CN112359099A

CN112359099A - Methods and systems for nucleic acid amplification

Info

Publication number: CN112359099A
Application number: CN202011164325.5A
Authority: CN
Inventors: 李响
Original assignee: Coyote Bioscience Co ltd
Current assignee: Coyote Bioscience Co ltd
Priority date: 2013-12-25
Filing date: 2014-12-25
Publication date: 2021-02-12
Also published as: CN105121663A; CN105121663B

Abstract

The present invention relates to a method for amplifying and analyzing a nucleic acid sample, the method comprising providing a reaction vessel comprising a biological sample and reagents necessary for nucleic acid amplification to obtain a reaction mixture, and subjecting the reaction mixture in the reaction vessel to a plurality of cycles of a primer extension reaction, thereby amplifying a target nucleic acid. The application also relates to a system for carrying out the method. The methods and systems of the present invention enable rapid, accurate analytical detection of nucleic acids from complex sample types.

Description

Methods and systems for nucleic acid amplification

The present application is a divisional application of chinese patent application having application date of 2014 25/12, application No. 201480022014.1 entitled "method and system for nucleic acid amplification" (which corresponds to PCT application having application date of 2014 25/12, application No. PCT/CN 2014/094914).

Cross-referencing

This application claims priority to patent cooperation treaty application No. PCT/CN2013/090425 filed on 25.12.2013, which is incorporated by reference herein in its entirety for all purposes.

Background

Nucleic acid amplification methods allow for the selective amplification and identification of nucleic acids of interest from complex mixtures such as biological samples. To detect nucleic acids in a biological sample, the biological sample is typically processed to separate the nucleic acids from other components of the biological sample and other substances that may interfere with the nucleic acids 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, amplification methods known in the art, such as thermal cycle-based methods (e.g., Polymerase Chain Reaction (PCR)). After amplification of the nucleic acid of interest, the amplification product can be detected and the detection interpreted by the end user. However, extracting nucleic acids from a biological sample prior to nucleic acid amplification can be time consuming, resulting in a reduction in the time efficiency of the process as a whole.

Point of care (POC) testing has the potential to promote detection and treatment of infectious diseases under poor resource-constrained conditions of the laboratory infrastructure or in remote areas where receiving laboratory results delays and patient follow-up may be more complex. POC testing can also make existing level health care facilities more able to provide sample-to-answer (sample-to-answer) results to patients during a single visit. However, inefficiencies of POC methods and apparatus limit the goals that can be achieved. For example, the preparation of nucleic acids (e.g., of pathogens) from complex sample types (e.g., biological samples) requires a skilled technician to manually perform multiple processing steps and subsequent detection in a dedicated laboratory space, and thus results are often reported after hours or even days.

Thus, there is a need for a fast, accurate method and apparatus for analyzing nucleic acids from complex sample types. Such methods and devices are useful, for example, to enable rapid sample-feedback detection and treatment of diseases detectable via their nucleic acids.

Disclosure of Invention

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

In one aspect, the present invention provides a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. In one embodiment, the method comprises: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for a target RNA; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of cycles of primer extension reactions to generate amplified DNA products indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at the denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (ii) incubating the reaction mixture at the extension temperature for an extension duration of less than or equal to 60 seconds, thereby amplifying the target RNA. In another embodiment, the method comprises: (a) receiving a biological sample that has been obtained from a subject; (b) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase and (ii) a primer set for a target RNA, to obtain a reaction mixture; (c) subjecting the reaction mixture to a plurality of 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; (d) detecting the amount of amplified DNA product of (c); and outputting information to a recipient regarding the amount of amplified DNA product, wherein 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.

In some embodiments, the reagents further comprise a reporter that produces 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 comprises one or more primers. In some embodiments, the primer set comprises a first primer for generating a strand complementary to the target RNA. In some embodiments, the primer set comprises 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, dengue, orthomyxovirus, hepatitis virus (hepevirus), and/or hepatitis a, b, c (e.g., RNA-HCV with a), delta, and penta viruses.

In some embodiments, the reaction vessel comprises 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 (pipette tip). In some embodiments, the reaction vessel is part of an array of reaction vessels. In some embodiments, the reaction vessel portions of the reaction vessel array are individually addressable by the liquid handling device. In some embodiments, the reaction vessel comprises two or more thermal zones. In some embodiments, the reaction vessel is sealed, optionally hermetically sealed.

In some embodiments, the denaturation temperature is from about 90 ℃ to 100 ℃, or from about 92 ℃ to 95 ℃. In some embodiments, the extension temperature is about 35 ℃ to 72 ℃, or about 45 ℃ to 65 ℃. 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.

In some embodiments, the target RNA is not subjected to concentration prior to providing a reaction vessel comprising the biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification. In some embodiments, the biological sample is not subjected to RNA extraction when a reaction vessel is provided that comprises the biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification. In some embodiments, the method further comprises the step of adding a lysing agent to the reaction vessel prior to or during providing a reaction vessel comprising the biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification. 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.

In some embodiments, the biological sample is a biological fluid from a subject. In some embodiments, the biological sample is selected from the group consisting of exhaled breath, blood, urine, feces, saliva, cerebrospinal fluid, and sweat.

In some embodiments, the DNA amplification is performed via polymerase chain reaction. In some embodiments, the polymerase chain reaction is a nested polymerase chain reaction. In some embodiments, the DNA amplification is linear amplification. In some embodiments, the amplification produces a detectable amount of DNA product indicative of the presence of the target RNA in the biological sample at a cycle threshold (Ct) of less than 50, less than 40, less than 30, less than 20, less than 10, or less than 5. In some embodiments, the amplification produces a detectable amount of DNA product indicative of the presence of the target RNA in the biological sample over 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.

In some embodiments, the recipient is a physician, pharmaceutical company, or subject undergoing treatment. In some embodiments, subjecting the reaction mixture to a plurality of 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 is performed for 30 cycles or less, 20 cycles or less, or 10 cycles or less. In some embodiments, the detection is optical, electrostatic, or electrochemical. In some embodiments, the method comprises providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification and deoxyribonucleic acid (DNA) amplification.

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

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 comprises the following steps: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a deoxyribonucleic acid (DNA) polymerase and optionally a reverse transcriptase, and (ii) a primer set for a target nucleic acid, to obtain a reaction mixture; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein an individual series is different from at least one other individual series in the plurality of series with respect to the denaturing conditions and/or the extending conditions.

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 deoxyribonucleic acid 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 prior to (b). In some embodiments, the one or more denaturing conditions are selected from the group consisting of a denaturation temperature profile (profile) and a denaturing agent.

In some embodiments, the biological sample is diluted. This may help to minimize inhibition. In some embodiments, the biological sample is concentrated. This may help to increase or otherwise improve sensitivity.

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

In some embodiments, the plurality of series of primer extension reactions comprises a first series comprising more than 10 cycles, each cycle of the first series comprising (i) incubating the reaction mixture at about 92-95 ℃ for no more than 30 seconds, followed by (ii) incubating the reaction mixture at about 35-65 ℃ for no more than 1 minute, and a second series comprising more than 10 cycles, each cycle of the second series comprising: (i) incubating the reaction mixture at about 92-95 ℃ for no more than 30 seconds, followed by (ii) incubating the reaction mixture at about 40-60 ℃ for no more than 1 minute.

In some embodiments, the plurality of series of primer extension reactions produces a detectable amount of amplification product indicative of the presence of the target nucleic acid in the biological sample at a lower cycle threshold than a single series of primer extension reactions under comparable denaturing and extension conditions. In some embodiments, the method further comprises, prior to (b), pre-heating the biological sample at a pre-heating temperature of 90 ℃ to 100 ℃ for a pre-heating time of no more than 10 minutes, 2 minutes, or 1 minute. In some embodiments, the pre-heating temperature is from 92 ℃ to 95 ℃. In some embodiments, the duration of preheating is no more than about 30 seconds.

In another aspect, the present invention provides a system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. In one embodiment, the system comprises: (a) an input module that receives a user request to amplify a target RNA in a biological sample; (b) an amplification module that, in response to the user request: receiving in a reaction vessel a reaction mixture comprising a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for a target RNA; and, subjecting the reaction mixture in the reaction vessel to a plurality of cycles of a primer extension reaction to generate an amplified DNA product indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (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; and (c) an output module operably coupled to the amplification module, wherein the output module outputs information about the target RNA or DNA product to a recipient.

In another embodiment, the system comprises: (a) an input module that receives a user request to amplify a target RNA in a biological sample; (b) an amplification module that, in response to the user request: (i) receiving in a reaction vessel a reaction mixture comprising a biological sample that has been obtained from a subject and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising: (1) a reverse transcriptase, and (2) a primer set for a target RNA; and (ii) subjecting the reaction mixture to a plurality of 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; (iii) (iv) detecting the amount of amplified DNA product of (iii); and (iv) outputting information to the recipient regarding the amount of amplified DNA product, 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. In some embodiments, the output module is an electronic display. In some embodiments, the electronic display comprises a user interface. In some embodiments, the output module is a communication interface operatively coupled to a computer network.

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 comprises: (a) an input module that receives a user request to amplify a target RNA in a biological sample; (b) an amplification module that, in response to the user request: receiving in a reaction vessel a reaction mixture comprising a biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for a target nucleic acid; and, subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein an individual series is different from at least one other individual series in the plurality of series with respect to the denaturing conditions and/or the extending conditions; and (c) an output module operably coupled to the amplification module, wherein the output module outputs information about the nucleic acid or the amplification product to a recipient.

In another aspect, the present invention provides a computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. In one embodiment, the method comprises: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for a target RNA; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of cycles of primer extension reactions to generate amplified DNA products indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (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.

In another embodiment, the method comprises: (a) receiving a biological sample that has been obtained from a subject; (b) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase, and (ii) a primer set for a target RNA, to obtain a reaction mixture; (c) subjecting the reaction mixture to a plurality of 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; (d) detecting the amount of the DNA product of (c); and (e) outputting information about the amount of the DNA product to a recipient, wherein the amount of time for completing (a) - (e) is less than or equal to about 30 minutes.

In another aspect, the invention provides a computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target nucleic acid present in a biological sample obtained from a subject. In one embodiment, the method comprises: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for a target nucleic acid, to obtain a reaction mixture; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products from the target nucleic acid, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein an individual series is different from at least one other individual series in the plurality of series with respect to the denaturing conditions and/or the extending conditions.

Another aspect of the invention provides a system for amplifying a target nucleic acid in a biological sample obtained from a subject. The system can include an electronic display screen including a user interface displaying graphical elements that can be accessed by a user to perform an amplification protocol to amplify a target nucleic acid in a biological sample. The system may also include a computer processor coupled to the electronic display screen and programmed to execute the amplification protocol upon user selection of a graphical element. The amplification protocol can include subjecting a reaction mixture comprising the biological sample and reagents necessary to perform nucleic acid amplification to a plurality of series of primer extension reactions to generate amplification products 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: the reaction mixture is incubated under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by incubation of the reaction mixture under extending conditions characterized by an extending temperature and an extending duration. The individual series may differ from at least one other individual series in the plurality of series with respect to denaturing conditions and/or extension conditions.

In some embodiments, the amplification protocol can further comprise selecting a primer set for the target nucleic acid. In some embodiments, the reagents can comprise a deoxyribonucleic acid (DNA) polymerase, optionally a reverse transcriptase, and a primer set for the target nucleic acid. In some embodiments, the user interface may display a plurality of graphical elements. Each graphical element may be associated with a given amplification protocol of a plurality of amplification protocols. In some embodiments, each graphical element may be associated with a disease. A given amplification protocol of the plurality of amplification protocols can be directed to determining the presence of a disease in a subject. In some embodiments, the disease may be associated with a virus such as, for example, an RNA virus or a DNA virus. In some embodiments, the virus may be selected from human immunodeficiency virus i (hiv i), human immunodeficiency virus ii (hiv ii), orthomyxovirus, ebola virus, dengue virus, influenza virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis d virus, hepatitis e virus, hepatitis g virus, EB virus, mononucleosis virus, cytomegalovirus, SARS virus, west nile virus, poliovirus, measles virus, herpes simplex virus, smallpox virus, adenovirus and varicella virus. In some embodiments, the influenza virus may be selected from the group consisting of H1N1 virus, H3N2 virus, H7N9 virus, and H5N1 virus. In some embodiments, the adenovirus may be adenovirus type 55 (ADV55) or adenovirus type 7 (ADV 7). In some embodiments, the hepatitis c virus can be an RNA-hepatitis c virus with a (RNA-HCV). In some embodiments, the disease can be associated with a pathogenic bacterium (e.g., Mycobacterium tuberculosis) or a pathogenic protozoan (e.g., Plasmodium).

In some embodiments, the target nucleic acid can be associated with a disease. In some embodiments, the amplification protocol can be directed to determining the presence of a disease based on the presence of an amplification product. In some embodiments, the disease may be associated with a virus such as, for example, an RNA virus or a DNA virus. In some embodiments, the virus may be selected from human immunodeficiency virus i (hiv i), human immunodeficiency virus ii (hiv ii), orthomyxovirus, ebola virus, dengue virus, influenza virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis d virus, hepatitis e virus, hepatitis g virus, EB virus, mononucleosis virus, cytomegalovirus, SARS virus, west nile virus, poliovirus, measles virus, herpes simplex virus, smallpox virus, adenovirus and varicella virus. In some embodiments, the influenza virus may be selected from the group consisting of H1N1 virus, H3N2 virus, H7N9 virus, and H5N1 virus. In some embodiments, the adenovirus may be adenovirus type 55 (ADV55) or adenovirus type 7 (ADV 7). In some embodiments, the hepatitis c virus can be an RNA-hepatitis c virus with a (RNA-HCV). In some embodiments, the disease may be associated with a pathogenic bacterium (e.g., mycobacterium tuberculosis) or a pathogenic protozoan (e.g., plasmodium).

Other aspects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the invention are shown and described. As will be realized, the disclosure is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features believed characteristic of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures"), of which:

FIG. 1 is a schematic diagram depicting an exemplary system.

FIGS. 2A and 2B are graphs depicting the results of an exemplary nucleic acid amplification reaction described in example 1.

FIGS. 3A and 3B are graphs depicting the results of an exemplary nucleic acid amplification reaction described in example 1.

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

FIG. 5 is a graph depicting the results of an exemplary nucleic acid amplification reaction described in example 3.

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

FIGS. 7A and 7B are graphs depicting the results of an exemplary nucleic acid amplification reaction described in example 4.

FIGS. 8A and 8B are graphs depicting the results of an exemplary nucleic acid amplification reaction described in example 4.

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

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

FIG. 11 is a graph depicting the results of an exemplary nucleic acid amplification reaction described in example 5.

FIG. 12 is a graph depicting the results of an exemplary nucleic acid amplification reaction described in example 5.

FIG. 13 is a graph depicting the results of an exemplary nucleic acid amplification reaction described in example 7.

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

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

FIGS. 16A and 16B are graphs depicting the results of an exemplary nucleic acid amplification reaction described in example 10.

FIG. 17 is a graph depicting the results of the nucleic acid amplification reaction described in example 11.

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

FIG. 19A and FIG. 19B are graphs depicting the results of the nucleic acid amplification reaction described in example 13.

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

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

FIG. 22A and FIG. 22B are graphs depicting the results of the nucleic acid amplification reaction described in example 17.

FIG. 23A, FIG. 23B and FIG. 23C are graphs depicting the results of the nucleic acid amplification reaction described in example 18.

FIGS. 24A and 24B are graphs depicting the results of the nucleic acid amplification reaction described in example 19.

FIGS. 25A and 25B are graphs depicting the results of the nucleic acid amplification reaction described in example 19.

FIG. 26A and FIG. 26B are graphs depicting the results of the nucleic acid amplification reaction described in example 20.

FIG. 27 is a graph depicting the results of the nucleic acid amplification reaction described in example 21.

FIG. 28A is a schematic illustration of an exemplary electronic display with an exemplary user interface.

FIG. 28B is a schematic illustration of an exemplary electronic display with an exemplary user interface.

Detailed Description

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used in this specification and the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

As used herein, the terms "amplifying" and "amplification" are used interchangeably and generally refer to the generation of one or more copies of a nucleic acid or "amplification product". The term "DNA amplification" generally refers to the generation of one or more copies of a DNA molecule or an "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 a reverse transcriptase.

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

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, the unwinding of the secondary structure of a single-stranded nucleic acid. Denaturation may include inactivation of the pathogen cell wall or viral coat, as well as inactivation of inhibitor proteins. Conditions at which denaturation can occur include a "denaturation temperature," which generally refers to the temperature at which denaturation is permitted to occur, and a "denaturation duration," which generally refers to the amount of time allotted for denaturation to occur.

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

As used herein, the term "nucleic acid" generally refers to a polymeric form of nucleotides of any length (deoxyribonucleotides (dntps) or ribonucleotides (rNTP)) or analogs thereof. The nucleic acid can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of nucleic acids include DNA, RNA, coding or non-coding regions of a gene or gene fragment, 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), micro-RNA (mirna), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be made before or after nucleic acid assembly. The nucleotide sequence of a nucleic acid may be interrupted by non-nucleotide components. The nucleic acid may be further modified after polymerization, for example by coupling or binding to a reporter agent.

As used herein, the term "primer extension reaction" generally refers to the denaturation of double-stranded nucleic acids, the binding of a primer to one or both strands of the denatured nucleic acid, followed by primer extension.

As used herein, the term "reaction mixture" generally refers to a composition comprising reagents necessary to accomplish nucleic acid amplification (e.g., DNA amplification, RNA amplification), non-limiting examples of such reagents include primer sets specific for a target RNA or target DNA, DNA resulting from reverse transcription of RNA, DNA polymerase, reverse transcriptase (e.g., for reverse transcription of RNA), suitable buffers (including zwitterionic buffers), cofactors (e.g., divalent and monovalent cations), dntps, and other enzymes (e.g., uracil-DNA glycosylase (UNG), and the like). In some cases, the reaction mixture may further comprise one or more reporter agents.

As used herein, "reporter agent" 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 amplification product.

As used herein, the term "target nucleic acid" generally refers to a nucleic acid molecule having a nucleotide sequence whose presence, amount, and/or sequence, or a change in one or more thereof, is to be determined in a starting population of nucleic acid molecules. The target nucleic acid can be any type of nucleic acid, including DNA, RNA, and analogs thereof. As used herein, "target ribonucleic acid (RNA)" generally refers to a target nucleic acid that is an RNA. As used herein, "target deoxyribonucleic acid (DNA)" generally refers to a target nucleic acid that is DNA.

As used herein, the term "subject" generally refers to an entity or medium having genetic information that is testable or detectable. The subject may be a human or an individual. The subject can be a vertebrate, e.g., a mammal. Non-limiting examples of mammals include mice, simians, humans, livestock, sport animals (sport animals) and pets. Other examples of subjects include food, plants, soil, and water.

In one aspect, the present invention provides a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The method comprises the following steps: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for a target RNA; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of cycles of primer extension reactions to generate amplified DNA products indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at the denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (ii) incubating the reaction mixture at the extension temperature for an extension duration of less than or equal to 60 seconds, thereby amplifying the target RNA.

In another aspect, the present invention provides a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The method comprises the following steps: (a) receiving a biological sample that has been obtained from a subject; (b) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase and (ii) a primer set for a target RNA, to obtain a reaction mixture; (c) subjecting the reaction mixture to a plurality of 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; (d) detecting the amount of amplified DNA product of (c); and (e) outputting information about the amount of amplified DNA product to a recipient, wherein the amount of time for completing (a) - (e) is less than or equal to about 30 minutes.

In one aspect, the invention provides a method of amplifying a target nucleic acid present in a biological sample obtained from a subject. The method comprises the following steps: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a deoxyribonucleic acid (DNA) polymerase and optionally a reverse transcriptase, and (ii) a primer set for a target nucleic acid, to obtain a reaction mixture; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein the individual series is different from at least one other individual series in the plurality of series with respect to the denaturing conditions and/or the extending conditions.

In any of the 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, it is not further processed, except for any means for collecting a biological sample from the subject for further processing. For example, blood is obtained directly from a subject by: into the circulatory system of the subject, removing blood from the subject (e.g., through a needle), and passing the removed blood into a reservoir. The reservoir may contain reagents (e.g., anticoagulants) to make the blood sample available for further analysis. In another example, a swab may be used to access epithelial cells on the oropharyngeal surface of a subject. After obtaining a biological sample from a subject, a swab containing the biological sample can be contacted with a fluid (e.g., a buffer) to collect the biological fluid from the swab.

In some embodiments, the biological sample has not been purified when provided in the reaction vessel. In some embodiments, when the biological sample is provided to the reaction vessel, the nucleic acid of the biological sample has not been extracted. For example, when a biological sample is provided into a reaction vessel, RNA or DNA in the biological sample may not be extracted from the biological sample. Furthermore, in some embodiments, the target nucleic acid (e.g., target RNA or target DNA) present in the biological sample may not be concentrated prior to providing the biological sample into the reaction vessel.

Any suitable biological sample comprising nucleic acids may be obtained from a subject. The biological sample may be a solid substance (e.g., biological tissue) or may be a fluid (e.g., biological fluid). In general, a biological fluid may include any fluid associated with a living organism. Non-limiting examples of biological samples include blood (or components of blood-e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location of a subject (e.g., tissue, circulatory system, bone marrow), cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, interstitial fluid derived from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, luminal fluid, sputum, pus, microbiota (micropipota), meconium, milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric juice and digestive fluid, tears, ocular fluid, sweat, mucus, cerumen, oil, glandular secretions, spinal fluid, hair, Nails, skin cells, plasma, nasal swabs or nasopharyngeal washes, spinal fluid, cord blood, emphatic fluid, and/or other excretions or body tissues.

The biological sample may be obtained from the subject by any means known in the art. Non-limiting examples of means for obtaining a biological sample directly from a subject include: access the circulatory system (e.g., intravenous or intra-arterial access via a syringe or other needle), collection of secreted biological samples (e.g., stool, urine, sputum, saliva, etc.), surgery (e.g., biopsy), swabbing (e.g., oral swab, oropharyngeal swab), pipetting, and respiration. In addition, the biological sample may be obtained from any anatomical site in the subject where the desired biological sample is located.

In any of the aspects, the target nucleic acid is amplified to generate an amplification product. The target nucleic acid may be a target RNA or a 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 RNAs 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 (e.g., RNA-HCV with a virus), hepatitis d virus, hepatitis e virus, hepatitis g virus, EB virus, mononucleosis virus, cytomegalovirus, SARS virus, west nile virus, poliovirus, and measles virus.

Where the target nucleic acid is a 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, smallpox virus, adenoviruses (e.g., adenovirus type 55, adenovirus type 7), and varicella virus (e.g., fowlpox). In some cases, the target DNA may be bacterial DNA. The bacterial DNA may be from a bacterium that is pathogenic to the subject, e.g., mycobacterium tuberculosis, a bacterium known to cause tuberculosis. In some cases, the target DNA may be DNA from a pathogenic protozoan (e.g., one or more plasmodium-type protozoa that can cause malaria).

In any of the various aspects of the invention, a biological sample obtained from a subject is provided with reagents necessary for performing nucleic acid amplification in a reaction vessel to obtain a reaction mixture. Any suitable reaction vessel may be used. In some embodiments, the reaction vessel includes a body that may include an inner surface, an outer surface, an open end, and an opposing closed end. In some embodiments, the reaction vessel may comprise a lid. The lid may be configured to contact the body at its open end such 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 such that it remains attached to the reaction vessel in the open and closed configurations. In some cases, the lid is removable so that the lid is separated from the reaction vessel when the reaction vessel is opened. In some embodiments, the reaction vessel may be sealed, optionally hermetically sealed.

The reaction vessels may be of different sizes, shapes, weights and configurations. In some examples, the reaction vessel may be a circular or oval tube. In some embodiments, the reaction vessel may be rectangular, square, diamond, circular, oval, or triangular. The reaction vessel may be of regular 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, wells, capillaries, cartridges, dishes, centrifuge tubes, or pipette tips. The reaction vessel may be constructed of any suitable material, non-limiting examples of which include glass, metal, plastic, and combinations thereof.

In some embodiments, the reaction vessel is part of an array of reaction vessels. The array of reaction vessels is particularly useful for automated methods and/or for processing multiple samples simultaneously. For example, the reaction vessel may be a well of a microplate consisting of a plurality of wells. In another example, the reaction vessel can be housed in a well of a thermal block of a thermal cycler instrument, wherein the thermal block comprises a plurality of wells that are each capable of receiving a sample vessel. An array of reaction vessels may comprise any suitable number of reaction vessels. For example, an array can 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 vessel portions of the reaction vessel array may also be individually addressed by the fluid handling device so that the fluid handling device can properly identify the reaction vessels and dispense the appropriate fluid materials into the reaction vessels. The fluid handling device may be used to automate the addition of fluid material to the reaction vessel.

In some embodiments, the reaction vessel may comprise a plurality of thermal zones. The hot zones within the reaction vessel may be achieved by exposing different regions 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 thermal zone is capable of receiving a biological sample and reagents necessary to obtain a reaction mixture for nucleic acid amplification. The reaction mixture may then be subjected to a first thermal cycling protocol. 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 cycling regime that is different from the regime of the upper hot zone. Such strategies may be particularly useful when using nested PCR to amplify DNA. In some embodiments, the thermal zone may be created within the reaction vessel with the aid of a heat sensitive layering material within the reaction vessel. In such cases, heating of the heat sensitive stratified material may be used to release the reaction mixture from one thermal zone into the next. In some embodiments, the reaction vessel comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more thermal zones.

In some embodiments, a reaction vessel comprising a thermal zone can be used to treat a biological sample prior to nucleic acid amplification. For example, a lysis agent may be added to the first thermal zone of the reaction vessel prior to the addition of the biological sample and reagents necessary for nucleic acid amplification. When a biological sample and reagents are added to a reaction vessel containing a lysing agent, a reaction mixture is obtained that is capable of lysing species (e.g., cells or viral particles) within the biological sample. Alternatively, the lysing agent can be added to the first thermal zone of the reaction mixture simultaneously with the biological sample and the reagents. Subjecting the first thermal zone to temperature conditions suitable for the action of the lysing agent can be used to lyse cells and viral particles in the biological sample in the first thermal zone such that nucleic acids in the biological sample are released into the reaction mixture. After lysis, the reaction mixture may then be passed into a second thermal zone of the reaction vessel for amplification of the released nucleic acids using the amplification methods described herein.

Where a lysing agent is desired, 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, dextranase (glucolase), protease E, viral endolysins, exolysin (exolysin), zymolase (zymolose), cytolysin (Iytase), protease K, endolysins and exolysins from bacteriophage, endolysins from bacteriophage PM2, endolysins from Bacillus subtilis bacteriophage SX, endolysins from Lactobacillus prophages Lj928, Lj965, bacteriophage 15Phiadh, endolysin from Streptococcus pneumoniae Cp-I, bifunctional peptidoglycan lysin from Streptococcus agalactis bacteriophage B30, endolysins and exolysins from prophage bacteria, endolysins from Listeria phage (Listeria), holin (hololysin) -endolysin, cytolysin 20 genes, Wolwy Staphyl (WM) phage WM, iy5WMY from Staphylococcus wowensis M phage varhiWMY, and combinations thereof. In some cases, the buffer can comprise a lysing agent (e.g., a lysis buffer). An example of a lysis buffer is sodium hydroxide (NaOH).

Any type of nucleic acid amplification reaction known in the art can be used to amplify the target nucleic acid and generate the amplification products. Further, amplification of nucleic acids can be linear, exponential, or a combination thereof. Amplification may be emulsion-based or may 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 a target RNA, DNA can be obtained by reverse transcription of the RNA and subsequent DNA amplification can be used to generate an amplified DNA product. The amplified DNA product may indicate the presence of the target RNA in the biological sample. In the case of amplifying DNA, any DNA amplification method known in the art may be used. Non-limiting examples of DNA amplification methods include Polymerase Chain Reaction (PCR), variations of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot-start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric staggered PCR (thermal asymmetric interlaced PCR), descending 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 using nested PCR, which can improve the sensitivity of detecting amplified DNA products.

In various aspects, these nucleic acid amplification reactions described herein can be performed in parallel. In general, 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: providing the reagents necessary for the two amplification methods in a reaction vessel to form and obtain a reaction mixture and subjecting the reaction mixture to conditions suitable for performing the two amplification reactions. DNA resulting from 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 are performed in parallel.

Advantages of performing nucleic acid amplification reactions in parallel may include rapid switching between coupled nucleic acid amplification reactions. For example, a target nucleic acid (e.g., target RNA, target DNA) can be extracted or released from a biological sample during a 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 released from the biological sample. The released target RNA can immediately begin reverse transcription (via reverse transcription amplification) to produce complementary DNA. The complementary DNA can then be amplified immediately, typically on the order of seconds. The short time interval between release of the target RNA from the biological sample and reverse transcription of the target RNA into complementary DNA can help to minimize the effects of inhibitors in the biological sample that may interfere with reverse transcription and/or DNA amplification.

In any of these various aspects, a nucleic acid amplification reaction can be performed using a primer set for a target nucleic acid. The primer set typically comprises one or more primers. For example, a primer set can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primers. In some cases, the primer set may comprise primers for different amplification products or different nucleic acid amplification reactions. For example, a primer set can comprise a first primer necessary to produce a first strand of a nucleic acid product that is complementary to at least a portion of a target nucleic acid and a second primer complementary to a nucleic acid strand product necessary to produce a second strand of the nucleic acid product that is complementary to at least a portion of the first strand of the nucleic acid product.

For example, the primer set can be directed against a target RNA. The primer set can comprise a first primer that can be used to generate a first strand of a nucleic acid product that is complementary to at least a portion of a target RNA. In the case of a reverse transcription reaction, the first strand of the nucleic acid product may be DNA. The primer set can further comprise a second primer operable to generate a second strand of the 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 can be one strand of a nucleic acid (e.g., DNA) product that is complementary to a DNA strand produced from an RNA template.

Any suitable number of primer sets can be used if desired. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more primer sets may be used. When multiple primer sets are used, one or more primer sets may each correspond to a particular nucleic acid amplification reaction or amplification product.

In some embodiments, a DNA polymerase is used. Any suitable DNA polymerase can be used, including commercially available DNA polymerases. A DNA polymerase generally refers to an enzyme that is capable of incorporating nucleotides into a DNA strand 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, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pmutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products, and derivatives thereof. For certain hot start polymerases, a denaturation step at 94 ℃ -95 ℃ for 2 minutes to 10 minutes may be required, which may change the heat profile depending on the polymerase.

In some embodiments, a reverse transcriptase is used. Any suitable reverse transcriptase may be used. Reverse transcriptase generally refers to an enzyme that is capable of incorporating nucleotides into a DNA strand when bound to an RNA template. Non-limiting examples of reverse transcriptase include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof.

In various aspects, a primer extension reaction is used to generate an amplification product. Primer extension reactions typically involve the following cycles: the reaction mixture is incubated at a denaturation temperature for a denaturation duration and the reaction mixture is incubated at an extension temperature for an extension duration.

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

The duration of denaturation can vary depending on, for example, the particular biological sample being analyzed, the particular source of the target nucleic acid in the biological sample (e.g., viral particles, bacteria), the reagents used, and/or the desired reaction conditions. For example, the denaturation duration can 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, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the denaturation duration can be no more than 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.

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

The extension duration can vary depending on, for example, the particular biological sample being analyzed, the particular source of the target nucleic acid in the biological sample (e.g., viral particles, bacteria), the reagents used, and/or the desired reaction conditions. For example, the extension 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, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the extension duration may be no more than 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.

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

The time required for amplification to produce a detectable amount of amplification product indicative of the presence of amplified target nucleic acid can vary depending on the biological sample from which the target nucleic acid is obtained, the particular nucleic acid amplification reaction to be performed, and the particular number of cycles of the amplification reaction desired. For example, amplification of the target nucleic acid can produce a detectable amount of amplification product indicative of the presence of the target nucleic acid in a time period of 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.

In some embodiments, amplification of the target RNA can produce a detectable amount of amplified DNA product indicative of the presence of the target RNA in a period of 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.

In some embodiments, the reaction mixture may be subjected to a plurality of series of primer extension reactions. Individual ones of the plurality of series may comprise a plurality of cycles of a particular primer extension reaction characterized by, for example, particular denaturing and extension conditions as described elsewhere herein. Typically, each individual series is different from at least one other individual series in the plurality of series, e.g., in terms of denaturing conditions and/or extension conditions. For example, a single series may differ from another single series of the plurality of series with respect to any one, two, three, or all four of denaturation temperature, denaturation duration, extension temperature, and extension duration. Further, 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.

For example, a plurality of series of primer extension reactions can include a first series and a second series. The first series, for example, can comprise more than ten cycles of a primer extension reaction, wherein each cycle of the first series comprises (i) incubating the reaction mixture at about 92 ℃ to about 95 ℃ for no more than 30 seconds, followed by (ii) incubating the reaction mixture at about 35 ℃ to about 65 ℃ for no more than about one minute. The second series, for example, can comprise more than ten cycles of primer extension reactions, wherein each cycle of the second series comprises (i) incubating the reaction mixture at about 92 ℃ to about 95 ℃ for no more than 30 seconds, followed by (ii) incubating the reaction mixture at about 40 ℃ to about 60 ℃ for no more than about 1 minute. In this particular 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.

In some embodiments, ramp time (i.e., the time it takes for the thermocycler to transition from one temperature to another) and/or ramp rate are important factors in amplification. For example, the temperature and time required for amplification to produce a detectable amount of amplification product indicative of the presence of a target nucleic acid can vary depending on the ramp rate and/or ramp time. The ramp rate can affect the temperature and time used for amplification.

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 may be adjusted based on the sample being processed.

In some cases, the ramp time between different temperatures may be determined, for example, based on the nature of the sample and the reaction conditions. The exact temperature and incubation time can also be determined based on the nature of the sample and the reaction conditions. In some embodiments, a single sample can be treated (e.g., subjected to amplification conditions) multiple times using multiple thermal cycles, each thermal cycle differing in, for example, ramp time, temperature, and/or incubation time. The best or optimal thermal cycle may then be selected for that particular sample. This provides a robust and efficient method of tailoring the thermal cycle to the particular sample or combination of samples being tested.

In some embodiments, the target nucleic acid can be subjected to denaturing conditions prior to initiation of the primer extension reaction. In the case of multiple series of primer extension reactions, the target nucleic acid may be subjected to denaturing conditions prior to performing the multiple series, or may be subjected to denaturing conditions between the multiple series. For example, the target nucleic acid can be subjected to denaturing conditions between a first series and a second series in the plurality of series. Non-limiting examples of such denaturing conditions include a denaturation temperature profile (e.g., one or more denaturation temperatures) and a denaturing agent.

An advantage of performing multiple series of primer extension reactions may be that the multiple series of methods produce detectable amounts of amplification products indicative of the presence of a target nucleic acid in a biological sample at a lower cycle threshold than a single series of primer extension reactions under comparable denaturing and extension conditions. The use of multiple series of primer extension reactions can reduce these cycling 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% as compared to a single series under comparable denaturing and extension conditions.

In some embodiments, the biological sample may be preheated prior to performing the primer extension reaction. The temperature (e.g., preheat temperature) and duration (e.g., preheat duration) of preheating the biological sample may vary depending on, for example, the particular biological sample being analyzed. In some examples, the biological sample can 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 can be preheated at a temperature of about 80 ℃ to about 110 ℃. In some examples, the biological sample can be preheated at a temperature of about 90 ℃ to about 100 ℃. In some examples, the biological sample can be preheated at a temperature of about 90 ℃ to about 97 ℃. In some examples, the biological sample can be preheated at a temperature of about 92 ℃ to about 95 ℃. In yet other examples, the biological sample can be preheated at a temperature of about 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃, 99 ℃, or 100 ℃.

In some embodiments, the reagents necessary for performing nucleic acid amplification (including the reagents necessary for performing parallel nucleic acid amplification) can further comprise a reporter agent that produces a detectable signal, the presence or absence of which indicates the presence or absence of an amplification product. The intensity of the detectable signal may be proportional to the amount of amplification product. In some cases, when the amplification product is generated from a different type of nucleic acid than the initially amplified target nucleic acid, the intensity of the detectable signal can be proportional to the amount of the initially amplified target nucleic acid. For example, in the case of amplification of a target RNA by reverse transcription and amplification of DNA obtained from reverse transcription in parallel, the reagents necessary for both reactions may also include a reporter agent that produces a detectable signal indicative of 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 a reporter also enables real-time amplification methods, including real-time PCR for DNA amplification.

The reporter agent may be linked to the nucleic acid, including the amplification product, by covalent or non-covalent means. Non-limiting examples of non-covalent means include ionic interactions, van der waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. In some embodiments, a reporter can be bound to the initial reactant and a change in the level of the reporter can be used to detect the amplification product. In some embodiments, the reporter may be detectable (or undetectable) only while nucleic acid amplification is in progress. In some embodiments, an optically active dye (e.g., a fluorescent dye) may be used as a reporter. Non-limiting examples of dyes include SYBR Green, SYBR blue, DAPI, propidium iodide (propidium iodide), Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acridine yellow, fluorescent coumarin (fluorocoumanin), ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, hominium bromide (hominium), mithramycin, ruthenium polypyridyl (ruthenium polypyridyl), amphenycin (anthramycin), phenanthridine and acridine, ethidium bromide, propidium iodide, hexidine iodide (hexidium iodide), ethidium dihydroethidium, ethidium homodimer-1 and ethidium homodimer-2, ethidium nitride (ethidium monozide) and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, hoechpi, DAHs 897-AAD, acridine AAD, stilbene monoacidine S (TOXO-O751, O-O751, oxy-Ylbine, oxy-O751, oxo-O3, oxo-O751, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBOBO-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, SYBR, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (Red), Fluorescein Isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (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 dimer I, ethidium dimer II, ethidium homodimer III, ethidium bromide, eosin, green umbelliferyl fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein, cascade blue (cascade blue), dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes (such as those including europium and terbium), carboxytetrachlorofluorescein, 5 and/or 6-carboxyfluorescein (FAM), 5- (or 6-) iodoacetamido-fluorescein, 5- { [2 (and 3) -5- (acetylmercapto) -succinyl ] amino } fluorescein (SAMSA-fluorescein), lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxyrhodamine (ROX), 7-amino-methyl-coumarin, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxypyrene-1, trisodium 3, 6-trisulfonate, 3, 6-disulfonic acid-4-amino-naphthalimide, 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.

In some embodiments, the reporter may be a sequence-specific oligonucleotide probe that is optically active upon hybridization to the amplification product. The use of oligonucleotide probes can improve the specificity and sensitivity of detection due to the sequence-specific binding of the probe to the amplification product. The probe can be attached to any optically active reporter (e.g., dye) described herein, and can further include a quencher capable of blocking the optical activity of the associated dye. Non-limiting examples of probes that can be used as a reporter include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

In some embodiments, the reporter may be an RNA oligonucleotide probe comprising an optically active dye (e.g., a fluorescent dye) and a quencher adjacently located on the probe. The close proximity of the dye to the quencher can block the optical activity of the dye. The probe can bind to the target sequence to be amplified. Once the probe is cleaved by the exonuclease activity of the DNA polymerase during amplification, the quencher separates from the dye, and the free dye regains its optical activity, which can then be detected.

In some embodiments, the reporter agent 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, e.g., a fluorescent dye. In the hairpin configuration, the optically active dye and the quencher are in sufficiently close proximity that the quencher is able to block the optical activity of the dye. However, once hybridized to the amplification product, the oligonucleotide assumes a linear conformation and hybridizes to the target sequence on the amplification product. Linearization of the oligonucleotide results in separation of the optically active dye from the quencher, allowing optical activity to recover and be detected. Sequence specificity of the molecular beacon for the target sequence on the amplification product can improve specificity and sensitivity of detection.

In some embodiments, the reporter may be a radioactive species. Non-limiting examples of radioactive species include¹⁴C、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、Tc99m、³⁵S or³H。

In some embodiments, the reporter agent may be an enzyme capable of producing a detectable signal. A detectable signal may be generated by the activity of the enzyme on its substrate, or on a particular substrate in the case of an enzyme having multiple substrates. Non-limiting examples of enzymes that can be used as reporter include alkaline phosphatase, horseradish peroxidase, I2-galactosidase, alkaline phosphatase, beta-galactosidase, acetylcholinesterase, and luciferase.

In various aspects, an amplification product (e.g., amplified DNA product, amplified RNA product) can be detected. Detection of the amplification products (including amplified DNA) can be accomplished using any suitable detection method known in the art. The particular type of detection method used may depend, for example, on the particular amplification product, the type of reaction vessel used for amplification, other reagents in the reaction mixture, whether a reporter is included in the reaction mixture, and the particular type of reporter used when using the reporter. 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, fluorimetry and ultraviolet-visible light absorption. Spectroscopic 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 amplification products after high performance liquid chromatography separation of the amplification products.

In any of the aspects, the time required to complete an element of a method may vary depending on the particular steps of the method. For example, the amount of time for completing an element of a method may be about 5 minutes to about 120 minutes. In other examples, the amount of time to complete an element of a method may be about 5 minutes to about 60 minutes. In other examples, the amount of time to complete an element of a method may be about 5 minutes to about 30 minutes. In other examples, the amount of time to complete an element of a method can 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.

In some embodiments, information regarding the presence and/or amount of amplification products (e.g., amplified DNA products) can be output to a recipient. Information about the amplification product can be output via any suitable method known in the art. In some embodiments, such information may be verbally provided to the recipient. In some embodiments, such information may be provided in a report. The report can include any number of desired elements, non-limiting examples of which include information about the subject's (e.g., gender, age, race, health, etc.), processed data (e.g., graphical displays (e.g., graphs, charts, data tables, data summaries), determined cycle thresholds, calculated values of the initial amount of the target polynucleotide), conclusions regarding the presence or absence of the target nucleic acid, diagnostic information, prognostic information, disease information, and the like, and combinations thereof. The report may be provided as a printed report (e.g., a hard copy) or may be provided as an electronic report. In some embodiments, including where an electronic report is provided, such information can be output via an electronic display (e.g., an electronic display screen), such as a monitor or television, a screen operatively connected to the means for obtaining amplification products, a tablet computer screen, a mobile device screen, or the like. Both the printed report and the electronic report may be stored separately in a file or database so that they may be accessed for comparison with a later report.

Further, the report may be sent to the recipient at a local or remote location using any suitable communication medium, including, for example, a network connection, a wireless connection, or an internet connection. In some embodiments, the report may be sent to a recipient's device, such as a personal computer, telephone, tablet, or other device. The report may 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 which include mailing the hardcopy report for receipt and/or viewing by the recipient.

Further, such information may be output to a variety of different types of recipients. Non-limiting examples of such recipients include the subject from which the biological sample was obtained, a physician treating the subject, a clinical monitor for clinical testing, a nurse, a researcher, a laboratory technician, a representative of a pharmaceutical company, a healthcare company, a biotechnology company, a hospital, a human assistance organization, a healthcare manager, an electronic system (e.g., one or more computers and/or one or more computer servers storing, for example, a medical record of the subject), a public health worker, other medical personnel, and other medical facilities.

In one aspect, the invention provides a system for performing a method according to any of the methods disclosed herein. In another aspect, the present invention provides a system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The system comprises: (a) an input module that receives a user request to amplify a target RNA in a biological sample; (b) an amplification module that, in response to the user request: receiving in a reaction vessel a reaction mixture comprising a biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for a target RNA; and, subjecting the reaction mixture in the reaction vessel to a plurality of cycles of a primer extension reaction to generate an amplified DNA product indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (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; and (c) an output module operably coupled to the amplification module, wherein the output module outputs information about the target RNA or DNA product to a recipient.

In another aspect, the present invention provides a system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject. The system comprises: (a) an input module that receives a user request to amplify a target RNA in a biological sample; (b) an amplification module that, in response to the user request: (i) receiving in a reaction vessel a reaction mixture comprising a biological sample that has been obtained from a subject and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (1) a reverse transcriptase, and (2) a primer set for a target RNA; and (ii) subjecting the reaction mixture to a plurality of 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; (iii) (iv) detecting the amount of amplified DNA product of (iii); and (iv) outputting information to the recipient regarding the amount of amplified DNA product, wherein the amount of time for completing (i) - (iv) is less than or equal to about 30 minutes; and (c) an output module operably coupled to the amplification module, wherein the output module transmits the information to a recipient.

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 comprises: (a) an input module that receives a user request to amplify a target RNA in a biological sample; (b) an amplification module that, in response to the user request: receiving in a reaction vessel a reaction mixture comprising a biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for a target nucleic acid; and, subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein an individual series is different from at least one other individual series in the plurality of series with respect to the denaturing conditions and/or the extending conditions; and (c) an output module operably coupled to the amplification module, wherein the output module outputs information about the target RNA or DNA product to a recipient.

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 can include an electronic display screen having a user interface displaying graphical elements that can be accessed by a user to perform an amplification protocol to amplify a target nucleic acid in a biological sample. The system may further include a computer processor (including any suitable device having a computer processor as described elsewhere herein) coupled to the electronic display screen and programmed to execute the amplification protocol upon user selection of the graphical element. The amplification protocol may comprise: a reaction mixture comprising a biological sample and reagents necessary for performing nucleic acid amplification is subjected to a plurality of series of primer extension reactions to generate amplification products. The amplification product may indicate the presence of the target nucleic acid in the biological sample. In addition, each series of primer extension reactions may include two or more of the following cycles: the reaction mixture is incubated under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by incubation under extension conditions characterized by an extension temperature and an extension duration. The individual series may differ from at least one other individual series in the plurality of series with respect to denaturing conditions and/or extension conditions.

In some embodiments, the target nucleic acid can be associated with a disease. For example, the disease may be associated with an RNA virus or a DNA virus. Examples of viruses are provided elsewhere herein. In some embodiments, the disease may be associated with a pathogenic bacterium (e.g., mycobacterium tuberculosis) or a pathogenic protozoan (e.g., such as plasmodium in malaria), including examples of such pathogens described elsewhere herein. In some embodiments, the amplification protocol can be directed to determining the presence of the disease based on the presence of the amplification product.

In some cases, the user interface may be a graphical user interface. Further, the user interface may include one or more graphical elements. The graphical elements may include image and/or textual information, such as pictures, icons, and text. The graphical elements may have different sizes and orientations on the user interface. Further, the electronic display screen may 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 implementations, the electronic display screen may include a touch screen (e.g., a capacitive or resistive touch screen) such that graphical elements displayed on a user interface of the electronic display screen may be selected via a user touching the electronic display screen.

In some embodiments, the amplification protocol can further comprise selecting a primer set for the target nucleic acid. In such cases, the primer set can be a primer set specifically designed for amplifying one or more sequences of a target nucleic acid molecule. In some embodiments, the amplification protocol can further include selecting a reporter specific for one or more sequences of the target nucleic acid molecule (e.g., an oligonucleotide probe comprising an optically active species or other type of reporter described elsewhere herein). Further, in some embodiments, the reagents can include any suitable reagents necessary for nucleic acid amplification as described elsewhere herein, such as deoxyribonucleic acid (DNA) polymerase, a primer set for a target nucleic acid, and (optionally) reverse transcriptase.

In some embodiments, the user interface may display a plurality of graphical elements. Each graphical element may be associated with a given amplification protocol of a plurality of amplification protocols. Each of the plurality of amplification protocols may comprise a different combination of series of primer extension reactions. However, in some cases, the user interface may display multiple graphical elements associated with the same amplification protocol. An example of a user interface having multiple graphical elements each associated with a given amplification scheme is shown in FIG. 28A. As shown in fig. 28A, an exemplary electronic display screen 2800 associated with a computer processor includes a user interface 2801. User interface 2801 includes a display of

graphical elements

2802, 2803, and 2804. Each graphical element may be associated with a particular augmentation scheme (e.g., graphical element 2802 is "scheme 1 (prot.1)", graphical element 2803 is "scheme 2 (prot.2)", and graphical element 2804 is "scheme 4 (prot.4)"). Upon user selection (e.g., user touching when electronic display 2800 includes a touch screen with a user interface) of a particular graphical element, the particular augmentation scheme associated with that graphical element may be executed by the associated computer processor. For example, when the user selects graphical element 2803, the amplification "scheme 2" is performed by the associated computer processor. Although only three graphical elements are shown in the exemplary user interface 2801 of fig. 28A, the user interface may have any suitable number of graphical elements. Further, although each graphical element displayed in user interface 2801 of fig. 28A is associated with only one amplification protocol, each graphical element of the user interface may be associated with one or more amplification protocols (e.g., a series of amplification protocols), such that an associated computer processor executes the series of amplification protocols upon user interaction with the graphical element.

In some embodiments, each graphical element and/or may be associated with a disease, and a given amplification protocol of the plurality of amplification protocols may be directed to determining the presence of a disease in a subject. Thus, in such cases, the user may select a graphical element to run an amplification protocol (or a series of amplification protocols) to analyze a particular disease. In some embodiments, the disease may be associated with a virus (e.g., any RNA virus or DNA virus, including 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 (e.g., RNA-hepatitis c virus with a (RNA-HCV)), hepatitis delta virus, hepatitis e virus, hepatitis g virus, EB virus, mononucleosis virus, cytomegalovirus, SARS virus, west nile virus, poliovirus, measles virus, herpes simplex virus, smallpox virus, adenoviruses (e.g., adenovirus 55 (ADV55), adenovirus 7 (ADV 7)), and varicella virus. In some embodiments, the disease may be associated with a pathogenic bacterium (e.g., mycobacterium tuberculosis) or a pathogenic protozoan (e.g., plasmodium in malaria), including examples of such pathogens described elsewhere herein.

An example of a user interface having 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. User interface 2811 includes display of

graphical elements

2812, 2813, and 2814. Each graphical element may be associated with a particular disease (e.g., graphical element 2812 is "ebola," graphical element 2813 is "H1N 1," and graphical element 2814 is "Hep C (hepatitis C)"), which in turn is associated with one or more amplification schemes directed to the particular disease. Once a user selects (e.g., when the electronic display screen 2810 includes a touch screen with a user interface, the user touches) a particular graphical element, the particular augmentation scheme associated with the disease associated with that graphical element may be executed by the associated computer processor. For example, when a user interacts with graphical element 2812, one or more amplification schemes associated with analyzing ebola virus are executed by an associated computer processor. 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 that each correspond to multiple diseases. Further, although each graphical element displayed in the user interface 2811 of fig. 28B is associated with only one disease, each graphical element of the user interface may be associated with one or more diseases, such that the associated computer processor performs a series of amplification schemes (e.g., various individual amplification schemes directed to a particular disease) when the user selects the graphical element. For example, the graphical elements may correspond to ebola virus and H1N1 virus, such that selection of the graphical element causes the associated computer processor to execute an amplification scheme for both ebola virus and H1N1 virus.

In various aspects, the system includes an input module that receives a user request to amplify a target nucleic acid (e.g., target RNA, target DNA) present in a biological sample obtained directly from a subject. Any suitable module capable of receiving such user requests may 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., a computer processor) include: desktop computers, laptop computers, tablet computers (e.g.,

iPad、

galaxy Tab), a cellular phone, a smart phone (e.g.,

iPhone、

a supported telephone), a Personal Digital Assistant (PDA), a video game console, a television, a music playing device (e.g.,

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 embedded in firmware as needed. If implemented in software, the routine (or program) may be stored in any computer readable memory such as RAM, ROM, flash memory, magnetic disk, laser disk, or other storage medium. Likewise, the software may 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 disk, a flash drive, etc. The steps can be taken as various groups and operationsA tool, module or technique, which in turn may be implemented in hardware, firmware, software, or any combination thereof. When implemented in hardware, some or all of these granules, operations, techniques, etc. may be implemented in, for example, a custom Integrated Circuit (IC), an Application Specific Integrated Circuit (ASIC), a field programmable logic array (FPGA), a Programmable Logic Array (PLA), etc.

In some embodiments, the input module is configured to receive a user request to perform amplification of a target nucleic acid. The input module may receive the user request directly (e.g., through an input device such as a keyboard, mouse, or touch screen operated by the user) or indirectly (e.g., through a wired or wireless connection, including via the internet). The input module may provide the user's request to the amplification module via the output electronics. In some embodiments, the input module can include a User Interface (UI), such as a Graphical User Interface (GUI), 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 comprising a display of a device containing a computer processor. Such displays may include resistive or capacitive touch screens.

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, among others.

In aspects, the system includes an amplification module for performing a nucleic acid amplification reaction on a target nucleic acid or 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 any of a fluid processing device, one or more thermal cyclers, a device for receiving one or more reaction vessels (e.g., wells of a thermally cycled thermal block), a detector (e.g., an optical detector, a spectroscopic detector, an electrochemical detector) capable of detecting an amplification product, and a device for outputting information (e.g., raw data, processed data, or any other type of information described herein) to a recipient regarding the presence and/or amount of an amplification product (amplified DNA product). In some cases, the amplification module may comprise a device having a computer processor as described elsewhere herein, and may also be capable of analyzing raw data obtained from the detection with the aid of suitable software. Further, 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.

In some embodiments, one or more steps of providing materials to the reaction vessel, amplifying the nucleic acid, detecting the amplification product, and outputting information may be automated by the amplification module. In some embodiments, the automated operations may include the use of one or more fluidic processors and associated software. Several commercially available fluid handling systems may be employed to run the automated operation of such processes. Non-limiting examples of such fluidic processors include fluidic processors from Perkin-Elmer, Caliper Life Sciences, Tecan, Eppendorf, Apricot Design, and Velocity 11.

In some embodiments, the amplification module may comprise a real-time detection instrument. Non-limiting examples of such instruments include real-time PCR thermal cyclers, ABIs

7000 sequence detection system, ABI

7700 sequence detection system, Applied Biosystems 7300 real-time PCR system, Applied Biosystems 7500 real-time PCR system, Applied Biosystems 7900HT fast real-time PCR system (all from Applied Biosystems); LightCycler^TMSystem (Roche Diagnostics GmbH); mx3000P^TMReal-time PCR System, Mx3005P^TMReal-time PCR system and

multiplex quantitative PCR system(

Multiplex Quantitative PCR System) (Stratagene, La Jolla, Calif); and a Smart Cycler System (Cepheid, distributed by Fisher Scientific). In some embodiments, the amplification module may comprise another automated instrument, for example,

AmpliPrep/

systems (Roche Molecular Systems), TIGRIS DTS system (Hologic Gen-Probe, San Diego, Calif.), PANTHER system (Hologic Gen-Probe, San Diego, Calif.), BD MAX^TMSystems (Becton Dickinson), GeneXpert systems (Cepheid),

(BioFire Diagnostics) System, iCubate System, IDBox System (Luminex), Encompass MDx^TM(Rheonix) System, Liat^TMAanlyzer (IQuum) System, Molecular Diagnostic Platform System from Biocartis,

ML systems (Enigma Diagnostics),

Systems (T2 Biosystems),

System (NanoSphere), Diagnostic System of Great Basin, Unyvero^TMSystems (Curetis), PanNAT systems (Micronics) or Spartan^TMRX systems (Spartan Bioscience).

In various aspects, the system comprises an output module operably connected to an amplification module. In some embodiments, the output module may comprise 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, in some cases, the electronic display including 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 may 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 is capable of analyzing data received from the amplification module. In some cases, the output module comprises a report generator capable of generating and transmitting a report to a recipient, wherein 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 by software contained in the amplification module. Alternatively, the output module may transmit information after receiving a user instruction. The information conveyed by the output module can be viewed electronically or printed out by a printer.

One or more of the input module, amplification module, and output module may be contained in the same device, or may contain one or more of the same components. For example, an amplification module may also comprise an input module, an output module, or both. In other examples, a device including a processor may be included in both an input module and an output module. The user can use the device to request amplification of a target nucleic acid and can also use the device as a means to transmit information about the amplification product to a recipient. In some cases, a device containing a processor may be included in all three modules, such that the device containing a processor may also be used to control an instrument (e.g., a thermal cycler, a detector, a fluid handling device) included in an amplification module or any other module, provide instructions to the instrument, and receive information back from the instrument.

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 act as part of both an input module and an output module. The user places a reaction vessel 102 containing a reaction mixture ready for nucleic acid amplification into an amplification module 104. The amplification module includes a thermal cycler 105 and a detector 106. The input module 107 includes a computer 101 and associated input device 103 (e.g., keyboard, mouse, etc.), and the input device 103 can accept a user request for amplification of a target nucleic acid in a reaction mixture. Input module 107 communicates the user's request to amplification module 104 and nucleic acid amplification is initiated in thermal cycler 105. As the amplification proceeds, the detector 106 of the amplification module detects the amplification product. Information about the amplification product (e.g., raw data obtained by the detector) is transmitted from the detector 106 back to the computer 101, the computer 101 also serving as a component of the output module 108. Computer 101 receives information from 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 to its ultimate recipient 109 over a computer network (e.g., intranet, internet) via a computer network interface 110, in hard copy form via a printer 111, or via an electronic display 112 operatively connected to the computer 101. In some cases, electronic display 112

In one aspect, the invention provides a computer-readable medium containing machine-executable code that, when executed by one or more processors, performs a method according to any of the methods disclosed herein. In another aspect, the present invention provides a computer-readable medium comprising machine-executable code, which when executed by one or more computer processors, performs a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, the method comprising: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for a target RNA; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of cycles of primer extension reactions to generate amplified DNA products indicative of the presence of the target RNA, each cycle comprising (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (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.

In another aspect, the present invention provides a computer-readable medium comprising machine-executable code, which when executed by one or more computer processors, performs a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, the method comprising: (a) receiving a biological sample that has been obtained from a subject; (b) providing a reaction vessel comprising a biological sample and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase and (ii) a primer set for a target RNA, to obtain a reaction mixture; (c) subjecting the reaction mixture to a plurality of 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; (d) detecting the amount of the DNA product of (c); and (e) outputting information about the amount of the DNA product to a recipient, wherein the amount of time for completing (a) - (e) is less than or equal to about 30 minutes.

In one aspect, the present invention provides a computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained from a subject, the method comprising: (a) providing a reaction vessel comprising a biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for a target nucleic acid, to obtain a reaction mixture; and (b) subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products from the target nucleic acid, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein the individual series is different from at least one other individual series in the plurality of series with respect to the denaturing conditions and/or the extending conditions.

A 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, any storage device in any computer, etc., such as may be used to perform computing steps, processing steps, etc. Volatile storage media include dynamic memory, such as the main memory of a computer. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves such as those generated during Radio Frequency (RF) and Infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD, or DVD-ROM, any other optical medium, punch paper tape, any other physical storage medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link (link) carrying such a carrier wave, or any other medium from which a computer can read program code and/or data. Many of these computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The present invention provides embodiments including, but not limited to:

1. a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(a) providing a reaction vessel comprising the biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for the target RNA; and

(b) subjecting the reaction mixture in the reaction vessel to a plurality of cycles of a primer extension reaction to generate an amplified DNA product indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (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.

2. The method of embodiment 1, wherein the reagents further comprise a reporter that produces a detectable signal indicative of the presence of the amplified DNA product.

3. The method of embodiment 2, wherein the intensity of the detectable signal is proportional to the amount of the amplified DNA product or target RNA.

4. The method of embodiment 2, wherein the reporter is a dye.

5. The method of embodiment 1, wherein the primer set comprises one or more primers.

6. The method of embodiment 5, wherein the primer set comprises a first primer for generating a strand complementary to the target RNA.

7. The method of embodiment 1, wherein the target RNA is viral RNA.

8. The method of embodiment 1, wherein the reaction vessel comprises a body and a lid.

9. The method of embodiment 1, wherein the reaction vessel takes the form of a pipette tip.

10. The method of embodiment 1, wherein the reaction vessel is part of an array of reaction vessels.

11. The method of embodiment 10, wherein the reaction vessels are individually addressable by a liquid handling device.

12. The process of embodiment 1, wherein the reaction vessel comprises two or more hot zones.

13. The method of embodiment 1, wherein the denaturation temperature is between about 90 ℃ and 100 ℃.

14. The method of embodiment 13, wherein the denaturation temperature is about 92 ℃ to 95 ℃.

15. The method of embodiment 1, wherein the extension temperature is about 35 ℃ to 72 ℃.

16. The method of embodiment 15, wherein the extension temperature is about 45 ℃ to 65 ℃.

17. The method of embodiment 1, wherein the denaturation duration is less than or equal to 30 seconds.

18. The method of embodiment 1, wherein the extension duration is less than or equal to 30 seconds.

19. The method of embodiment 1, wherein the target RNA has not undergone concentration prior to step (a).

20. The method of embodiment 1, wherein, in step (a), the biological sample has not undergone RNA extraction.

21. The method of embodiment 1, further comprising the step of adding a lysing agent to the reaction vessel prior to or during step (a).

22. The method of embodiment 1, wherein the biological sample is a biological fluid from the subject.

23. The method of embodiment 1, wherein the amplifying produces a detectable amount of DNA product indicative of the presence of the target RNA in the biological sample at a cycle threshold (Ct) of less than 40.

24. The method of embodiment 1, wherein the amplifying produces a detectable amount of DNA product indicative of the presence of the target RNA in the biological sample over a period of 10 minutes or less.

25. The method of embodiment 1, wherein the reaction vessel is sealed.

26. The method of embodiment 1, wherein the DNA amplification is polymerase chain reaction.

27. The method of embodiment 26, wherein the polymerase chain reaction is a nested polymerase chain reaction.

28. The method of embodiment 1, wherein the target RNA is released from the biological sample during one or more cycles of the primer extension reaction.

29. A method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(a) receiving the biological sample that has been obtained from the subject;

(b) providing a reaction vessel comprising the biological sample and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase and (ii) a primer set for the target RNA, to obtain a reaction mixture;

(c) subjecting the reaction mixture to a plurality of 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;

(d) detecting the amount of amplified DNA product of (c); and

(e) outputting information about the amount of amplified DNA product to a recipient,

wherein the amount of time to complete (a) - (e) is less than or equal to about 30 minutes.

30. The method of embodiment 29, wherein the recipient is a physician, pharmaceutical company, or the subject undergoing treatment.

31. The method of embodiment 29, wherein (b) comprises DNA amplification.

32. The method of embodiment 29, wherein (c) is performed for 30 cycles or less.

33. The method of embodiment 29, wherein the information is output as a report.

34. The method of embodiment 29, wherein the message is output to an electronic display.

35. The method of embodiment 29, wherein the amount of time is less than or equal to 10 minutes.

36. A method of amplifying a target nucleic acid present in a biological sample obtained from a subject, comprising:

(a) providing a reaction vessel comprising the biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a deoxyribonucleic acid (DNA) polymerase and optionally a reverse transcriptase, and (ii) a primer set for the target nucleic acid, to obtain a reaction mixture; and

(b) subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein a single series is different from at least one other single series in the plurality of series with respect to the denaturing conditions and/or the extending conditions.

37. The method of embodiment 36, wherein the target nucleic acid is a ribonucleic acid.

38. The method of embodiment 36, wherein the reagents are necessary for reverse transcription amplification in parallel with deoxyribonucleic acid amplification.

39. The method of embodiment 36, wherein, in (b), the amplification product is an amplified deoxyribonucleic acid product.

40. The method of embodiment 36, wherein, in (a), the biological sample is not purified.

41. The method of embodiment 36, wherein, in (a), the biological sample is concentrated.

42. The method of embodiment 36, wherein in (a), the biological sample is diluted.

43. The method of embodiment 36, further comprising subjecting the target nucleic acid to one or more denaturing conditions prior to (b).

44. The method of embodiment 43, wherein the one or more denaturing conditions are selected from the group consisting of a denaturing temperature profile and a denaturing agent.

45. The method of embodiment 36, further comprising subjecting the target nucleic acid to one or more denaturing conditions between the first and second series of the plurality of series of primer extension reactions.

46. The method of embodiment 36, wherein the individual series differ with respect to at least any one of a rate of ramping between the denaturation temperature and the extension temperature, a denaturation duration, an extension temperature, and an extension duration.

47. The method of embodiment 46, wherein the single series differs with respect to at least any two of ramp rate between denaturation temperature and extension temperature, denaturation duration, extension temperature, and extension duration.

48. The method of embodiment 36, wherein the plurality of series of primer extension reactions comprises a first series comprising more than 10 cycles, each cycle of the first series comprising (i) incubating the reaction mixture at about 92-95 ℃ for no more than 30 seconds, followed by (ii) incubating the reaction mixture at about 35-65 ℃ for no more than 1 minute, and a second series comprising more than 10 cycles, each cycle of the second series comprising (i) incubating the reaction mixture at about 92-95 ℃ for no more than 30 seconds, followed by (ii) incubating the reaction mixture at about 40-60 ℃ for no more than 1 minute.

49. The method of embodiment 36, wherein the plurality of series of primer extension reactions produce a detectable amount of amplification product indicative of the presence of the target nucleic acid in the biological sample at a lower cycle threshold than a single series of primer extension reactions under comparable denaturing and extension conditions.

50. The method of embodiment 36, further comprising, prior to (b), pre-heating the biological sample at a pre-heating temperature of 90 ℃ to 100 ℃ for a pre-heating duration of no more than 10 minutes.

51. The method of embodiment 50, wherein the duration of preheating is no more than about 1 minute.

52. A system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(a) an input module that receives a user request to amplify the target RNA in the biological sample;

(b) an amplification module that, in response to the user request:

receiving in a reaction vessel a reaction mixture comprising the biological sample and reagents necessary for reverse transcription amplification in parallel with deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for the target RNA; and

subjecting the reaction mixture in the reaction vessel to a plurality of cycles of a primer extension reaction to generate an amplified DNA product indicative of the presence of the target RNA, each cycle comprising: (i) incubating the reaction mixture at a denaturation temperature for a denaturation duration of less than or equal to 60 seconds, followed by (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; and

(c) an output module operably coupled to the amplification module, wherein the output module outputs information about the target RNA or the DNA product to a recipient.

53. A system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(b) an amplification module that, in response to the user request:

(i) receiving in a reaction vessel a reaction mixture comprising said biological sample that has been obtained from said subject and reagents necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, said reagents comprising (1) a reverse transcriptase, and (2) a primer set for said target RNA; and

(ii) subjecting the reaction mixture to a plurality of 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;

(iii) (iv) detecting said amount of amplified DNA product of (iii); and

(iv) (iii) outputting information about the amount of amplified DNA product to a recipient, wherein the amount of time for completing (i) - (iv) is less than or equal to about 30 minutes; and

(c) an output module operably coupled to the amplification module, wherein the output module communicates the information to a recipient.

54. A system for amplifying a target nucleic acid present in a biological sample obtained from a subject, comprising:

(a) an input module that receives a user request to amplify the target nucleic acid in the biological sample;

(b) an amplification module that, in response to the user request:

receiving in a reaction vessel a reaction mixture comprising the biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for the target nucleic acid; and

subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more of the following cycles: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein a single series is different from at least one other single series in the plurality of series with respect to the denaturing conditions and/or the extending conditions; and

(c) an output module operably coupled to the amplification module, wherein the output module outputs information about the target nucleic acid or the amplification product to a recipient.

55. A computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target nucleic acid (RNA) present in a biological sample obtained directly from a subject, the method comprising:

56. A computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, the method comprising:

(a) receiving the biological sample that has been obtained from the subject;

(d) detecting said amount of the DNA product of (c); and

(e) outputting information about the amount of DNA product to a recipient,

57. A computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target nucleic acid present in a biological sample obtained from a subject, the method comprising:

(a) providing a reaction vessel comprising the biological sample and reagents necessary for performing nucleic acid amplification, the reagents comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for the target nucleic acid, to obtain a reaction mixture; and

58. A system for amplifying a target nucleic acid in a biological sample obtained from a subject, comprising:

an electronic display screen comprising a user interface displaying graphical elements accessible by a user to perform an amplification protocol to amplify the target nucleic acid in the biological sample; and

a computer processor coupled to the electronic display screen and programmed to execute the augmentation protocol upon the user selecting the graphical element, the augmentation protocol comprising:

subjecting a reaction mixture comprising the biological sample and reagents necessary for performing nucleic acid amplification to a plurality of series of primer extension reactions to generate amplification products indicative of the presence of the target nucleic acid in the biological sample, each series comprising two or more cycles of: (i) incubating the reaction mixture under denaturing conditions characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under extending conditions characterized by an extending temperature and an extending duration, wherein a single series is different from at least one other single series in the plurality of series with respect to the denaturing conditions and/or the extending conditions.

59. The system of embodiment 58, wherein the amplification protocol further comprises selecting a primer set for the target nucleic acid.

60. The system of embodiment 58, wherein the reagents comprise (i) a deoxyribonucleic acid (DNA) polymerase and optionally a reverse transcriptase, and (ii) a primer set for the target nucleic acid.

61. The system of embodiment 58, wherein the user interface displays a plurality of graphical elements, wherein each of the graphical elements is associated with a given amplification protocol of a plurality of amplification protocols.

62. The system of embodiment 61, wherein each of said graphical elements is associated with a disease, and wherein a given amplification protocol of said plurality of amplification protocols is directed to determining the presence of said disease in said subject.

63. The system of embodiment 62, wherein the disease is associated with a virus.

64. The system of embodiment 63, wherein the virus is an RNA virus.

65. The system of embodiment 63, wherein the virus is a DNA virus.

66. The system of embodiment 63, wherein the virus is 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 A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, EB virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile fever virus, poliovirus, measles virus, herpes simplex virus, smallpox virus, adenovirus, and varicella virus.

67. The system of embodiment 66, wherein the influenza virus is selected from the group consisting of a H1N1 virus, a H3N2 virus, a H7N9 virus, and a H5N1 virus.

68. The system of embodiment 66, wherein the adenovirus is adenovirus type 55 (ADV55) or adenovirus type 7 (ADV 7).

69. The system of embodiment 66, wherein the hepatitis C virus is an RNA-hepatitis C virus with A (RNA-HCV).

70. The system of embodiment 61, wherein the disease is associated with a pathogenic bacterium or a pathogenic protozoan.

71. The system of embodiment 68, wherein the pathogenic bacteria is Mycobacterium tuberculosis.

72. The system of embodiment 68, wherein the pathogenic protozoan is a Plasmodium.

73. The system of embodiment 58, wherein the target nucleic acid is associated with a disease.

74. The system of embodiment 73, wherein said amplification protocol is directed to determining the presence of said disease based on the presence of said amplification product.

75. The system of embodiment 73, wherein the disease is associated with a virus.

76. The system of embodiment 75, wherein the virus is an RNA virus.

77. The system of embodiment 75, wherein the virus is a DNA virus.

78. The system of embodiment 75, wherein the virus is 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 A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, EB virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile fever virus, poliovirus, measles virus, herpes simplex virus, smallpox virus, adenovirus, and varicella virus.

79. The system of embodiment 78, wherein the influenza virus is selected from the group consisting of an H1N1 virus, an H3N2 virus, an H7N9 virus, and an H5N1 virus.

80. The system of embodiment 78, wherein the adenovirus is adenovirus type 55 (ADV55) or adenovirus type 7 (ADV 7).

81. The system of embodiment 78, wherein the hepatitis c virus is an RNA-hepatitis c virus with a (RNA-HCV).

82. The system of embodiment 73, wherein the disease is associated with a pathogenic bacterium or a pathogenic protozoan.

83. The system of embodiment 82, wherein the pathogenic bacteria is Mycobacterium tuberculosis.

84. The system of embodiment 82, wherein the pathogenic protozoan is a plasmodium.

Examples

Example 1: amplification and detection of nucleic acids in viral stock samples and biological samples

Amplification and detection experiments were performed to compare the results obtained from the virus standard sample and the biological sample. Subjecting a biological sample comprising an RNA viral pathogen and a standard sample of the viral pathogen to amplification conditions, thereby amplifying RNA of the pathogen. A set of experiments was performed on each of 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 a stock solution containing the virus. H3N2 and H1N1(2007) at a concentration of 10⁶IU/mL. Dilutions of 1/2, 1/20, 1/200, 1/2000 and 1/20000 were amplified for H5N1 and H1N1 (2007). In each experimental group, negative controls (e.g., samples that did not contain viral RNA) were also amplified.

5 microliters of each sample was combined in a 25 μ L reaction tube with the reagents necessary to perform reverse transcription of viral RNA and the reagents necessary to complete amplification of complementary DNA obtained from reverse transcription (e.g., parallel nucleic acid amplification). The reagents necessary for performing reverse transcription and DNA amplification are provided as a commercially available pre-mix (e.g., Qiagen One-Step RT-PCR or One-Step RT-qPCR kit) comprising reverse transcriptase (e.g., Sensiscript and Omniscript transcriptases), DNA polymerase (e.g., HotStarTaq DNA polymerase) and dNTPs. In addition, the reaction tube contains a TaqMan probe containing FAM dye for detecting amplified DNA products. 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 comprising 5 minutes at 95 ℃, followed by 20 minutes at 45 ℃, then 2 minutes at 95 ℃, and then 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 55 ℃. Detection of the amplification product is performed during the incubation.

The amplification results for H3N2 are graphically shown in fig. 2 (fig. 2A for each viral standard sample, fig. 2B for the biological sample), while the amplification results for H1N1(2007) are graphically shown in fig. 3 (fig. 3A for each viral standard sample, fig. 3B for the biological sample). The fluorescence of the recorded FAM dyes was plotted against cycle number.

As shown in fig. 2A, each H3N2 virus standard sample showed a detectable signal relative to the negative control, with a Ct value ranging from 18 to 32. As shown in FIG. 2B, each of the virus H3N2 biological samples showed detectable signal relative to the negative control, with Ct values ranging from 29 to 35.

As shown in fig. 3A and with the exception of the 1/20000 dilution, each H1N1(2007) virus standard sample showed a detectable signal relative to the negative control with a Ct value ranging from 24-35. As shown in FIG. 3B, each H1N1(2007) biological sample showed a detectable signal relative to the negative control, with a Ct value ranging from 28 to 35.

Overall, the data shown in fig. 2 and 3 indicate that the tested virus is detectable via amplified DNA products at concentrations as low as 50IU/mL and over a 4-log concentration range with good sensitivity, with a cycle threshold of no more than about 40. Furthermore, the data also indicate that detection of viral RNA obtained from a biological sample obtained from a subject can also be detected in a similar manner.

Example 2: amplification and detection of viral nucleic acids in different buffer systems

Amplification and detection experiments were performed to compare the results obtained using different buffer systems for amplification. One set of experiments was performed for two different buffer systems (S1 and S2). The S1 buffer contained zwitterionic buffer and BSA, while the S2 buffer contained zwitterionic bufferAn agent and sodium hydroxide. Experiments for each buffer were completed using a set of H5N1 influenza virus standard samples obtained as serial dilutions of stock solutions containing the virus. The concentration of H5N1 was 10⁶IU/mL. Dilutions of 1/2, 1/20, 1/200, 1/2000, 1/20000, 1/200000 and negative controls were amplified.

5 microliters of each sample was combined in a 25 μ L reaction tube with the reagents necessary to perform reverse transcription of viral RNA and the reagents necessary to complete amplification of complementary DNA obtained from reverse transcription (e.g., parallel nucleic acid amplification). Reagents necessary for reverse transcription and DNA amplification include reverse transcriptase, DNA polymerase, dNTPs and appropriate S1 or S2 buffer. In addition, the reaction tube contains a TaqMan probe containing FAM dye for detecting amplified DNA products. 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 comprising 5 minutes at 95 ℃, followed by 20 minutes at 45 ℃, then 2 minutes at 95 ℃, followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 55 ℃. Detection of the amplification product is performed during the incubation.

The amplification results for buffer system S1 are shown graphically in FIG. 4A, while the amplification results for buffer system S2 are shown graphically in FIG. 4B. The fluorescence of the recorded FAM dyes was plotted against cycle number.

As shown in fig. 4A, each virus standard sample amplified in buffer system S1 showed a detectable signal relative to the negative control, with a Ct value ranging from 25 to 36. As shown in FIG. 4B, each of the virus standard samples amplified in buffer S2 showed a detectable signal with a Ct value ranging from 25 to 35 relative to the negative control.

Overall, the data shown in figure 4 indicate that the tested virus is detectable via amplified DNA products at concentrations as low as 50IU/mL and over a 5-log concentration range with good sensitivity, with a cycle threshold of no more than about 40. Furthermore, the data also show that similar amplification results can be obtained using different buffer systems.

Example 3: amplification and detection of Hepatitis B Virus (HBV) in plasma samples

Amplification experiments were performed to determine the robustness of amplification methods for detecting target nucleic acids in biological samples. The amplification reactions were performed separately on diluted human plasma samples containing different concentrations of Hepatitis B Virus (HBV), e.g., 50 infectious units/mL (IU/mL), 200IU/mL, 2000IU/mL, 20000 IU/mL. HBV is a DNA virus that replicates via an RNA intermediate. HBV is detectable via direct PCR of DNA viruses. In addition to negative control samples (e.g., plasma containing no HBV), samples at various concentrations (n-2-4) were also tested.

mu.L of each sample was placed in a 50. mu.L reaction tube with reagents necessary for carrying out reverse transcription of RNA and reagents necessary for completing amplification of complementary DNA obtained from reverse transcription (e.g., parallel nucleic acid amplification) to obtain a reaction mixture. The reagents necessary for performing reverse transcription and DNA amplification are provided as a commercially available pre-mix (e.g., Qiagen One-Step RT-PCR or One-Step RT-qPCR kit) comprising reverse transcriptase (e.g., Sensiscript and Omniscript transcriptases), DNA polymerase (e.g., HotStarTaq DNA polymerase) and dNTPs. In addition, the mixture also contains a TaqMan probe that contains FAM dye for detecting amplified DNA products. The reaction mixture also contains a zwitterionic buffer and uracil-DNA glycosylase (UNG) to prevent the inhibitory effect of amplification inhibitors found in plasma. Each reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol for denaturation and extension conditions comprising 1 minute at 94 ℃, followed by 10 minutes at 50 ℃, then 2 minutes at 94 ℃, followed by 50 cycles of 5 seconds at 94 ℃ and 35 seconds at 58 ℃. Detection of the amplification product is performed during the incubation.

The amplification results are graphically shown in FIG. 5, and the Ct values determined are tabulated in Table 1. In fig. 5, the Relative Fluorescence Units (RFU) of the FAM dyes recorded are plotted against cycle number. As shown in fig. 5 and table 1, HBV was detectable at each concentration tested, with cycling thresholds ranging from 28.99 to 39.39. Generally, a higher concentration of sample corresponds to a lower cycling threshold.

Overall, the data shown in figure 5 and table 1 indicate that HBV is detectable via amplified DNA product at concentrations as low as 50IU/mL (lowest value tested) with good sensitivity, with a cycle threshold of no more than about 40. Although the highest concentration tested (20000IU/mL) was 400 times more concentrated than the lowest concentration tested (50IU/mL), the cycle threshold was only about 25% higher for the lower concentrations, indicating that the amplification protocol is overall robust.

Table 1: ct results for the experiment in example 3

Example 4: preheating a biological sample and a series of amplification reactions prior to amplifying nucleic acids in the biological sample

Amplification experiments were performed to determine the effect of preheating the biological sample on detection sensitivity and also to determine the effect of using multiple series of amplification reactions on detection sensitivity.

20 aliquots of 25 μ L of reaction mix were prepared, each containing 1 μ L of the pathogenic species, the reagents necessary to complete the appropriate nucleic acid amplification reaction (e.g., reverse transcription and DNA amplification of RNA species, and DNA amplification of DNA species), and a TaqMan probe containing a FAM dye. Wherein four reaction mixtures comprise H1N1(2007) (i.e., RNA viruses), four reaction mixtures comprise H3N2 (i.e., RNA viruses), four reaction mixtures comprise H1N1(2009), four reaction mixtures comprise mycobacterium Tuberculosis (TB) (i.e., bacterial samples), and four reaction mixtures comprise Aleutian Disease Virus (ADV) (i.e., DNA viruses). H1N1(2007), H1N1(2009), H3N2, and ADV pathogenic species were derived from oropharyngeal swabs obtained from subjects. TB was obtained from a bacterial stock.

Various combinations of preheat and amplification protocols were used and are summarized in table 2. For the first reaction mixture of various pathogenic species, the pathogenic species were preheated at 95 ℃ for 10 minutes before being added to the reaction mixture. After the addition of the pathogenic species to the reaction mixture, the reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol for denaturation and extension conditions comprising 2 minutes at 95 ℃ followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 55 ℃. Detection of the amplification product is performed during the incubation. These reaction mixtures are referred to as pH-1 mixtures.

For the second reaction mixture of each pathogenic species, the pathogenic species was preheated at 50 ℃ for 30 minutes before being added to the reaction mixture. After the addition of the pathogenic species to the reaction mixture, the reaction mixture was incubated in a real-time PCR thermal cycler according to a protocol for denaturation and extension conditions comprising 2 minutes at 95 ℃ followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 55 ℃. Detection of the amplification product is performed during the incubation. These reaction mixtures are referred to as pH-2 mixtures.

For the third reaction mixture of each pathogenic species, the pathogenic species is not preheated prior to addition to the reaction mixture. The reaction mixtures were incubated in a real-time PCR thermal cycler according to a protocol for denaturation and extension conditions comprising 1 minute at 95 ℃, followed by 10 minutes at 55 ℃, then 2 minutes at 95 ℃, followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 55 ℃. Detection of the amplification product is performed during the incubation. These reaction mixtures are referred to as PTC-1 mixtures.

For the fourth reaction mixture of each pathogenic species, the pathogenic species is not preheated prior to addition to the reaction mixture. These reaction mixtures are subjected to a protocol comprising a plurality of series of amplification reactions, each series comprising a plurality of cycles of denaturing and extension conditions. The reaction mixture was incubated in a real-time PCR thermal cycler according to this protocol, which included 1 minute at 95 ℃, followed by 10 cycles in series 1 (5 seconds at 95 ℃, 20 seconds at 60-50 ℃ (step down at 1 ℃/cycle), and 10 seconds at 60 ℃), then 2 minutes at 95 ℃, followed by 40 cycles in series 2 (5 seconds at 95 ℃, 30 seconds at 55 ℃). Series 1 and series 2 differ in their extension temperature and extension duration. Detection of the amplification product is performed during the incubation. These reaction mixtures are referred to as PTC-2 mixtures.

Table 2: experimental conditions for example 4

The results of the various pathogenic species are graphically shown in fig. 6(H1N1(2007)), fig. 7(H3N2), fig. 8(H1N1(2009)), fig. 9(TB), and fig. 10 (ADV). Item A in each of FIGS. 6 to 10 represents the results obtained for the reaction mixtures PH-1 and PH-2, and item B in each of FIGS. 6 to 10 represents the results obtained for the reaction mixtures PTC-1 and PTC-2. The Ct values determined for each experiment are summarized in table 3. Ct values could not be determined for the pH-1 and pH-2ADV reaction mixtures, which correspond to the data shown in FIG. 10A.

According to the data presented in Table 3, Ct values between the PH-1 and PH-2 reaction mixtures are very similar, which indicates that pathogenic species (or biological samples containing pathogenic species) can be preheated under a range of conditions to achieve similar detection sensitivity. In addition, the PTC-1 reaction mixture has a Ct value similar to that determined for the pH-1 and pH-2 reaction mixtures. PTC-1 is similar to the PH-1/PH-2 protocol, except that PTC-1 does not include a pre-heating step. Thus, comparison of the PTC-1 data with the PH-1/PH-2 data shows 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 were used, preheating may be worse than not preheating.

However, the Ct value for PTC-2 was lower than that of any of pH-1, pH-2 or PTC-1 for all pathogenic species tested. Comparison of the PTC-1 and PTC-2 data shows that: subjecting the reaction mixture to a plurality of series of amplification reactions (each series comprising a plurality of cycles of denaturing and extension conditions) may improve detection sensitivity.

Table 3: ct results for the experiment in example 4

Example 5: multiplexing of samples (Multiplexing)

Amplification and detection experiments were performed to benchmark (benchmark) various amplification protocols and determine whether multiplexing could be achieved. Biological samples comprising RNA (e.g., H1N1(2007), H1N1(2009), H3N2) or DNA (e.g., ADV, human bocavirus (HBoV) viral pathogen, or DNA bacterial pathogen (e.g., TB)) are subjected to various amplification conditions. Each biological sample was obtained directly from the subject via an oropharyngeal swab, except for TB samples from bacterial stocks. 1 microliter of each sample was combined in a 25. mu.L reaction tube with the reagents necessary to perform nucleic acid amplification and detection of the amplification product as described herein to obtain a reaction mixture.

To evaluate the multiplexing capacity of the amplification protocol, three reaction mixtures (each comprising one of H3N2, ADV, or a mixture of H3N2 and ADV) were incubated according to an amplification protocol comprising 50 cycles of 2 minutes at 94 ℃, 20 minutes at 45 ℃, 1 minute at 94 ℃, followed by 5 seconds at 94 ℃ and 35 seconds at 55 ℃ in a real-time PCR thermal cycler. Detection of the amplification product is performed during the incubation.

The results of the experiment are graphically shown in fig. 11 and in table 4 below. As shown in fig. 11, both H3N2 and TB can be similarly detected when combined together or when the other is not present. A Ct value of 26.03 was recorded for the H3N2 reaction mixture when ADV was not present, and 30.5 was recorded for the ADV reaction mixture when H3N2 was not present. When H3N2 and ADV were combined into a single reaction mixture, Ct values of 26(H3N2) and 30(ADV) were obtained. The Ct values of the combined reaction mixtures were almost identical compared to the single component reaction mixtures. The results show that: multiplexing can be achieved with good sensitivity, and both RNA and DNA species can be detected.

Table 4: results of H3N2 and ADV multiplexing experiments in example 5

Type (B)	Sample (I)	Ct
			RNA virus	H3N2	26.03
DNA virus	ADV	30.5
			RNA and DNA viruses	H3N2 and ADV	26(H3N2) and 30(ADV)

In another experiment to evaluate the multiplexing capacity of the amplification protocol, three reaction mixtures (each comprising one of H3N2, TB or a mixture of H3N2 and TB) were incubated according to the amplification protocol in a real-time PCR thermal cycler (including 2 minutes at 95 ℃, followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 55 ℃). Detection of the amplification product is performed during the incubation.

The results of the experiment are graphically shown in fig. 12 and in table 5 below. As shown in fig. 12, both H3N2 and TB can be similarly detected when combined together or when the other is not present. A Ct value of 32 was recorded for the H3N2 reaction mixture when TB was absent and 32 for the TB reaction mixture when H3N2 was absent. When H3N2 and TB were combined into a single reaction mixture, Ct values were obtained for 29(H3N2) and 30 (TB). The Ct values for the combined reaction mixtures were similar compared to the single component reaction mixtures. The results show that: multiplexing can be achieved with good sensitivity, and in a multiplexing scheme, both RNA and DNA species can be detected.

Table 5: results of H3N2 and TB multiplex experiments in example 5

Example 6: benchmarking of multiple series of amplification reactions

Amplification and detection experiments were performed to benchmark various amplification protocols including multiple series of amplification reactions. Biological samples comprising RNA (e.g., H1N1(2007), H1N1(2009), H3N2) or DNA (e.g., ADV, human bocavirus (HBoV) viral pathogen, or DNA bacterial pathogen (e.g., TB)) are subjected to various amplification conditions. Each biological sample was obtained directly from the subject via an oropharyngeal swab, except for TB samples from bacterial stocks. 1 microliter of each sample was combined in a 25. mu.L reaction tube with the reagents necessary to perform nucleic acid amplification and detection of the amplification product as described herein to obtain a reaction mixture.

In one set of experiments, the amplification mixture was subjected to an amplification protocol comprising two series of amplification reactions, each series comprising different denaturation and extension conditions. Six reaction mixtures (two containing H3N2, two containing ADV, two containing HBoV) were incubated in a real-time PCR thermal cycler according to an amplification protocol comprising 1 second at 94 ℃, followed by 11 cycles of series 1 (1 second at 94 ℃ and 10 seconds at 45 ℃) followed by 40 cycles of series 2 (5 seconds at 95 ℃ and 30 seconds at 55 ℃). Detection of the amplification product is performed during the incubation.

The results of the experiment are shown in table 6 below. As shown in table 6, the Ct values determined ranged from 8.35 to 23. The results show that: protocols involving multiple series of amplification reactions can be used to achieve good sensitivity. Furthermore, the results also show that: both RNA and DNA species can be detected using a protocol that includes multiple series of amplification reactions.

Table 6: results of H3N2, ADV and HBoV experiments in example 6

In another set of experiments, the amplification mixture was subjected to an amplification protocol comprising three series of amplification reactions, each series differing from each other with respect to their denaturing and/or extension conditions. Five reaction mixtures (one containing sH1N1(2007), one containing H3N2, one containing pH1N1(2009), one containing ADV, and one containing TB) were incubated in a real-time PCR thermal cycler according to an amplification protocol comprising 1 minute at 94 ℃, followed by 5 cycles of series 1 (5 seconds at 94 ℃, and 30 seconds at 60-50 ℃ (stepped down at 1 ℃/cycle)), followed by 5 cycles of series 2 (5 seconds at 94 ℃ and 30 seconds at 50 ℃), followed by 2 minutes at 95 ℃, followed by 40 cycles of series 3 (5 seconds at 95 ℃ and 30 seconds at 55 ℃). Detection of the amplification product is performed during the incubation.

The results of the experiment are shown in table 7 below. As shown in table 7, the Ct values determined range from 20 to 30. The results show that: protocols involving multiple series of amplification reactions can be used to achieve good sensitivity. Furthermore, the results also show that: both RNA and DNA species can be detected using a protocol that includes multiple series of amplification reactions.

Table 7: results of sH1N1(2007), H3N2, pH1N1(2009), ADV, and TB experiments in example 6

Example 7: benchmarking of multiple series of amplification reactions

Amplification and detection experiments were performed to benchmark various amplification protocols including multiple series of amplification reactions. Biological samples comprising H3N2 were subjected to various amplification conditions. Each biological sample was obtained directly from the subject via an oropharyngeal swab. 1 microliter of each sample was combined in a 25. mu.L reaction tube with the reagents necessary to perform nucleic acid amplification and detection of the amplification product as described herein to obtain a reaction mixture.

Subjecting the amplification mixture to an amplification protocol, some protocols including one of three different first series of amplification reactions comprising different denaturing and extension conditions than the second series, and a second series that is the same. Each of the first series and the second series includes a plurality of cycles. Another experiment was performed without the first series, which included only the second series. Each of four aliquots of the reaction mixture containing H3N2 were incubated in a real-time PCR thermal cycler according to one of the amplification protocols shown in table 8 below:

table 8: experimental protocol in example 7

The results of the experiment are graphically shown in fig. 13 and are listed below in table 9. As shown in fig. 13, reaction mixture 3 had the highest Ct value (28.59). Other reaction mixtures comprising multiple series have lower values ranging from 8.5 to 26.5. The results show that: protocols involving multiple series of amplification reactions can be used to achieve good sensitivity. Furthermore, the results also show that: protocols involving multiple series of amplification reactions can achieve better sensitivity than protocols with only a single series.

Table 9: experimental results of example 7

Reaction mixture	Ct
			1	22.97
2	26.5
		3	28.59
4	8.5

Example 8: benchmarking of multiple series of amplification reactions

Subjecting the amplification mixture to an amplification protocol, some protocols comprising one of six first series of amplification reactions comprising different denaturing and extension conditions than the second series, and a second series that are identical. Six additional experiments were performed without the first series. Each of twelve H3N 2-containing reaction mixtures was incubated in a real-time PCR thermal cycler according to one of the amplification protocols shown in table 10 below:

table 10: experimental protocol in example 8

The results of the experiments are listed in table 11 below. Ct values ranged from 14.53 to 27.28, and no product was detected in reaction mixtures 2-5. In general, reaction mixtures that have not undergone multiple series of amplification reactions either have no detectable product or have a higher Ct value than reaction mixtures that have undergone multiple series of amplification reactions. The results show that: protocols involving multiple series of amplification reactions can be used to achieve good sensitivity. Furthermore, the results also show that: protocols involving multiple series of amplification reactions can achieve better sensitivity than protocols with only a single series. In some cases, multiple series of amplification reactions may be necessary to produce detectable amounts of amplification product.

Table 11: experimental results of example 8

Example 9: results comparing purified and unpurified samples

Amplification and detection experiments were performed to compare the results obtained with purified and unpurified samples. Purified and unpurified biological samples comprising H1N1 were subjected to an amplification protocol. Each biological sample was obtained directly from the subject via an oropharyngeal swab. 1 microliter of each sample was combined in a 25. mu.L reaction tube with the reagents necessary to perform nucleic acid amplification and detection of the amplification product as described herein to obtain a reaction mixture. Three reaction mixtures were generated, wherein two reaction mixtures contained samples purified by one of column purification or magnetic purification. The third reaction mixture contained an unpurified sample.

The reaction mixture was incubated in a real-time PCR thermal cycler according to an amplification protocol comprising 50 cycles of 2 minutes at 94 ℃, 20 minutes at 45 ℃, 1 minute at 94 ℃ followed by 5 seconds at 94 ℃ and 35 seconds at 55 ℃. Detection of the amplification product is performed during the incubation.

The results of the experiment are graphically shown in fig. 14 and are shown below in table 12. As shown in table 12, the Ct values determined range from 27 to 31 and are similar between the unpurified samples and the samples purified by various means. The results show that: purification of the sample may not be necessary to achieve similar detection sensitivity.

Table 12: experimental results of example 9

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. Both samples contained one of a whole blood or saliva sample, and both samples contained a 10-fold dilution (in PBS) of one of a whole blood or saliva sample. Each of these four samples was combined with the reagents necessary to perform reverse transcription of viral RNA and the reagents necessary to complete amplification of the complementary DNA obtained from reverse transcription. The reagents necessary for performing reverse transcription and DNA amplification are provided as a commercially available pre-mix (e.g., Takara One-Step RT-PCR or One-Step RT-qPCR kit) comprising reverse transcriptase (e.g., Sensiscript and Omniscript transcriptases), DNA polymerase (e.g., HotStarTaq DNA polymerase) and dNTPs. In addition, the reaction tube contains a TaqMan probe containing FAM dye for detecting amplified DNA products. 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 comprising 20 minutes at 45 ℃, followed by 2 minutes at 94 ℃, followed by 42 cycles of 5 seconds at 94 ℃ and 35 seconds at 55 ℃. Detection of the amplification product is performed during the incubation.

The amplification results of H3N2 are graphically shown in fig. 15 (fig. 15A corresponding to undiluted blood, fig. 15B corresponding to diluted blood) and fig. 16 (fig. 16A corresponding to undiluted saliva, fig. 16B corresponding to diluted saliva). The fluorescence of the recorded FAM dyes was plotted against cycle number.

As shown in FIGS. 15 and 16, both the undiluted and diluted blood and saliva reaction mixtures showed detectable signals with Ct values ranging from 24 to 33. Thus, the data shown in fig. 15 and 16 indicate that: undiluted biological samples can be analyzed with good sensitivity and with Ct values not greater than about 40. Furthermore, the data also show that: in cases where dilution of the sample is necessary for analysis, the amplification product can also be detected in a similar manner. In some cases, dilution may be another way to eliminate inhibition from a sample (e.g., whole blood) if there is too much inhibitor from the sample.

Example 11: nested PCR

Amplification and detection experiments were performed on samples containing H1N1 virus. Eight samples were tested. Each sample included a H1N1(2007) viral stock. The samples were diluted in PBS at the dilutions shown in table 13 below. Viral stock concentration 1x10⁶IU/mL. To generate amplified DNA products, a reaction mixture containing a given sample is incubated according to a protocol of denaturing and extension conditions. The scheme comprises the following steps: (i) in the first run, the mixture was heated in a thermal cycler at 94 ℃ for 1 minute, followed by 10 or 15 cycles as follows (as shown in table 13 below): 5 seconds at 94 ℃ and 10 seconds at 57 ℃; and (ii) in a second run, the mixture was heated in a thermocycler at 94 ℃ for 1 minute, followed by 35 cycles of: 5 seconds at 94 ℃ and 30 seconds at 57 ℃. Add 1. mu.L of serially diluted samples to Takar in a reaction volume of 25. mu.La One-step qPCR premix. After some cycles of the first run, 1 μ Ι _ from the reaction was added to the reaction mixture of the second run. The amplification results for H1N1 are graphically shown in FIG. 17. The graph shows the Relative Fluorescence Units (RFU) recorded as a function of cycle number. The graph for each of the 8 samples (1-8) has been shown in the figure. The samples with detectable signals had Ct values as shown in table 13.

Table 13: experimental results of example 11

Example 12: amplification and detection of Ebola recombinant plasmids

Amplification and detection experiments were performed on human whole blood samples containing different copy numbers of recombinant plasmids corresponding to Zaire ebola virus (Zaire-EBOV). Eight samples were tested. Six of the samples contained specific copy numbers (250000, 25000, 2500, 250, 25, and 2.5 copies) of recombinant plasmid, while two samples (one containing only blood and one containing only water) served as control samples. The whole blood sample was analyzed without sample purification.

Each sample is combined into a reaction mixture with reagents necessary for nucleic acid amplification (e.g., DNA polymerase, dntps, primers, cofactors, suitable buffers, primers, etc.) and a reporter (e.g., an oligonucleotide probe comprising a FAM dye). A summary of the reaction mixtures (including the copy number of the recombinant plasmid) according to the sample numbers is shown in Table 14. To generate the amplification 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 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 45 cycles of 5 seconds at 95 ℃ and 10 seconds at 55 ℃ are carried out. During the second series, the signal from the reporter was recorded to generate an amplification curve and Ct values were obtained. The amplification curves for this experiment are shown graphically in fig. 18, each curve being labeled with a sample number corresponding to the sample number shown in table 14. The results shown in fig. 18 show the Relative Fluorescence Units (RFU) recorded as a function of cycle number. The Ct values obtained from the curves shown in fig. 18 are summarized in table 15.

As shown in FIG. 18, recombinant plasmids were detected via the amplification products for all samples containing recombinant plasmids except for sample 6. In addition, no recombinant plasmid was detected in any of the control samples (samples 7 and 8). Thus, the results shown in fig. 18 indicate that in some cases, multiple series of denaturation and extension conditions were used, and that without sample purification, a detection sensitivity of 25 plasmid copies/reaction (rxn) could be obtained.

Table 14: experimental reaction mixture of example 12

Sample (I)	Plasmid (copy/reaction)
		1	250000
2	25000
		3	2500
4	250
		5	25
6	2.5
		7	0 (blood only)
8	0 (Water only)

Table 15: ct values determined by the experiment in example 12

Sample (I)	Copying/reacting	Ct
			1	250000	26.12
2	25000	33.61
			3	2500	37.61
4	250	40.61
			5	25	42.97
6	2.5	---
			7	0	---
8	0	---

Example 13: amplification and detection of Ebola virus

Amplification and detection experiments were performed on human whole blood samples containing varying copy numbers of Zaire ebola virus (Zaire-EBOV) pseudovirus. Eight samples (replicate 1 and replicate 2) were tested in duplicate for a total of 16 samples. Six of the samples contained a specific copy number (2500000, 250000, 25000, 2500, 250 and 25 copies) of pseudovirus, while two samples (one containing only blood and one containing only water) served as control samples. The whole blood sample was analyzed without sample purification.

Each sample is combined with reagents necessary for reverse transcription and nucleic acid amplification (e.g., reverse transcriptase, DNA polymerase, dntps, cofactors, primers, appropriate buffers, etc.) and a reporter (e.g., an oligonucleotide probe containing a FAM dye) into a 30 μ L reaction mixture. A summary of the reaction mixtures by sample number (including the copy number of the pseudovirus) is shown in Table 16. To generate amplification products from pseudoviruses, each reaction mixture was subjected to two series of denaturation and extension conditions. These two series are as follows: (i) in the first series, 15 cycles of 1 second at 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 45 cycles of 5 seconds at 95 ℃ and 10 seconds at 55 ℃ are carried out. During the second series, the signal from the reporter was recorded to generate an amplification curve and Ct values were obtained. Amplification curves for this experiment are graphically shown in fig. 19A (replicate 1) and fig. 19B (replicate 2), each labeled with sample numbers corresponding to those shown in table 16. The results shown in fig. 19A and 19B show the Relative Fluorescence Units (RFU) recorded as a function of cycle number. The Ct values obtained from the curves shown in fig. 19A and 19B are summarized in table 17, where "Ct 1" corresponds to repeat group 1 and "Ct 2" corresponds to repeat group 2.

As shown in fig. 19A and 19B, for all samples containing pseudoviruses (samples 1-6), pseudoviruses were detected in both replicate groups via the amplification products. In addition, no pseudovirus was detected in any of the control samples (samples 7 and 8). Thus, the results shown in fig. 19A and 19B indicate that, in some cases, multiple series of denaturation and extension conditions can be used to obtain detection sensitivity of 25 virus copies/reaction without sample purification.

Table 16: experimental reaction mixture for example 13

Sample (I)	Pseudovirus (copy/response)
		1	2500000
2	250000
		3	25000
4	2500
		5	250
6	25
		7	0 (blood only)
8	0 (Water only)

Table 17: ct values determined by the experiment in example 13

Example 14: amplification and detection of Ebola virus

Amplification and detection experiments were performed on human whole blood samples containing varying copy numbers of Zaire ebola virus (Zaire-EBOV) pseudovirus. Eight samples were tested. Six of the samples contained a specific copy number (2500000, 250000, 25000, 2500, 250 and 25) of pseudovirus, while two samples (one containing 20000 copies of a pseudovirus positive control and one containing only water) served as control samples. The whole blood sample was analyzed without sample purification.

Each sample is combined with reagents necessary for reverse transcription and nucleic acid amplification (e.g., reverse transcriptase, DNA polymerase, primers, dntps, cofactors, appropriate buffers, etc.) and a reporter (e.g., an oligonucleotide probe containing a FAM dye) into a 30 μ L reaction mixture. A summary of the sample numbers for each reaction mixture (including the copy number of the pseudovirus) is shown in Table 18. To generate amplification products from pseudoviruses, each reaction mixture was subjected to two series of denaturation and extension conditions. These two series are as follows: (i) in the first series, 15 cycles of 1 second at 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 35 cycles of 5 seconds at 95 ℃ and 10 seconds at 55 ℃ are carried out. During the second series, the signal from the reporter was recorded to generate an amplification curve and Ct values were obtained. The amplification curves for this experiment are graphically shown in fig. 20, with each curve labeled with sample numbers corresponding to those shown in table 18. The results shown in fig. 20 show the Relative Fluorescence Units (RFU) recorded as a function of cycle number. The Ct values obtained from the curves shown in fig. 20 are summarized in table 19.

As shown in FIG. 20, for all samples containing pseudoviruses (samples 1-6), including the sample containing the positive control pseudovirus (sample 7), the pseudovirus was detected via the amplification product. In addition, no pseudovirus was detected in the control sample containing only water (sample 8). Thus, the results shown in fig. 20 indicate that in some cases, multiple series of denaturation and extension conditions can be used to obtain detection sensitivity of 25 virus copies/reaction without sample purification.

Table 18: experimental reaction mixture for example 14

Sample (I)	Pseudovirus (copy/response)
		1	2500000
2	250000
		3	25000
4	2500
		5	250
6	25
		7	20000 (positive control pseudovirus)
8	0 (Water only)

Table 19: ct values determined by the experiment in example 14

Sample (I)	Copying/reacting	Ct
			1	2500000	10.44
2	250000	13.30
			3	25000	16.14
4	2500	19.62
			5	250	22.92
6	25	30.00
			7	20000 (Positive control)	15.94
8	0 (Water only)	---

Example 15: amplification and detection of Ebola virus

Amplification and detection experiments were performed on human whole blood samples containing one of two copy numbers (250 copies/reaction or 25 copies/reaction) of Zaire ebola virus (Zaire-EBOV) pseudovirus. For a total of eight samples, each whole blood sample was tested using one of the four reagent systems. Each reagent system (B-1, B-2, B-3, and B-4) comprises reagents necessary for reverse transcription and nucleic acid amplification (e.g., reverse transcriptase, DNA polymerase, primers, dNTPs, cofactors, appropriate buffers, etc.) and a reporter (e.g., an oligonucleotide probe comprising a FAM dye). The various reagent systems contain various components in the reagent system at different concentrations. Each whole blood sample was combined with its appropriate reagent system into 30. mu.L of reaction mixture. A summary of the sample numbers for each reaction mixture (including the copy number of the pseudovirus and the reagent system) is shown below in Table 20. To generate amplification products from pseudoviruses, each reaction mixture was subjected to two series of denaturation and extension conditions. These two series are as follows: (i) in the first series, 15 cycles of 1 second at 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 40 cycles of 5 seconds at 95 ℃ and 10 seconds at 55 ℃ are carried out. During the second series, the signal from the reporter was recorded to generate an amplification curve and Ct values were obtained. The amplification curves for this experiment are shown graphically in fig. 21, each curve being labeled with sample numbers corresponding to those shown in table 20. The results shown in fig. 21 show the Relative Fluorescence Units (RFU) recorded as a function of cycle number. The Ct values obtained from the curves shown in fig. 21 are summarized in table 21.

As shown in fig. 21, pseudovirus was detected via the amplification product for all samples, including samples containing 25 copies/reaction. Thus, the results shown in fig. 21 indicate that in some cases, multiple series of denaturation and extension conditions can be used, different reagent systems can be used, and detection sensitivity of 25 virus copies/reaction can be obtained without sample purification.

Table 20: experimental reaction mixture of example 15

Table 21: ct values determined by the experiment in example 15

Sample (I)	Copying/reacting	Ct
			1	250	20.38
2	25	24.82
			3	250	20.62
4	25	24.05
			5	250	20.26
6	25	25.09
			7	250	19.86
8	25	24.00

Example 16: real-time PCR detection of Zaire Ebola virus

One-step qPCR methods of the invention were used to analyze serum samples from patients for zaire ebola virus. The sample was not purified. The samples included nine positive samples of zaire ebola virus and seven negative samples of zaire ebola virus. The Roche LC96 real-time PCR system was used.

The procedure for analyzing the samples in this example is shown in table 22.

Table 22: thermal cycling program

The results of this one-step qPCR method are shown in table 23. This one-step qPCR method showed 100% consistency in testing zaire ebola virus compared to validated reagents and methods.

Table 23: results

Example 17: amplification and detection of malaria

Amplification and detection experiments were performed on human whole blood samples containing unknown concentrations of malaria pathogens. Two sets of experiments were completed. In the first set of experiments, 1: 4 dilutions (in 1X PBS) duplicate experiments were completed; the experiment was completed on a sample containing whole blood and a plasmid corresponding to a malaria pathogen; and the experiment was completed for the water only control. In a second set of experiments, experiments were completed on samples containing multiple dilutions (1: 4, 1: 40, 1: 400, 1: 4000, 1: 40000, and 1: 400000) of human whole blood samples in 1 × PBS with control samples of blood only and water only. Whole blood samples were analyzed without sample purification.

Each sample is combined with reagents necessary for nucleic acid amplification (e.g., DNA polymerase, primers, dntps, cofactors, appropriate buffers, etc.) and a reporter (e.g., an oligonucleotide probe containing a FAM dye) into a 30 μ L reaction mixture. The reaction mixtures for the first set of experiments are shown in table 24 as a summary of sample numbers (including dilutions). The reaction mixtures for the second set of experiments are shown in table 25 for a summary of sample numbers (including dilutions). To produce an amplification product from a malaria pathogen, each reaction mixture was subjected to two series of denaturing and extension conditions. These two series are as follows: (i) in the first series, 13 cycles of 1 second at 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 45 cycles of 5 seconds at 95 ℃ and 10 seconds at 55 ℃ are carried out. During the second series, the signal from the reporter is recorded to generate an amplification curve. The amplification curves for the first set of experiments are shown graphically in FIG. 22A, while the amplification curves for the second set of experiments are shown graphically in FIG. 22B. Each curve is labeled with its corresponding sample number in table 24 and table 25, respectively. The results shown in fig. 22A and 22B show the Relative Fluorescence Units (RFU) recorded as a function of cycle number.

As shown in fig. 22A, malaria pathogens were detected via the amplification products for both reaction mixtures containing whole blood samples (samples 1 and 2) and for the positive control containing recombinant plasmids (sample 3). Furthermore, no malaria pathogens were detected in the water only control sample (sample 4). Thus, the results shown in fig. 22A indicate that in some cases, malaria pathogens can be detected without sample purification using multiple series of denaturing and extension conditions.

As shown in fig. 22B, malaria pathogens were detected via amplification products for all reaction mixtures containing whole blood samples (samples 1-6). Furthermore, no malaria pathogens were detected in the control samples of water only and blood only (samples 7 and 8). Thus, the results shown in fig. 22B indicate that, in some cases, multiple series of denaturation and extension conditions can be used and without sample purification, at up to 1: 400000 pathogens, including malaria pathogens, were detected at dilutions.

Table 24: experimental reaction mixtures for the first set of experiments in example 17

Sample (I)	Degree of dilution
		1	1：4
2	1：4
		3	1: 2 (plasmid in Whole blood control)
4	Zero (only water)

Table 25: experimental reaction mixtures for the second set of experiments in example 17

Sample (I)	Degree of dilution
		1	1：4
2	1：40
		3	1：400
4	1：4000
		5	1：40000
6	1：400000
		7	0 (blood only)
8	0 (Water only)

Example 18: amplification and detection of dengue virus

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

mu.L of each sample is combined with reagents necessary for reverse transcription and nucleic acid amplification (e.g., reverse transcriptase, DNA polymerase, primers, dNTPs, cofactors, appropriate buffers, etc.) and a reporter (e.g., an oligonucleotide probe containing a FAM dye) into 30. mu.L of a reaction mixture. For a summary of the reaction mixtures (including dilutions), the first set of experiments is shown in table 26, the second set of experiments is shown in table 27, and the third set of experiments is shown in table 28. To generate amplification products from the virus, each reaction mixture was subjected to two series of denaturation and extension conditions. These two series are as follows: (i) in the first series, 10 cycles of 5 seconds at 95 ℃ and 10 seconds at 45 ℃ were carried out at 42 ℃ for 1 minute, followed by 1 minute at 95 ℃; and (ii) in the second series, 45 cycles of 5 seconds at 95 ℃ and 10 seconds at 55 ℃ are carried out. During the second series, the signal from the reporter is recorded to generate an amplification curve. The amplification curves for the first set of experiments are shown graphically in FIG. 23A, the amplification curves for the second set of experiments are shown graphically in FIG. 23B, and the amplification curves for the third set of experiments are shown graphically in FIG. 23C. Each curve is labeled with its corresponding sample number in table 26, table 27, and table 28, respectively. The results shown in fig. 23A, 23B, and 23C show the Relative Fluorescence Units (RFU) recorded as a function of cycle number. Ct values obtained from the curves shown in fig. 23A, 23B and 23C are shown in table 26, table 27 and table 28, respectively.

As shown in FIG. 23A, for the three reaction mixtures (samples 1-3) containing virus, virus was detected via the amplification products. In addition, no virus was detected in the control sample with only water (sample 4). Thus, the results shown in fig. 23A indicate that, in some cases, dengue virus can be detected using multiple series of extension and denaturation conditions.

As shown in fig. 23B, for dengue virus-containing samples that were undiluted (sample 1) or diluted up to 1: 1000 (

samples

2, 3 and 4), virus was detected via the amplification product. However, 1: ct value of 1000 reaction mixture (sample 4). No virus was detected at the higher dilutions (

samples

5, 6 and 7) or in the water only control sample (sample 8). Thus, the results shown in fig. 23B indicate that, in some cases, multiple series of denaturation and extension conditions can be used and without sample purification, at a ph of up to 1: virus was detected at a dilution of 1000, where up to 1: a dilution of 100 may yield a Ct value.

As shown in fig. 23C, for dengue virus-containing samples that were undiluted (sample 1) or diluted up to 1: 1000 (

samples

2, 3 and 4), virus was detected via the amplification product. However, 1: ct value of 1000 reaction mixture. No virus was detected in either the higher dilution (sample 5) or the water only control sample (sample 6). Thus, the results shown in fig. 23C indicate that, in some cases, multiple series of denaturation and extension conditions can be used and without sample purification, at a ph of up to 1: virus was detected at a dilution of 1000, where up to 1: a dilution of 100 may yield a Ct value.

Table 26: example 18 Experimental reaction mixtures and Ct values determined for the first set of experiments

Sample (I)	Degree of dilution	Ct value
				1	Undiluted	19.32
2	Undiluted	20.40
			3	1：10	23.23
4	No virus (water only)	---

Table 27: example 18 Experimental reaction mixtures and Ct values determined for the second set of experiments

Sample (I)	Degree of dilution	Ct value
				1	Undiluted	20.85
2	1：10	25.14
			3	1：100	31.57
4	1：1000	---
			5	1：10000	---
6	1：100000	---
			7	1：1000000	---
8	No virus (water only)	---

Table 28: experimental reaction mixtures and Ct values determined for the third set of experiments in example 18

Sample (I)	Degree of dilution	Ct value
				1	Undiluted	19.22
2	1：10	22.43
			2	1：100	26.55
4	1：1000	---
			5	1：10000	---
6	No virus (water only)	---

Example 19: detection of Single Nucleotide Polymorphisms (SNPs)

Amplification and detection experiments were performed on samples of human oropharyngeal swabs or blood containing cytochrome P4502C 19, CYP2C19 x 2 (with the "GA" genotype) or CYP2C19 x3 (with the "GG" genotype) of a specific genotype. Two sets of experiments were performed-one set for samples obtained from human pharyngeal swabs and one set for samples obtained from blood. In the first set of experiments, seven different samples obtained from human pharyngeal swabs were analyzed without sample purification. In the second set of experiments, five different blood samples were analyzed without sample purification.

Each sample is combined into a reaction mixture with reagents necessary for nucleic acid amplification (e.g., DNA polymerase, primers, dntps, cofactors, suitable buffers, etc.) and two reporter agents (e.g., an oligonucleotide probe comprising a FAM dye for detecting nucleic acid amplification, an oligonucleotide probe comprising a texas red dye for detecting the "GA" genotype). To generate the amplification products, each reaction mixture was subjected to a thermal cycling protocol comprising 5 minutes at 95 ℃ followed by 50 cycles of 5 seconds at 95 ℃ and 10 seconds at 49 ℃. During thermocycling, the signal from the reporter is recorded to generate an amplification curve. Amplification curves for the first set of experiments (oropharyngeal swabs) are shown graphically in fig. 24A (corresponding to the signal of the FAM oligonucleotide probes) and fig. 24B (corresponding to the signal of the texas red oligonucleotide probes). The amplification curves for the second set of experiments (blood samples) are shown graphically in fig. 25A (corresponding to the signal of the FAM oligonucleotide probe) and fig. 25B (corresponding to the signal of the texas red oligonucleotide probe). The results shown in fig. 24A, 24B, 25A, and 25B show the Relative Fluorescence Units (RFU) recorded as a function of cycle number. Each curve is labeled with its corresponding reaction mixture number in table 29 (oropharyngeal swab experiment) or table 30 (blood experiment). The Ct values determined from the amplification curves are also shown in table 29 or table 30 together with the determined genotypes. In FIG. 24B or FIG. 25B, in the amplification curve in which the signal from Texas Red was observed, the corresponding reaction mixture was determined to have the "GA" genotype. Further, in fig. 24B or fig. 25B, in the amplification curve in which no signal from texas red was observed, it was determined that the corresponding reaction mixture had the "GG" genotype.

As shown in fig. 24A, amplification products were observed for each reaction mixture containing samples obtained from oropharyngeal swabs, indicating that amplification of nucleic acids occurred. However, as shown in fig. 24B, amplification products were observed only in some of the reaction mixtures containing samples obtained from oropharyngeal swabs (

reaction mixtures

1, 4, 6 and 7), which correspond to the "GA" genotype. No amplification product was observed in the other reaction mixtures (

reaction mixtures

2, 3 and 5), which correspond to the "GG" genotype. The results shown in fig. 24A and 24B were validated by amplification and detection experiments using DNA extracted from buccal swab samples (data not shown). Thus, the results shown in fig. 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.

As shown in FIG. 25A, an amplification product was observed for each reaction mixture containing a sample obtained from blood, indicating that amplification of nucleic acid occurred. However, as shown in fig. 25B, amplification products were observed only in some of the reaction mixtures (

reaction mixtures

1, 2 and 5) containing samples obtained from blood, which correspond to the "GA" genotype. No amplification products were observed in the other reaction mixtures (reaction mixtures 3 and 4), which correspond to the "GG" genotype. The results shown in fig. 25A and 25B were verified using nucleic acid sequencing. Thus, the results shown in fig. 25A and 25B indicate that, in some cases, SNPs can be detected by real-time amplification in a sample obtained from blood without sample purification.

Table 29: ct values and genotypes determined for oropharyngeal swab experiments in example 19

Table 30: ct values and genotypes determined for the blood experiments in example 19

Example 20: amplification and detection of adenovirus type 55 (ADV55) and adenovirus type 7 (ADV7)

Amplification and detection experiments were performed on samples obtained from oropharyngeal swabs containing different copy numbers of adenovirus type 55 (ADV55) or unknown concentrations of adenovirus type 7 (ADV 7). Two sets of experiments were completed-one for samples with ADV55 and one for experiments with ADV 7. In the first set of experiments, six different experiments with samples containing different copy numbers (1, 10, 100, 1000, 10000 and 100000 copies) of ADV55 were completed without sample purification and together completed negative control experiments. In the second set of experiments, eight different experiments were completed with samples containing unknown copy numbers of ADV7 without sample purification.

Each sample is combined into a reaction mixture with reagents necessary for nucleic acid amplification (e.g., DNA polymerase, primers, dntps, cofactors, suitable buffers, etc.) and a reporter (e.g., an oligonucleotide probe comprising a FAM dye). A summary of the reaction mixtures for the first set of experiments (including copy numbers of ADV55) is shown in table 31. To generate amplification products from the virus, each reaction mixture was subjected to two series of denaturation and extension conditions. These two series are as follows: (i) in the first series, 20 cycles of 1 second at 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 35 cycles of 5 seconds at 95 ℃ and 34 seconds at 60 ℃ were carried out. During the second series, the signal from the reporter was recorded to generate an amplification curve and Ct values were obtained. The amplification curves for the first set of experiments are shown graphically in fig. 26A, each curve being labeled with reaction mixture numbers corresponding to those shown in table 31. The amplification curves for the second set of experiments are shown graphically in fig. 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 indicated. The results shown in fig. 26A and 26B show the Relative Fluorescence Units (RFU) recorded as a function of cycle number.

As shown in fig. 26A, ADV55 was detected via the amplification product for all reaction mixtures (reaction mixtures 1-6) containing virus-containing samples. In addition, no virus was detected in the negative control reaction mixture (reaction mixture 7). Thus, the results shown in fig. 26A indicate that, in some cases, ADV55 virus can be detected without sample purification and at various dilution levels using multiple series of extension and denaturation conditions.

As shown in fig. 26B, ADV7 was detected via the amplification product for all reaction mixtures. Thus, the results shown in fig. 26B indicate that, in some cases, ADV7 virus can be detected using multiple series of extension and denaturation conditions and without sample purification.

Table 31: experimental reaction mixture for ADV55 experiment in example 20

Table 32: ct values determined for the ADV7 experiment in example 20

Reaction mixture	Ct value
			1	5.12
2	7.16
		3	10.97
4	14.15
		5	17.58
6	20.29
		7	22.13
8	17.66

Example 21: amplification and detection of RNA hepatitis C Virus with A (RNA-HCV)

Amplification and detection experiments were performed on plasma samples containing different copy numbers of RNA hepatitis c virus with a (RNA-HCV). Three different experiments with samples containing different copy numbers (10, 100 and 500 copies) of RNA-HCV were done without sample purification and together experiments against negative controls were done.

Each sample is combined into a reaction mixture with reagents necessary for reverse transcription and nucleic acid amplification (e.g., reverse transcriptase, DNA polymerase, primers, dntps, cofactors, suitable buffers, etc.) and a reporter (e.g., an oligonucleotide probe comprising a FAM dye). A summary of the reaction mixtures (including RNA-HCV copy number) is shown in Table 33. To generate amplified DNA products from the virus, each reaction mixture was subjected to two series of denaturation and extension conditions. These two series are as follows: (i) in the first series, 20 cycles of 1 second at 95 ℃ and 1 second at 45 ℃ were carried out, followed by 1 minute at 95 ℃; and (ii) in the second series, 55 cycles of 5 seconds at 95 ℃ and 34 seconds at 60 ℃ are carried out. During the second series, the signal from the reporter is recorded to generate an amplification curve. The amplification curves for the first set of experiments are shown graphically in fig. 27, with each curve labeled with a number corresponding to the reaction mixture number shown in table 33. The results shown in fig. 27 show the Relative Fluorescence Units (RFU) recorded as a function of cycle number.

As shown in FIG. 27, RNA-HCV was detected via the amplification products for all reaction mixtures (reaction mixtures 1-3) containing virus-containing samples. In addition, no RNA-HCV was detected in the negative control reaction mixture (reaction mixture 4). Thus, the results shown in fig. 27 indicate that in some cases, multiple series of extension and denaturation conditions can be used to detect RNA-HCV without sample purification. Detection sensitivity of 10 copies/reaction can also be achieved.

Table 33: experimental reaction mixture for RNA-HCV experiment in example 21

Reaction mixture	RNA-HCV copy number/response
		1	10
2	100
		3	500
4	0 (negative control)

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

2. A method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(a) receiving the biological sample that has been obtained from the subject;

(d) detecting the amount of amplified DNA product of (c); and

3. A method of amplifying a target nucleic acid present in a biological sample obtained from a subject, comprising:

4. A system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(b) an amplification module that, in response to the user request:

5. A system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, comprising:

(b) an amplification module that, in response to the user request:

(iii) (iv) detecting said amount of amplified DNA product of (iii); and

(iv) outputting information about the amount of amplified DNA product to a recipient,

wherein the amount of time to complete (i) - (iv) is less than or equal to about 30 minutes; and

6. A system for amplifying a target nucleic acid present in a biological sample obtained from a subject, comprising:

(b) an amplification module that, in response to the user request:

7. A computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target nucleic acid (RNA) present in a biological sample obtained directly from a subject, the method comprising:

8. A computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject, the method comprising:

(a) receiving the biological sample that has been obtained from the subject;

(d) detecting said amount of the DNA product of (c); and

(e) outputting information about the amount of DNA product to a recipient,

9. A computer-readable medium comprising machine-executable code which, when executed by one or more computer processors, performs a method of amplifying a target nucleic acid present in a biological sample obtained from a subject, the method comprising:

10. A system for amplifying a target nucleic acid in a biological sample obtained from a subject, comprising: