protocol, which includes 1 minute at 95 C, followed by series 1 (5 seconds at 95C, 20 seconds at 60-50C (in 1C/cycle steps) Decrease), and 10 cycles at 60C for 10 seconds), then at 95C for 2 minutes, followed by 40 cycles of series 2 (5 seconds at 95C, 30 seconds at 55C). Series 1 and Series 2 differ in their extension temperature and extension duration. During incubation

Perform detection of amplified products. These reaction mixtures are called PTC-2 mixtures.

Table 2: Experimental conditions of Example 4

<td>Reaction mixture type</td><td>Program</td>

<td>PH-1</td><td>The pathogenic species was preheated at 95°C for 10 minutes before being added to the reaction mixture, followed by 2 minutes at 95Y, (5 seconds at 95°C, 30 seconds at 55°C) X 40 cycles</td>

<td>PH-2</td><td>The pathogenic species were preheated at 50% for 30 minutes before being added to the reaction mixture, followed by 2 minutes at 95°C, (5 seconds at 95°C, 30 seconds at 55°C) X 40 cycles</td>

<td>PTC-1</td><td>At 95. 1 minute at ©, 10 minutes at 55H, then 95. (: 2 minutes, (5 seconds at 95°C, 30 seconds at 55°C) x 40 cycles</td>

<td>PTC-2</td><td>At 95 for 1 minute, (5 seconds at 95°C, 20 seconds at 60-50°C (step down at 1. (7 cycles), 10 seconds at 60 gas) X 10 cycles, then 2 minutes at 95°C, (5 seconds at 95°C, 30 seconds at 55°C) x 40 cycles</td>

[0181] The results of various pathogenic types are shown graphically in Figure 6 (H1N1 (2007)), Figure 7 (H3N2), Figure 8 (H1N1 (2009)), Figure 9 (TB) and Figure 10 (ADV) . Item A in each of FIGS. 6 to 10 represents the results obtained for the reaction mixtures PHT and PH-2, and item B in each of FIGS. 6 to 10 represents the results obtained for the reaction mixtures PTCT and PTC-2 the result of. The Ct values determined for each experiment are summarized in Table 3. The Ct value could not be determined for the PHT and PH-2ADV reaction mixture, which corresponds to the data shown in Figure 10A.

[0182] According to the data shown in Table 3, the Ct values between PHT and PH-2 reaction mixtures are very similar, which indicates that pathogenic species (or biological samples containing pathogenic species) can be preheated under a series of conditions, To obtain similar detection sensitivity. In addition, the PTCT reaction mixture has a Ct value similar to the Ct value determined for the PHT and PH-2 reaction mixture. PTC1 is similar to PHT/PH-2, the difference is that PTCT does not include a pre-heating step. Therefore, a comparison of PTCT data with PHT/PH-2 data indicates that preheating the pathogenic species before providing it to the reaction mixture may not be necessary to obtain results with good sensitivity. However, in some cases where TB and ADV samples are used, preheating may be worse than no preheating.

[0183] However, for all pathogenic species tested, the Ct value of PTC-2 was lower than the Ct value of any of PHT, PH-2, or PTCT. Comparison of PTCT and PTC-2 data shows that subjecting the reaction mixture to multiple series of amplification reactions (each series includes multiple cycles of denaturation and extension conditions) can improve detection sensitivity.

Table 3: Ct results of the experiment in Example 4

[0185]

<td>Types of</td><td>sample</td><td>PH-1 (Ct)</td><td>PH-2 (Ct)</td><td>PTC-1 (Ct)</td><td>PTC-2 (Ct)</td>

<td>RNA virus</td><td>H1N1(2007)</td><td>27</td><td>30</td><td>28</td><td>22</td>

<td>RNA virus</td><td>H3N2</td><td>34</td><td>33</td><td>32</td><td>23</td>

<td>RNA virus</td><td>H1N1(2009)</td><td>32</td><td>32</td><td>32</td><td>24</td>

<td>DNA bacteria</td><td>TB</td><td>34</td><td>32</td><td>26</td><td>20</td>

<td>DNA virus</td><td>ADV</td><td>-</td><td>-</td><td>36</td><td>30</td>

Example 5: Multiplexing of samples (Multiplexing)

[0187] Perform amplification and detection experiments to benchmark various amplification schemes and determine whether multiplexing can be achieved. Biological samples containing RNA (for example, H1N1 (2007), H1N1 (2009), H3N2) or DNA (for example, ADV, human Boca virus (HBoV) viral pathogens or DNA bacterial pathogens (for example, TB)) are subjected to various Amplification conditions. With the exception of TB samples from bacterial stocks, each biological sample was obtained directly from the subject via an oropharyngeal swab. Combine 1 microliter of each sample in a 25uL reaction tube with the reagents necessary for nucleic acid amplification and detection of amplified products as described herein to obtain a reaction mixture.

[0188] In order to evaluate the multiplexing ability of the amplification scheme, three reaction mixtures (each containing H3N2, ADV or a mixture of H3N2 and ADV) were incubated in a real-time PCR thermal cycler according to the amplification scheme. The protocol included 2 minutes at 94°C, 20 minutes at 45C, 1 minute at 94C, followed by 50 cycles of 5 seconds at 94C and 35 seconds at 55C. During the incubation period, the amplification product is detected.

[0189] The experimental results are shown graphically in FIG. 11 and in Table 4 below. As shown in Figure 11, H3N2 and TB can be similarly detected when combined together or when the other is not present. When ADV is not present, a Ct value of 26.03 is recorded for the H3N2 reaction mixture, and when H3N2 is not present, a Ct value of 30.5 is recorded for the ADV reaction mixture. When H3N2 and ADV are combined into a single reaction mixture, Ct values of 26 (H3N2) and 30 (ADV) are obtained. The combined reaction mixture has almost the same Ct value compared to the single-component reaction mixture. The results show that multiplexing can be achieved with good sensitivity, and both RNA and DNA species can be detected.

[0190] Table 4: The results of the H3N2 and ADV multiplexing experiment in Example 5

<td>Types of</td><td>sample</td><td>Ct</td>

<td>RNA virus</td><td>H3N2</td><td>26.03</td>

<td>DNA virus</td><td>ADV</td><td>30.5</td>

<td>RNA and DNA viruses</td><td>H3N2 and ADV</td><td>26 (H3N2) and 30 (ADV)</td>

[0192] In another experiment to evaluate the multiplexing ability of the amplification scheme, in a real-time PCR thermal cycler according to the amplification scheme (including 2 minutes at 95C, followed by 5 seconds at 95C and 30 seconds at 55C) Three reaction mixtures (each containing one of H3N2, TB, or a mixture of H3N2 and TB) were incubated. During the incubation period, the amplification product is detected.

[0193] The experimental results are graphically shown in FIG. 12 and shown in Table 5 below. As shown in Figure 12, H3N2 and TB can be similarly detected when combined together or when the other is not present. When TB is not present, a Ct value of 32 is recorded for the H3N2 reaction mixture, and when H3N2 is not present, a Ct value of 32 is recorded for the TB reaction mixture. When H3N2 and TB are combined into a single reaction mixture, Ct values of 29 (H3N2) and 30 (TB) are obtained. The combined reaction mixture has a similar Ct value compared to the single component reaction mixture. The results show that multiplexing can be achieved with good sensitivity, and in the multiplexing scheme, both RNA and DNA species can be detected.

[0194]

[0195] Table 5: Results of the H3N2 and TB multiplexing experiment in Example 5

<td>Types of</td><td>sample</td><td>Ct</td>

<td>RNA virus</td><td>H3N2</td><td>32</td>

<td>DNA virus</td><td>TB</td><td>32</td>

<td>RNA and DNA viruses</td><td>H3N2 and TB</td><td>29 (H3N2) and 30 (TB)</td>

[0196]

[0197] Example 6: Standardization of multiple series of amplification reactions Amplification and detection experiments were performed to standardize various amplification schemes including multiple series of amplification reactions. Subjecting biological samples containing RNA (for example, H1N1 (2007), H1N1 (2009), H3N2) or DNA (for example, ADV, human Boca virus (HBoV) viral pathogens, or DNA bacterial pathogens (for example, TB)) to various Amplification conditions. With the exception of TB samples from bacterial stocks, each biological sample was obtained directly from the subject via an oropharyngeal swab. Combine 1 microliter of each sample in a 25uL reaction tube with the reagents necessary for nucleic acid amplification and detection of amplified products as described herein to obtain a reaction mixture. [0198] In a set of experiments, the amplification mixture was subjected to an amplification protocol that included two series of amplification reactions, each series including different denaturation and extension conditions. Incubate six reaction mixtures (two containing H3N2, two containing ADV, and two containing HBoV) in a real-time PCR thermal cycler according to the amplification protocol. The amplification protocol includes 1 second at 94C, followed by series 1 (in 11 cycles of 1 second at 94C and 10 seconds at 45C) followed by 40 cycles of series 2 (5 seconds at 95C and 30 seconds at 55C). The amplification product is detected during the incubation.

[0199] The experimental results are shown in Table 6 below. As shown in Table 6, the range of the determined Ct value is 8.35-23. The results show that a protocol including multiple series of amplification reactions can be used to achieve good sensitivity. In addition, the results also show that a protocol including multiple series of amplification reactions can detect both RNA and DNA species.

[0201] Table 6: Results of H3N2, ADV and HBoV experiments in Example 6

<td>Types of</td><td>sample</td><td>Ct</td>

<td>RNA virus</td><td>H3N2-1</td><td>17</td>

<td>RNA virus</td><td>H3N2-2</td><td>20</td>

<td>DNA virus</td><td>ADV-1</td><td>18.8</td>

<td>DNA virus</td><td>ADV-2</td><td>23</td>

<td>DNA virus</td><td>HBoV-1</td><td>8.35</td>

<td>DNA virus</td><td>HBoV-2</td><td>18.37</td>

[0202] In another set of experiments, the amplification mixture was subjected to an amplification protocol including three series of amplification reactions, each series being different from each other in terms of its denaturation and/or extension conditions. Incubate five reaction mixtures in a real-time PCR thermal cycler according to the amplification protocol (one containing sH1N1 (2007), one containing H3N2, one containing pH1N1 (2009), one containing ADV, and one containing TB), The amplification protocol consists of 5 cycles at 94°C for 1 minute, followed by series 1 (5 seconds at 94°C, and 30 seconds at 60-50C (decrease at 1°C/cycle)). , And then perform 5 cycles of series 2 (5 seconds at 94°C and 30 seconds at 50°C), followed by 95. . Next 2 minutes, followed by series 3 (5 seconds at 95°C and 30 seconds at 55°C)

Of 40 cycles. During the incubation period, the amplification product is detected.

[0203] The experimental results are shown in Table 7 below. As shown in Table 7, the range of the determined Ct value is 20-30. The results show that a protocol including multiple series of amplification reactions can be used to achieve good sensitivity. In addition, the results also show that a protocol including multiple series of amplification reactions can detect both RNA and DNA species.

Table 7: Results of sHIN1 (2007), H3N2, pH1N1 (2009), ADV and TB experiments in Example 6

<td>Types of</td><td>sample</td><td>Ct</td>

<td>RNA virus</td><td>sHINl (2007)</td><td>22</td>

<td>RNA virus</td><td>H3N2</td><td>23</td>

<td>RNA virus</td><td>pHINl (2009)</td><td>24</td>

<td>DNA virus</td><td>ADV</td><td>30</td>

<td>DNA bacteria</td><td>TB</td><td>20</td>

[0206] Example 7: Standardization of multiple series of amplification reactions

[0207] Perform amplification and detection experiments to standardize various amplification schemes including multiple series of amplification reactions. The biological sample containing H3N2 is subjected to various amplification conditions. Each biological sample was obtained directly from the subject via an oropharyngeal swab. Combine 1 microliter of each sample in a 25uL reaction tube with the reagents necessary for nucleic acid amplification and detection of amplified products as described herein to obtain a reaction mixture.

[0208] The amplification mixture is subjected to an amplification protocol, some protocols include one of three different first series of amplification reactions and the same second series, the three first series including denaturation and denaturation different from the second series Extension conditions. Each of the first series and the second series includes multiple cycles. Another experiment was performed without the first series, which included only the second series. In the real-time PCR thermal cycler, incubate each of the four H3N2 reaction mixtures according to one of the amplification schemes shown in Table 8 below:

[0209] Table 8: Experimental protocol in Example 7

<td>Reaction mixture</td><td>Program</td>

<td>1</td><td>1 minute at 94 ku, (series 1A-at 94. (: next 1 second, at 45. (: next 2 minutes) x 5 cycles, 1 minute at 95H, (series 2-5 seconds at 95H, at 30 seconds at 55°C) X 50 cycles</td>

<td>2</td><td>2 minutes at 80°C, (series 1B-at 80. (2 times for 1 second, at 45°C for 2 minutes) x 5 cycles, at 95. (: next minute, (series 2-at 95. ( : Next 5 seconds, 30 seconds at 55°C) X 50 cycles</td>

<td>3</td><td>At 80 for 2 minutes, at 45. (: next 30 minutes, 95 minutes, 1 minute, (series 2-5 seconds at 95°C, 30 seconds at 55°C) x 50 cycles</td>

<td>4</td><td>At 94. (2 times for 1 second, (line || 1C-at 94. (: Next for 1 second, at 45°C for 30 seconds) x 50 cycles, (series 2-at 95°C for 5 seconds, at 55. ( : Next 30 seconds) x 50 cycles</td>

[0211] The experimental results are shown graphically in Figure 13 and in Table 9 below. As shown in Figure 13, reaction mixture 3 has the highest Ct value (28.59). Other reaction mixtures including multiple series have lower values ranging from 8.5 to 26.5. Knot

The results show that a protocol including multiple series of amplification reactions can be used to achieve good sensitivity. In addition, the results also show that the scheme including multiple series of amplification reactions can achieve better sensitivity than the scheme with only a single series.

[02 Table 9: Experimental results of Example 7

<td>Reaction mixture</td><td>Ct</td>

<td>1</td><td>22.97</td>

<td>2</td><td>26.5</td>

<td>3</td><td>28.59</td>

<td>4</td><td>8.5</td>

[0214] Example 8: Benchmarking of multiple series of amplification reactions

[0215] Perform amplification and detection experiments to benchmark various amplification schemes including multiple series of amplification reactions. The biological sample containing H3N2 is subjected to various amplification conditions. Each biological sample was obtained directly from the subject via an oropharyngeal swab. Combine 1 microliter of each sample in a 25uL reaction tube with the reagents necessary for nucleic acid amplification and detection of amplified products as described herein to obtain a reaction mixture.

[0216] The amplification mixture is subjected to an amplification protocol, some protocols include one of the six first series of amplification reactions and the same second series, the six first series including denaturation and extension different from the second series condition. The other six experiments were conducted without the first series. In the real-time PCR thermal cycler, incubate each of the twelve H3N2 reaction mixtures according to one of the amplification schemes shown in Table 10 below:

[0217] Table 10: Experimental protocol in Example 8

<td>Reaction mixture</td><td>Program</td>

<td>1</td><td>3 minutes at 95 years, 5 minutes at 45 years, 1 minute at 95H, "Series 2-5 seconds at 95°C, 30 seconds at 55°C) x 40 cycles</td>

<td>2</td><td>10 minutes at 95°C, 5 minutes at 45°C, 1 minute at 95°C, (series 2-5 seconds at 95°C, 30 seconds at 55°CT) x 40 cycles</td>

<td>3</td><td>3 minutes at 95°C, 20 minutes at 45°C, 1 minute at 95°C, (series 2-5 seconds at 95 ku, 30 seconds at 55H) x 40 cycles</td>

<td>4</td><td>At 95. (: next 10 minutes, at 45. (: next 20 minutes, 1 minute at 95H, (series 2-5 seconds at 95°C, 30 seconds at 55°CT) x 40 cycles</td>

<td>5</td><td>10 minutes at 95°C, 3 minutes at 45°C, 1 minute at 95°C, (series 2-5 seconds at 95 ku, 30 seconds at 55H) x 40 cycles</td>

<td>6</td><td>At 45. (: Next 20 minutes, 1 minute at 95H, (Series 2-5 seconds at 95 hours, 30 seconds at the next) X 40 cycles</td>

[0219]

<td>7</td><td>At 94 for 2 minutes, (series 1A-at 94 for 1 second, at 45 for 10 seconds) X 10 cycles, at 95°CT for 1 minute, (series 2-at 95°C for 5 seconds, 30 seconds at 55°C) X 50 cycles</td>

<td>8</td><td>At 94 for 10 seconds, (series 1B-at 94 for 1 second, at 45. (: for 10 seconds) x 10 cycles, at 95°C for 1 minute, (series 2 at 95°C for 5 Seconds, 30 seconds at 55°C) X 50 cycles</td>

<td>9</td><td>At 94. (: next 2 minutes, (series 1C-10 seconds at 94P, 20 seconds at 45P) X 10 cycles, 1 minute at 95, (series 2-at 95. (: next 5 seconds, at 55 °C 30 seconds) X 50 cycles</td>

<td>10</td><td>At 94. (: next 10 seconds, (series 1D-at 94. (: next 10 seconds, at 45. (: next 20 seconds) X 10 cycles, at 95°C for 1 minute, (series 2-at 95°C 5 seconds at 55°C, 30 seconds at 55°C) X 50 cycles</td>

<td>11</td><td>At 94%: for 2 minutes, (line 1E-at 94. (2 times for 30 seconds, at 45°C for 60 seconds) X 10 cycles, at 95 degrees for 1 minute, (line for line 2-at 95 .(: Next 5 seconds, 30 seconds at 55°C) X 50 cycles</td>

<td>12</td><td>At 94. (: next 10 seconds, (series 1F-30 seconds at 94P, 60 seconds at 45P) x 10 cycles, 1 minute at 95H, (series 2-at 95. (: next 5 seconds, at 55 steam Next 30 seconds) X 50 cycles</td>

[0220] The experimental results are listed in Table 11 below. The Ct value ranged from 14.53 to 27.28, and no product was detected in the reaction mixture 2-5. Generally speaking, a reaction mixture that has not undergone multiple series of amplification reactions either has no detectable product, or has a higher Ct value than a reaction mixture that has undergone multiple series of amplification reactions. The results show that a protocol including multiple series of amplification reactions can be used to achieve good sensitivity. In addition, the results also show that the scheme including multiple series of amplification reactions can achieve better sensitivity than the scheme with only a single series. In some cases, multiple series of amplification reactions may be necessary to produce a detectable amount of amplification product.

[0221] Table 11: Experimental results of Example 8

[0222]

<td>Reaction mixture</td><td>Ct</td>

<td>1</td><td>26.03</td>

<td>2</td><td>-</td>

<td>3</td><td>-</td>

<td>4</td><td>-</td>

<td>5</td><td>-</td>

<td>6</td><td>27.28</td>

<td>7</td><td>21.64</td>

<td>8</td><td>19.56</td>

<td>9</td><td>17.2</td>

<td>10</td><td>14.53</td>

<td>11</td><td>19.2</td>

<td>12</td><td>-</td>

[0223] Example 9: Comparison of the results of purified and unpurified samples

[0224] Amplification and detection experiments were performed to compare the results obtained with purified and unpurified samples. The purified and unpurified biological samples containing H1N1 are subjected to an amplification protocol. Each biological sample was obtained directly from the subject via an oropharyngeal swab. Combine 1 microliter of each sample in a 25uL reaction tube with the reagents necessary for nucleic acid amplification and detection of amplified products as described herein to obtain a reaction mixture. Three reaction mixtures were generated, two of which contained samples purified by one of column purification or magnetic purification. The third reaction mixture contains an unpurified sample.

[0225] The reaction mixture was incubated in a real-time PCR thermal cycler according to an amplification protocol that included 2 minutes at 94°C and 45°C. . 20 minutes at 94 °C, 1 minute at 94 °C, then at 94 °C. . Next 5 seconds and at 55. . 50 cycles of the next 35 seconds. The amplification product is detected during the incubation.

[0226] The experimental results are shown graphically in FIG. 14 and in Table 12 below. As shown in Table 12, the determined Ct values ranged from 27 to 31, and were similar between unpurified samples and samples purified by various means. The results indicate that sample purification may not be necessary to achieve similar detection sensitivity.

[0227]

[0228] Table 12: Experimental results of Example 9

<td>Sample type</td><td>Ct</td>

<td>Column purification</td><td>31</td>

<td>Magnetic bead purification</td><td>27</td>

<td>Unpurified</td><td>28</td>

[0229]

[0230] Example 10: Analysis of whole blood and saliva samples Amplification and detection experiments were performed on blood and saliva samples containing H3N2 virus. Four different samples were tested. The two samples contain one of the whole blood or saliva samples, and the two samples contain a 10-fold dilution (in PBS) of one of the whole blood or saliva samples. Each of these four samples was combined with the reagents necessary for reverse transcription of viral RNA and the reagents necessary to complete the amplification of complementary DNA obtained from reverse transcription. The reagents necessary for reverse transcription and DNA amplification are provided as a commercially available premix (for example, Takara One-Step RT-PCR or One-Step RT-qPCR kit), which contains reverse transcriptase (for example, Sensiscript and Omniscript transcriptase), DNA polymerase (for example, HotStarTaq DNA polymerase), and dNTP. In addition, the reaction tube also contains a TaqMan probe that contains FAM dye for detecting the amplified DNA product. In order to generate amplified DNA products, each reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol of denaturation and extension conditions, which included 20 minutes at 45C, followed by 2 minutes at 94C, followed by 5 seconds at 94C And 42 cycles of 35 seconds at 55C. During the incubation period, the amplification product is detected.

[0231] The amplification results of H3N2 are graphically shown in Figure 15 (Figure 15A corresponds to undiluted blood, Figure 15B corresponds to diluted blood) and Figure 16 (Figure 16A corresponds to undiluted saliva, and Figure 16B corresponds to diluted Saliva). The recorded fluorescence of the FAM dye is plotted against cycle number.

[0232] As shown in FIG. 15 and FIG. 16, both the undiluted and diluted blood and saliva reaction mixtures showed detectable signals, with Ct values ranging from 24-33. Therefore, the data shown in Figure 15 and Figure 16 show that the undiluted biological sample can be analyzed with good sensitivity, and the Ct value is not greater than about 40. In addition, the data also shows that when the dilution of the sample is necessary for analysis, amplification products can also be detected in a similar manner. In some cases, if there is too much inhibitor from the sample, dilution can be another way to eliminate the inhibition from the sample (eg, whole blood).

Example 11: Nested PCR

[0234] Amplification and detection experiments were performed on samples containing H1N1 virus. Eight samples were tested. Each sample includes H1N1 (2007) virus stock. The samples were diluted in PBS at the dilution shown in Table 13 below. The concentration of the virus stock is lxl() 6iu/mL. In order to generate amplified DNA products, the reaction mixture containing a given sample is incubated according to a protocol of denaturation and extension conditions. The protocol includes: (i) In the first run, the mixture is heated at 94° in a thermal cycler Heat for 1 minute at C, followed by 10 or 15 cycles as follows (as shown in Table 13 below): 5 seconds at 94C and at 57°C. . And (ii) in the second run, place the mixture at 94 in the thermal cycler. . Heat for 1 minute, followed by 35 cycles as follows: 94 °C for 5 seconds and 57C for 30 seconds. Add UL serial dilution samples to a 25uL reaction volume of Takara One-step qPCR premix. After some cycles in the first run, 1 PL from the reaction was added to the reaction mixture of the second run. The amplification results of H1N1 are graphically shown in Figure 17. The graph shows the recorded relative fluorescence units (RFU) as a function of cycle number. The graphs of each of the 8 samples (1-8) have been shown in this figure. The samples with detectable signals have the Ct values shown in Table 13.

[0235] Table 13: Experimental results of Example 11

<td>#</td><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td>

<td>Sample dilution</td><td>1/10</td><td>1/100</td><td>1/1000</td><td>0</td><td>1/10</td><td>1/100</td><td>1/1000</td><td>0</td>

<td>Ct</td><td>18</td><td>21</td><td>27</td><td>-</td><td>11</td><td>17</td><td>24</td><td>-</td>

<td>First run loop</td><td colspan="4">10 cycles</td><td colspan="4">15 cycles</td>

[0237] Example 12: Amplification and detection of Ebola recombinant plasmid

[0238] Amplification and detection experiments were performed on a human whole blood sample, the sample containing different copy numbers of recombinant plasmids corresponding to the Zaire-EBOV virus (Zaire-EBOV). Eight samples were tested. Six of the samples contained recombinant plasmids with specific copy numbers (250,000, 25,000, 2500, 250, 25, and 2.5 copies), and two samples (one containing only blood and one containing only water) were used as control samples. The whole blood sample was analyzed without sample purification.

[0239] Combine each sample with reagents necessary for nucleic acid amplification (for example, DNA polymerase, dNTP, primers, cofactors, suitable buffers, primers, etc.) and reporters (for example, oligonucleotides containing FAM dyes). Glycolic acid probe) is combined into the reaction mixture. The summary of each reaction mixture (including the copy number of the recombinant plasmid) according to the sample number is shown in Table 14. In order to generate amplified products, each reaction mixture was subjected to two series of denaturation and extension conditions. The two series are as follows: (i) in the first series, 15 cycles of 1 second at 95C and 1 second at 45C are performed, followed by 1 minute at 95C; and (ii) in the second series, Perform 45 cycles of 5 seconds at 95C and 10 seconds at 55C. During the second series, the signal from the reporter was recorded to generate the amplification curve and obtain the Ct value. The amplification curve of this experiment is shown graphically in Figure 18, and each curve is marked with a sample number corresponding to the sample number shown in Table 14. The results shown in Figure 18 show the recorded relative fluorescence units (RFU) as a function of cycle number. The Ct values obtained from the curve shown in FIG. 18 are summarized in Table 15. [0240] As shown in FIG. 18, except for sample 6, for all samples containing recombinant plasmids, recombinant plasmids were detected through amplification products. In addition, no recombinant plasmid was detected in any control samples (samples 7 and 8). Therefore, the knot shown in Figure 18 The results show that, in some cases, multiple series of denaturation and extension conditions are used, and without sample purification, a detection sensitivity of 25 plasmid copies/reaction (rxn) can be obtained.

[0241]

Table 14: Experimental reaction mixture of Example 12

<td>sample</td><td>Plasmid (copy/reaction)</td>

<td>1</td><td>250000</td>

<td>2</td><td>25000</td>

<td>3</td><td>2500</td>

<td>4</td><td>250</td>

<td>5</td><td>25</td>

<td>6</td><td>2.5</td>

<td>7</td><td>0 (Only blood)</td>

<td>8</td><td>0 (Only water)</td>

Table 15: Ct values determined by experiments in Example 12

[0243]

[0244]

<td>sample</td><td>Copy/reaction</td><td>Ct</td>

<td>1</td><td>250000</td><td>26.12</td>

<td>2</td><td>25000</td><td>33.61</td>

<td>3</td><td>2500</td><td>37.61</td>

<td>4</td><td>250</td><td>40.61</td>

<td>5</td><td>25</td><td>42.97</td>

<td>6</td><td>2.5</td><td>---</td>

<td>7</td><td>0</td><td>---</td>

<td>8</td><td>0</td><td>---</td>

Example 13: Amplification and detection of Ebola virus. Human whole blood samples containing different copy numbers of Zaire-EBOV pseudoviruses were carried out.

[0245]

[0246] Amplification and detection experiments. Eight samples (repeat group 1 and repetition group 2) were tested in duplicate, for a total of 16 samples. Six of the samples contained pseudoviruses with specific copy numbers (2.500000, 250,000, 25000, 2500, 250, and 25 copies), and two samples (one containing only blood and one containing only water) were used as control samples. The whole blood sample was analyzed without sample purification.

[0247] Combine each sample with reagents necessary for reverse transcription and nucleic acid amplification (for example, reverse transcriptase, DNA polymerase, dNTP, cofactors, primers, suitable buffers, etc.) and reporters (for example, containing FAM dye oligonucleotide probes) were combined into a 30UL reaction mixture. A summary of the reaction mixtures by sample number (including the copy number of the pseudovirus) is shown in Table 16. In order to generate amplification products from pseudoviruses, each reaction mixture is subjected to two series of denaturation and extension conditions. The two series are as follows: (i) In the first series, 15 cycles of 1 second at 95°C and 1 second at 45°C are performed, followed by 1 minute at 95°C; and (ii) In the second series, 45 cycles of 5 seconds at 95°C and 10 seconds at 55°C were performed. During the second series, the signal from the reporter is recorded to generate the amplification curve and obtain the Ct value. The amplification curves of this experiment are graphically shown in Fig. 19A (repetition group 1) and Fig. 19B (repetition group 2), and each curve is marked with a sample number corresponding to those sample numbers shown in Table 16. The results shown in Figures 19A and 19B show the recorded relative fluorescence units (RFU) as a function of cycle number. The Ct values obtained from the curves shown in FIG. 19A and FIG. 19B are summarized in Table 17, where "Ct 1" corresponds to repetition group 1, and "Ct 2" corresponds to repetition group 2.

[0248] As shown in FIG. 19A and FIG. 19B, for all samples containing pseudoviruses (samples 1-6), pseudoviruses were detected in both replicate groups via amplification products. In addition, no false virus was detected in any control samples (samples 7 and 8). because

Therefore, the results shown in FIGS. 19A and 19B indicate that, in some cases, multiple series of denaturation and extension conditions can be used to obtain a detection sensitivity of 25 virus copies/reaction without sample purification.

<td>[0249]</td><td colspan="5">Table 16: Experimental reaction mixture of Example 13</td>

<td rowspan="9">[0250]</td><td colspan="2">sample</td><td colspan="3">Fake virus (copy/reaction)</td>

<td colspan="2">1</td><td colspan="3">2500000</td>

<td colspan="2">2</td><td colspan="3">250000</td>

<td colspan="2">3</td><td colspan="3">25000</td>

<td colspan="2">4</td><td colspan="3">2500</td>

<td colspan="2">5</td><td colspan="3">250</td>

<td colspan="2">6</td><td colspan="3">25</td>

<td colspan="2">7</td><td colspan="3">0 (Only blood)</td>

<td colspan="2">8</td><td colspan="3">0 (Only water)</td>

<td>[0251]</td><td colspan="5">Table 17: Ct values determined by experiments in Example 13</td>

<td rowspan="9">[0252]</td><td>sample</td><td colspan="2">Copy/reaction</td><td>Ct 1</td><td>Ct 2</td>

<td>1</td><td colspan="2">2500000</td><td>8.57</td><td>8.44</td>

<td>2</td><td colspan="2">250000</td><td>12.09</td><td>11.27</td>

<td>3</td><td colspan="2">25000</td><td>15.03</td><td>14.99</td>

<td>4</td><td colspan="2">2500</td><td>18.90</td><td>18.87</td>

<td>5</td><td colspan="2">250</td><td>21.71</td><td>21.71</td>

<td>6</td><td colspan="2">25</td><td>27.86</td><td>39.42</td>

<td>7</td><td colspan="2">0 (Only blood)</td><td>---</td><td>---</td>

<td>8</td><td colspan="2">0 (Only water)</td><td>---</td><td>---</td>

<td>[0253][0254]</td><td colspan="5">Example 14: Amplification and detection of Ebola virus. Human whole blood samples containing different copy numbers of Zaire-EBOV pseudovirus were carried out.</td>

Amplification and detection experiments. Eight samples were tested. Six of the samples contained pseudoviruses with specific copy numbers (250,000, 250,000, 25,000, 2500, 250, and 25), and two samples (one containing 20,000 copies of the pseudovirus positive control, and one containing only water) were used as control samples. The whole blood sample was analyzed without sample purification.

[0255] Combine each sample with reagents necessary for reverse transcription and nucleic acid amplification (for example, reverse transcriptase, DNA polymerase, primers, dNTPs, cofactors, suitable buffers, etc.) and reporters (for example, containing FAM dye oligonucleotide probes) were combined into a 30UL reaction mixture. The summary of each reaction mixture by sample number (including the copy number of pseudovirus) is shown in Table 18. In order to generate amplification products from pseudoviruses, each reaction mixture is subjected to two series of denaturation and extension conditions. The two series are as follows: (i) In the first series, 15 cycles of 1 second at 95°C and 1 second at 45°C are performed, followed by 1 minute at 95°C; and (ii) In the second series, 35 cycles of 5 seconds at 95°C and 10 seconds at 55°C were performed. During the second series, the signal from the reporter was recorded to generate the amplification curve and obtain the Ct value. The amplification curves of this experiment are graphically shown in Figure 20, and each curve is marked with a sample number corresponding to those sample numbers shown in Table 18. The results shown in Figure 20 show the recorded relative fluorescence units (RFU) as a function of cycle number. The Ct values obtained from the curve shown in FIG. 20 are summarized in Table 19.

[0256] As shown in FIG. 20, for all samples containing pseudoviruses (samples 1-6), including the samples containing positive control pseudoviruses (sample 7), pseudoviruses were detected through amplification products. In addition, it was not detected in the control sample (sample 8) containing only water

To the fake virus. Therefore, the results shown in Figure 20 indicate that, in some cases, multiple series of denaturation and extension conditions can be used to obtain a detection sensitivity of 25 virus copies/reaction without sample purification.

<td rowspan="7">[0257][0258]</td><td colspan="4">Table 18: Experimental reaction mixture of Example 14</td>

<td colspan="2">sample</td><td colspan="2">Fake virus (copy/reaction)</td>

<td colspan="2">1</td><td colspan="2">2500000</td>

<td colspan="2">2</td><td colspan="2">250000</td>

<td colspan="2">3</td><td colspan="2">25000</td>

<td colspan="2">4</td><td colspan="2">2500</td>

<td colspan="2">5</td><td colspan="2">250</td>

<td>[0259]</td><td colspan="2">6</td><td colspan="2">25</td>

<td></td><td colspan="2">7</td><td colspan="2">20000 (Positive control false virus)</td>

<td></td><td colspan="2">8</td><td colspan="2">0 (Only water)</td>

<td>[0260]</td><td colspan="3">Table 19: Ct values determined by experiments in Example 14</td><td></td>

<td>[0261]</td><td>sample</td><td colspan="2">Copy/reaction</td><td>Ct</td>

<td></td><td>1</td><td colspan="2">2500000</td><td>10.44</td>

<td></td><td>2</td><td colspan="2">250000</td><td>13.30</td>

<td></td><td>3</td><td colspan="2">25000</td><td>16.14</td>

<td></td><td>4</td><td colspan="2">2500</td><td>19.62</td>

<td></td><td>5</td><td colspan="2">250</td><td>22.92</td>

<td></td><td>6</td><td colspan="2">25</td><td>30.00</td>

<td></td><td>7</td><td colspan="2">20000 (Positive control)</td><td>15.94</td>

<td></td><td>8</td><td colspan="2">0 (Only water)</td><td>---</td>

<td>[0262][0263]</td><td colspan="4">Example 15: Amplification and detection of Ebola virus. Zaire Ebola virus containing one of two copy numbers (250 copies/reaction or 25 copies/reaction)</td>

(Zaire-EBOV) Human whole blood samples of pseudovirus were amplified and detected. For a total of eight samples, each whole blood sample was tested using one of four reagent systems. Each reagent system (B-1, B-2, B-3 and B-4) contains the necessary reagents for reverse transcription and nucleic acid amplification (for example, reverse transcriptase, DNA polymerase, primers, dNTPs, auxiliary Factors, suitable buffers, etc.) and reporters (for example, oligonucleotide probes containing FAM dyes). Various reagent systems contain multiple components at different concentrations in the reagent system. Combine each whole blood sample with its appropriate reagent system into a 30UL reaction mixture. The summary of each reaction mixture by sample number (including copy number of pseudovirus and reagent system) is shown in Table 20 below. In order to generate amplification products from pseudoviruses, each reaction mixture is subjected to two series of denaturation and extension conditions. The two series are as follows: (i) In the first series, 15 cycles of 1 second at 95°C and 1 second at 45°C are performed, followed by 1 minute at 95°C; and (ii) In the second series, 40 cycles of 5 seconds at 95°C and 10 seconds at 55°C were performed. During the second series, the signal from the reporter was recorded to generate the amplification curve and obtain the Ct value. The amplification curves of this experiment are graphically shown in Figure 21, and each curve is marked with a sample number corresponding to those sample numbers shown in Table 20. As shown in Figure 21 The results show the recorded relative fluorescence units (RFU) as a function of cycle number. The Ct values obtained from the curve shown in FIG. 21 are summarized in Table 21.

[0264] As shown in FIG. 21, for all samples, including samples containing 25 copies/reactions, the amplification products were all detected

A fake virus was detected. Therefore, the results shown in Figure 21 indicate that in some cases, multiple series of denaturation and extension conditions can be used, different reagent systems can be used, and 25 virus copies/reactions can be obtained without sample purification. Detection sensitivity.

Table 20: Experimental reaction mixture of Example 15

<td>sample</td><td>Fake virus (copy/reaction)</td><td>Reagent system</td>

<td>1</td><td>250</td><td>B-1</td>

<td>2</td><td>25</td><td>B-1</td>

<td>3</td><td>250</td><td>B-2</td>

<td>4</td><td>25</td><td>B-2</td>

<td>5</td><td>250</td><td>B-3</td>

<td>6</td><td>25</td><td>B-3</td>

<td>7</td><td>250</td><td>B-4</td>

<td>8</td><td>25</td><td>B-4</td>

[0267] Table 21: Ct values determined from experiments in Example 15

<td>sample</td><td>Copy/reaction</td><td>Ct</td>

<td>1</td><td>250</td><td>20.38</td>

<td>2</td><td>25</td><td>24.82</td>

<td>3</td><td>250</td><td>20.62</td>

<td>4</td><td>25</td><td>24.05</td>

<td>5</td><td>250</td><td>20.26</td>

<td>6</td><td>25</td><td>25.09</td>

<td>7</td><td>250</td><td>19.86</td>

<td>8</td><td>25</td><td>24.00</td>

[0270] Example 16: Real-time PCR detection of Zaire Ebola virus

[0271] The one-step qPCR method of the present invention was used to analyze patient serum samples for Zaire Ebola virus. The sample is not purified. The samples include nine Zaire Ebola virus positive samples and seven Zaire Ebola virus negative samples. The Roche LC96 real-time PCR system is used.

[0272] The procedures for analyzing samples in this example are shown in Table 22.

[0273] Table 22: Thermal Cycle Program

<td>step</td><td>temperature</td><td>time</td><td>Number of cycles</td>

<td>1</td><td>42°C</td><td>1 minute</td><td>1 cycle</td>

<td rowspan="2">2</td><td>95°C</td><td>5 seconds</td><td rowspan="2">10 cycles</td>

<td>45°C</td><td>10 seconds</td>

<td>3</td><td>95°C</td><td>1 minute</td><td>1 cycle</td>

<td rowspan="2">4</td><td>95°C</td><td>5 seconds</td><td rowspan="2">40 cycles</td>

<td>55°C</td><td>10 seconds (read)</td>

[0275] The results of this one-step qPCR method are shown in Table 23. Compared with the validated reagents and methods, the one-step qPCR method showed 100% consistency in testing the Zaire Ebola virus.

[0276] Table 23: Results

<td>Sample serial number</td><td>Coyote one-step QPCR method (Cq)</td><td>Proven reagents and methods (Cq)</td><td>consistency</td>

<td>1</td><td>N/A</td><td>N/A</td><td>Yes</td>

<td>2</td><td>26.53</td><td>29.73</td><td>Yes</td>

<td>3</td><td>17.68</td><td>19.53</td><td>Yes</td>

<td>4</td><td>N/A</td><td>N/A</td><td>Yes</td>

<td>5</td><td>N/A</td><td>N/A</td><td>Yes</td>

<td>6</td><td>N/A</td><td>N/A</td><td>Yes</td>

<td>7</td><td>N/A</td><td>N/A</td><td>Yes</td>

<td>8</td><td>21.52</td><td>20.98</td><td>Yes</td>

<td>9</td><td>18.97</td><td>18.88</td><td>Yes</td>

<td>10</td><td>24.97</td><td>24.44</td><td>Yes</td>

<td>11</td><td>18.92</td><td>18.91</td><td>Yes</td>

<td>12</td><td>26.32</td><td>25.22</td><td>Yes</td>

<td>13</td><td>20.48</td><td>20.85</td><td>Yes</td>

<td>14</td><td>18.5</td><td>20.45</td><td>Yes</td>

<td>15</td><td>N/A</td><td>N/A</td><td>Yes</td>

[0278] Example 17: Amplification and detection of malaria

[0279] Amplification and detection experiments were performed on human whole blood samples containing unknown concentrations of malaria pathogens. Completed two sets of real

Test. In the first set of experiments, repeated experiments were completed on a 1:4 dilution of human whole blood samples (in 1X PBS); experiments were completed on samples containing whole blood and plasmids corresponding to the malaria pathogen; and for only The water-containing control completed the experiment. In the second set of experiments, samples containing multiple dilutions of human whole blood samples in 1X PBS (1:4, 1:40, 1:400, 1:4000, 1:40,000 and 1:400000) A control sample with only blood and only water completed the experiment. The whole blood sample was analyzed without sample purification.

[0280] Combine each sample with reagents necessary for nucleic acid amplification (for example, DNA polymerase, primers, dNTPs, cofactors, suitable buffers, etc.) and reporters (for example, oligonucleotide probes containing FAM dyes). Needle) Combine into 30UL reaction mixture. The reaction mixtures of the first set of experiments are summarized by sample number (including dilution) shown in Table 24. The reaction mixtures of the second set of experiments are summarized by sample number (including dilution) shown in Table 25. In order to generate amplified products from the malaria pathogen, each reaction mixture is subjected to two series of denaturation and extension conditions. The two series are as follows: (i) In the first series, 13 cycles of 1 second at 95°C and 1 second at 45°C are performed, followed by 1 minute at 95°C; and (ii) In the second series, 45 cycles of 5 seconds at 95C and 10 seconds at 55C were performed. During the second series, the signal from the reporter is recorded to generate an amplification curve. The amplification curve of the first set of experiments is graphically shown in Figure 22A, and the amplification curve of the second set of experiments is graphically shown in Figure 22B. Each curve is marked with its corresponding sample number in Table 24 and Table 25, respectively. The results shown in Figures 22A and 22B show the recorded relative fluorescence units (RFU) as a function of cycle number.

[0281] As shown in FIG. 22A, for the two reaction mixtures containing whole blood samples (samples 1 and 2) and for the positive control containing recombinant plasmids (sample 3), the malaria pathogen was detected through the amplified product. In addition, no malaria pathogen was detected in the water-only control sample (sample 4). Therefore, the results shown in Figure 22A indicate that, in some cases, multiple series of denaturation and extension conditions can be used to detect malaria pathogens without sample purification.

[0282] As shown in FIG. 22B, for all reaction mixtures containing whole blood samples (samples 1-6), the malaria pathogen was detected through the amplification product. In addition, no malaria pathogen was detected in the water-only and blood-only control samples (samples 7 and 8). Therefore, the results shown in Figure 22B indicate that, in some cases, multiple series of denaturation and extension conditions can be used and without sample purification, pathogens, including malaria pathogens, can be detected at a dilution as high as 1:400,000. .

<td>[0283]</td><td colspan="3">Table 24: Experimental reaction mixtures of the first set of experiments in Example 17</td>

<td>[0284]</td><td colspan="2">sample</td><td>Dilution</td>

<td></td><td colspan="2">1</td><td>1：4</td>

<td></td><td colspan="2">2</td><td>1：4</td>

<td>[0285]</td><td colspan="2">3</td><td>1:2 (plasmid in whole blood control)</td>

<td></td><td colspan="2">4</td><td>Zero (only water)</td>

<td>[0286]</td><td colspan="3">Table 25: Experimental reaction mixtures of the second set of experiments in Example 17</td>

<td>[0287]</td><td>sample</td><td colspan="2">Dilution</td>

<td></td><td>1</td><td>1：</td><td>4</td>

<td></td><td>2</td><td>1：</td><td>40</td>

<td></td><td>3</td><td>1：</td><td>400</td>

<td></td><td>4</td><td>1：</td><td>4000</td>

<td></td><td>5</td><td>1：</td><td>40000</td>

<td></td><td>6</td><td>1：</td><td>400000</td>

<td></td><td>7</td><td colspan="2">0 (Only blood)</td>

<td>8</td><td>0 (Only water)</td>

[0288] Example 18: Amplification and detection of dengue virus

[0289] Amplification and detection experiments were performed on samples obtained from cultures containing dengue virus at unknown concentrations. Completed three sets of experiments. In the first set of experiments, repeated experiments were completed for the undiluted culture; the experiment was completed for a 1:10 dilution of the culture; and the experiment was completed for the water-only control. In the second set of experiments, multiple dilutions of the culture (undiluted, 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000) and control samples with only water Completed the experiment. In the third set of experiments, experiments were performed on multiple dilutions of the culture (undiluted, 1:10, 1:100, 1:1000, and 1:10000) and a control sample with only water. The culture samples were analyzed without sample purification.

[0290] Combine 2uL of each sample with reagents necessary for reverse transcription and nucleic acid amplification (for example, reverse transcriptase, DNA polymerase, primers, dNTPs, cofactors, suitable buffers, etc.) and reporters (for example , Oligonucleotide probes containing FAM dye) are combined into a 30UL reaction mixture. Regarding the summary of the reaction mixture (including dilution), the first set of experiments are shown in Table 26, the second set of experiments are shown in Table 27, and the third set of experiments are shown in Table 28. In order to generate amplification products from the virus, each reaction mixture is subjected to two series of denaturation and extension conditions. The two series are as follows: (i) In the first series, at 42. . For the next 1 minute, perform 10 cycles of 5 seconds at 95°C and 10 seconds at 45°C, followed by 1 minute at 95°C; and (ii) in the second series, proceed at 95°C 45 cycles of 5 seconds and 10 seconds at 55°C. During the second series, the signal from the reporter is recorded to generate an amplification curve. The amplification curve of the first set of experiments is graphically shown in FIG. 23A, the amplification curve of the second set of experiments is graphically shown in FIG. 23B, and the amplification curve of the third set of experiments is graphically shown in FIG. 23C. Each curve is marked with its corresponding sample number in Table 26, Table 27 and Table 28. The results shown in Figure 23A, Figure 23B and Figure 23C show The recorded relative fluorescence units (RFU) as a function of cycle number are shown. The Ct values obtained from the curves shown in FIG. 23A, FIG. 23B, and FIG. 23C are shown in Table 26, Table 27, and Table 28, respectively.

[0291] As shown in FIG. 23A, for the three reaction mixtures containing viruses (samples 1-3), viruses were detected through amplification products. In addition, no virus was detected in the water-only control sample (sample 4). Therefore, the results shown in Figure 23A indicate that, in some cases, multiple series of extension and denaturation conditions can be used to detect dengue virus.

[0292] As shown in FIG. 23B, for a reaction mixture containing dengue virus and undiluted (sample 1) or diluted up to 1:1000 (samples 2, 3, and 4), virus was detected through the amplified product. However, the Ct value of the 1:1000 reaction mixture (Sample 4) has not been determined. No virus was detected in the higher dilutions (samples 5, 6 and 7) or in the water-only control sample (sample 8). Therefore, the results shown in Figure 23B indicate that in some cases, multiple series of denaturation and extension conditions can be used and without sample purification, virus can be detected at a dilution of up to 1:1000, where The Ct value can be generated up to a dilution of 1:100.

[0293] As shown in FIG. 23C, for a reaction mixture containing dengue virus and undiluted (sample 1) or diluted up to 1:1000 (samples 2, 3, and 4), virus was detected through the amplified product. However, the Ct value of the 1:1000 reaction mixture has not been determined. No virus was detected in the higher dilution (sample 5) or in the water-only control sample (sample 6). Therefore, the results shown in Figure 23C indicate that in some cases, multiple series of denaturation and extension conditions can be used and without sample purification, the virus can be detected at a dilution of up to 1:1000, where The Ct value can be generated up to a dilution of 1:100.

[0295] Table 26: Experimental reaction mixtures and determined Ct values of the first set of experiments in Example 18

<td>sample</td><td>Dilution</td><td>Ct value</td>

<td>1</td><td>Undiluted</td><td>19.32</td>

<td>2</td><td>Undiluted</td><td>20.40</td>

<td></td><td>3</td><td>1：10</td><td>23.23</td>

<td></td><td>4</td><td>No virus (water only)</td><td>---</td>

<td>[0296]</td><td colspan="3">Table 27: Experimental reaction mixtures and determined Ct values of the second set of experiments in Example 18</td>

<td>[0297]</td><td>sample</td><td>Dilution</td><td>Ct value</td>

<td></td><td>1</td><td>Undiluted</td><td>20.85</td>

<td></td><td>2</td><td>1：10</td><td>25.14</td>

<td></td><td>3</td><td>1：100</td><td>31.57</td>

<td></td><td>4</td><td>1：1000</td><td>---</td>

<td></td><td>5</td><td>1：10000</td><td>---</td>

<td></td><td>6</td><td>1：100000</td><td>---</td>

<td></td><td>7</td><td>1：1000000</td><td>---</td>

<td></td><td>8</td><td>No virus (water only)</td><td>---</td>

<td>[0298]</td><td colspan="3">Table 28: Experimental reaction mixtures and determined Ct values of the third set of experiments in Example 18</td>

<td>[0299]</td><td>sample</td><td colspan="2">Dilution Ct value</td>

<td></td><td>1</td><td>Undiluted 19</td><td>22</td>

<td></td><td>2</td><td colspan="2">1：10 22.43</td>

<td>[0300]</td><td>2</td><td>1：100 26</td><td>55</td>

<td></td><td>4</td><td>1：1000 ---</td><td></td>

<td></td><td>5</td><td>1：10000 ---</td><td></td>

<td></td><td>6</td><td>No virus (water only) ---</td><td></td>

<td>[0301]</td><td colspan="2">Example 19: Detection of Single Nucleotide Polymorphism (SNP)</td><td></td>

<td>[0302]</td><td colspan="3">For cytochrome P4502c19, CYP2C19*2 (with "GA" genotype) or</td>

CYP2C19*3 (with "GG" genotype) population throat swabs or blood samples for amplification and detection experiments. Two sets of experiments were performed, one set on samples obtained from human throat swabs and one set on samples obtained from blood. In the first set of experiments, seven different samples obtained from human throat swabs were analyzed without sample purification. In the second set of experiments, five different blood samples were analyzed without sample purification.

[0303] Combine each sample with the reagents necessary for nucleic acid amplification (for example, DNA polymerase, primers, dNTPs, cofactors, suitable buffers, etc.) and two reporters (for example, for detecting nucleic acid amplification) The oligonucleotide probe containing FAM dye and the oligonucleotide probe containing Texas Red dye used to detect the "GA" genotype) were combined into the reaction mixture. In order to generate amplified products, each reaction mixture is subjected to a thermal cycling protocol, which is included in 95. . Next 5 minutes, followed by 50 cycles of 5 seconds at 95°C and 10 seconds at 49°C. During thermal cycling, the signal from the reporter is recorded to generate an amplification curve. The amplification curves of the first set of experiments (population swabs) are graphically shown in Figure 24A (corresponding to the signal of the FAM oligonucleotide probe) and Figure 24B (corresponding to the signal of the Texas Red oligonucleotide probe) )in. The amplification curves of the second set of experiments (blood samples) are graphically shown in Figure 25A (corresponding to the signal of the FAM oligonucleotide probe) and Figure 25B (corresponding to the signal of the Texas Red oligonucleotide probe) . The results shown in Figures 24A, 24B, 25A, and 25B show the recorded relative fluorescence units (RFU) as a function of cycle number. Each curve is marked with its corresponding reaction mixture number in Table 29 (oropharyngeal swab test) or Table 30 (blood test). The Ct value determined from the amplification curve is also shown in the table along with the determined genotype 29 or Table 30. In Figure 24B or Figure 25B, in the amplification curve where the signal from Texas Red is observed, it is determined that the corresponding

CN 105121663 Β

The reaction mixture has the "GA" genotype. In addition, in FIG. 24B or FIG. 25B, in the amplification curve where the signal from Texas Red is not observed, it is determined that the corresponding reaction mixture has the "GG" genotype.

[0304] As shown in FIG. 24A, amplification products were observed for each reaction mixture containing a sample obtained from an oropharyngeal swab, indicating that nucleic acid amplification had occurred. However, as shown in Figure 24B, amplification products were only observed in some reaction mixtures (reaction mixtures 1, 4, 6 and 7) containing samples obtained from oropharyngeal swabs, which correspond to the "GA" gene type. In the other reaction mixtures (reaction mixtures 2, 3, and 5), no amplification products were observed, and these reaction mixtures corresponded to the "GG" genotype. The results shown in Figures 24A and 24B were verified through amplification and detection experiments using DNA extracted from buccal swab samples (data not shown). Therefore, the results shown in FIGS. 24A and 24B indicate that, in some cases, SNPs can be detected in samples obtained from oropharyngeal swabs by real-time amplification without sample purification.

[0305] As shown in FIG. 25A, amplification products were observed for each reaction mixture containing a sample obtained from blood, indicating that nucleic acid amplification had occurred. However, as shown in FIG. 25B, amplification products were only observed in some reaction mixtures (reaction mixtures 1, 2 and 5) containing samples obtained from blood, and these reaction mixtures corresponded to the "GA" genotype. In the other reaction mixtures (reaction mixtures 3 and 4), no amplification products were observed, and these reaction mixtures corresponded to the "GG" genotype. The results shown in Figures 25A and 25B were verified using nucleic acid sequencing. Therefore, the results shown in FIGS. 25A and 25B indicate that, in some cases, SNP can be detected in a sample obtained from blood by real-time amplification without sample purification.

[0306] Table 29: Ct values and genotypes determined for the oropharyngeal swab experiment in Example 19

[0307]

<td>Reaction compound</td><td>Ct-FAM reporter</td><td>Ct-Texas Red Reporter</td><td>genotype</td>

<td>1</td><td>38.70</td><td>40.25</td><td>GA</td>

<td>2</td><td>38.28</td><td>—</td><td>GG</td>

<td>3</td><td>34.16</td><td>—</td><td>GG</td>

<td>4</td><td>33.18</td><td>33.75</td><td>GA</td>

<td>5</td><td>35.20</td><td>—</td><td>GG</td>

[0308]

<td>6</td><td>33.08</td><td>33.59</td><td>GA</td>

<td>7</td><td>36.45</td><td>37.01</td><td>GA</td>

[0309] Table 30: Ct values and genotypes determined for the blood experiment in Example 19

[0310]

<td>Reaction mixture</td><td>Ct-FAM reporter</td><td>Ct-Texas Red Reporter</td><td>genotype</td>

<td>1</td><td>38.36</td><td>36.24</td><td>GA</td>

<td>2</td><td>39.97</td><td>39.67</td><td>GA</td>

<td>3</td><td>41.25</td><td>—</td><td>GG</td>

<td>4</td><td>33.96</td><td>—</td><td>GG</td>

<td>5</td><td>35.68</td><td>34.12</td><td>GA</td>

[0311] Example 20: Amplification and detection of adenovirus type 55 (ADV55) and adenovirus type 7 (ADV7)

[03 Perform amplification and detection experiments on samples containing different copy numbers of adenovirus type 55 (ADV55) or unknown concentration of adenovirus type 7 (ADV7) obtained from oropharyngeal swabs. Two sets of experiments were completed, one set for samples with ADV55 and one set for experiments with ADV7. In the first set of experiments, six different experiments using samples of ADV55 with different copy numbers (1, 10, 100, 1000, 10000, and 100,000 copies) were completed without sample purification, and completed together A negative control experiment. In the second set of experiments, without sample purification, eight different experiments with samples containing ADV7 of unknown copy number were completed.

[0313] Combine each sample with reagents necessary for nucleic acid amplification (for example, DNA polymerase, primers, dNTPs, cofactors, suitable buffers, etc.) and reporters (for example, oligonucleotide probes containing FAM dyes). Needle) merge into the reaction mixture. A summary of the reaction mixtures of the first set of experiments (including the copy number of ADV55) is shown in Table 31. In order to generate amplified products from the virus, each reaction mixture was subjected to two series of denaturation and extension conditions. The two series are as follows: (i) in the first series, perform 20 cycles of 1 second at 95C and 1 second at 45C, followed by 1 minute at 95C; and (ii) in the second series, perform 35 cycles of 5 seconds at 95C and 34 seconds at 60C. During the second series, the signal from the reporter was recorded to generate an amplification curve and obtain the Ct value. The amplification curves of the first set of experiments are graphically shown in FIG. 26A, and each curve is marked with a reaction mixture number corresponding to those reaction mixture numbers shown in Table 31. The amplification curves of the second set of experiments are graphically shown in Figure 26B and the corresponding Ct values are shown in Table 32. When the amplification curve in FIG. 26B corresponds to the reaction mixture number shown in Table 32, the amplification curve is marked. The results shown in Figure 26A and Figure 26B show the recorded relative fluorescence units (RFU) as a function of cycle number.

[0314] As shown in FIG. 26A, for all reaction mixtures (reaction mixtures 1-6) containing samples containing virus, ADV55 was detected through the amplified products. In addition, no virus was detected in the negative control reaction mixture (reaction mixture 7). Therefore, the results shown in Figure 26A indicate that, in some cases, multiple series of extension and denaturation conditions can be used to detect ADV55 virus without sample purification and at multiple dilution levels.

[0315] As shown in FIG. 26B, ADV7 was detected via the amplified product for all reaction mixtures. Therefore, the results shown in Figure 26B indicate that in some cases, multiple series of extension and denaturation conditions can be used and ADV7 virus can be detected without sample purification.

Table 31: Experimental reaction mixture of ADV55 experiment in Example 20

[0317]

<td>Reaction mixture</td><td>ADV55 copy number/reaction</td>

<td>1</td><td>1</td>

<td>2</td><td>10</td>

<td>3</td><td>100</td>

<td>4</td><td>1000</td>

<td>5</td><td>10000</td>

<td>6</td><td>100000</td>

<td>7</td><td>0 (Negative control)</td>

<td>[0318]</td><td colspan="2">Table 32: Ct value determined for the ADV7 experiment in Example 20</td>

<td rowspan="9">[0319]</td><td>Reaction mixture</td><td>Ct value</td>

<td>1</td><td>5.12</td>

<td>2</td><td>7.16</td>

<td>3</td><td>10.97</td>

<td>4</td><td>14.15</td>

<td>5</td><td>17.58</td>

<td>6</td><td>20.29</td>

<td>7</td><td>22.13</td>

<td>8</td><td>17.66</td>

<td>[0320][0321]</td><td colspan="2">Example 21: Amplification and detection of RNA hepatitis C virus (RNA-HCV) with A Amplification and detection of plasma samples containing different copy numbers of RNA hepatitis C virus (RNA-HCV)</td>

Detection experiment. Three different experiments using samples containing different copy numbers (10, 100, and 500 copies) of RNA-HCV were completed without sample purification, and the experiments for the negative control were completed together.

[0322] Combine each sample with reagents necessary for reverse transcription and nucleic acid amplification (for example, reverse transcriptase, DNA polymerase, primers, dNTPs, cofactors, suitable buffers, etc.) and reporters (for example, containing FAM dye oligonucleotide probes) are combined into the reaction mixture. The summary of the reaction mixture (including the copy number of RNA-HCV) is shown in Table 33. In order to produce amplified DNA products from the virus, each reaction mixture is subjected to two series of denaturation and extension conditions. The two series are as follows: (i) In the first series, 20 cycles of 1 second at 95°C and 1 second at 45°C are performed, followed by 1 minute at 95°C; and (ii) In the second series, 55 cycles of 5 seconds at 95°C and 34 seconds at 60°C were performed. During the second series, the signal from the reporter is recorded to generate an amplification curve. The amplification curves of the first set of experiments are graphically shown in Figure 27, and each curve is marked with a number corresponding to the reaction mixture number shown in Table 33. The results shown in Figure 27 show the recorded relative fluorescence units (RFU) as a function of cycle number.

[0323] As shown in FIG. 27, for all reaction mixtures (reaction mixtures 1-3) containing virus-containing samples, RNA-HCV was detected through the amplified products. In addition, RNAHCV was not detected in the negative control reaction mixture (reaction mixture 4). Therefore, the results shown in Figure 27 indicate that in some cases, multiple series of extension and denaturation conditions can be used to detect RNA-HCV without sample purification. The detection sensitivity of 10 copies/reaction can also be achieved.

[0324] Table 33: Experimental reaction mixture of RNA-HCV experiment in Example 21

[0325]

<td>Reaction mixture</td><td>RNA-HCV copy number/reaction</td>

<td>1</td><td>10</td>

<td>2</td><td>100</td>

<td>3</td><td>500</td>

<td>4</td><td>0 (Negative control)</td>

[0326] Although the preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now think of a large number of changes, changes and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The purpose is to define the scope of the present invention with the following claims, thereby covering methods and structures within the scope of these claims and their equivalents.