KR20160107208A - Methods and systems for nucleic acid amplification - Google Patents

Abstract

The present invention provides methods and systems for amplifying and analyzing nucleic acid samples.

Description

[0001] METHODS AND SYSTEMS FOR NUCLEIC ACID AMPLIFICATION [0002]

Cross-reference

The present application claims priority to Patent Cooperation Treaty application PCT / CN2013 / 090425, filed December 25, 2013, which is hereby incorporated by reference in its entirety for all purposes.

The nucleic acid amplification method enables selective amplification and identification of a desired nucleic acid from a complex mixture, e.g., a biological sample. To detect nucleic acids in a biological sample, the biological sample is typically treated to isolate the nucleic acid from other components of the biological sample and other agents that may interfere with nucleic acid and / or amplification. After isolation of the target nucleic acid from the biological sample, the target nucleic acid can be amplified, for example, by an amplification method known in the art, such as a thermocycling-based approach (e. G., Polymerase chain reaction (PCR)). After amplification of the target nucleic acid, the amplification product can be detected and the end result interpreted by the end user. However, extraction of the nucleic acid from the biological sample prior to amplification of the nucleic acid is time consuming and reduces the time efficiency for the whole process.

POC testing has the potential to improve the detection and management of infectious diseases in resource-limited situations where laboratory infrastructures are inadequate, or where there is potential complexity in the tracking of patients and delayed laboratory results reception. POC testing also allows for more patient-to-patient response to a patient during a single visit to a state-of-the-art health care facility. However, the inefficiency of POC methods and devices limits what can be achieved. For example, the preparation of a nucleic acid from a complex sample type (e. G., A biological sample) (e. G., Of a pathogen) may be performed manually, following a number of treatment steps and subsequent tests, To report, it involves a high level of expertise in dedicated laboratory space.

Thus, there is a need for a rapid, accurate method and apparatus for analyzing nucleic acids from complex sample types. Such methods and devices may be useful, for example, to recognize rapid sample-to-match detection and management of diseases that can be detected through their nucleic acids.

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 rapidly and with excellent 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 the steps of: (a) providing a reaction vessel comprising a reagent and a biological sample required to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, Wherein the reagent comprises a set of primers for (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a target RNA; And (b) performing a plurality of cycles of primer extension reaction on the reaction mixture in the reaction vessel to produce an amplified DNA product indicative of the presence of target RNA, wherein each cycle comprises the steps of: (i) Incubating the reaction mixture for an extension period of up to 60 seconds at an extension temperature to obtain the target RNA. ≪ RTI ID = 0.0 > In another embodiment, the method comprises the steps of: (a) receiving a biological sample from a subject; (b) obtaining a reaction mixture by providing a reaction container comprising a reagent and a biological sample necessary for performing reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, said reagent comprising (i) a reverse transcriptase and ii) comprises a primer set for the target RNA; (c) performing a multiple cycle primer extension reaction on the reaction mixture to produce a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample; (d) detecting the amount of the amplified DNA product of (c); And outputting information on the amount of the amplified DNA product to the receiver, wherein the amount of time for completing (a) - (e) is about 30 minutes or less. In some embodiments, the time is 20 minutes or less, 10 minutes or less, or 5 minutes or less.

In some embodiments, the reagent further comprises a reporter agent that produces a detectable signal indicative of the presence of the 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 agent is a dye. In some embodiments, the primer set comprises at least one primer. In some embodiments, the primer set comprises a first primer that produces strands complementary to the target RNA. In some embodiments, the primer set comprises a second primer that produces a strand complementary to a DNA product complementary to at least a portion of the target RNA. In some embodiments, the target RNA is a viral RNA. In some embodiments, the viral RNA is pathogenic to the subject. In some embodiments, the viral RNA is selected from the group consisting of: HIV I, HIV II, Ebola virus, Dengue virus, orthomyxovirus, Hepesvirus, and / or A, B, C (e.g. armored RNA- , D, and hepatitis E virus.

In some embodiments, the reaction vessel comprises a body and a cap. In some embodiments, the cap is removable. In some embodiments, the reaction vessel employs a pipette tip shape. In some embodiments, the reaction vessel is part of an array of reaction vessels. In some embodiments, the reaction vessel portion of the reaction vessel array is individually addressable by the fluid handling device. In some embodiments, the reaction vessel comprises two or more heat zones. In some embodiments, the reaction vessel is sealed and, in some cases, hermetically sealed.

In some embodiments, the denaturation temperature is between about 90 ° C and 100 ° C, or between about 92 ° C and 95 ° C. In some embodiments, the elongation temperature is from about 35 캜 to 72 캜, or from about 45 캜 to 65 캜. In some embodiments, the denaturation period is 30 seconds or less. In some embodiments, the stretching period is 30 seconds or less.

In some embodiments, the target RNA is not concentrated prior to providing the reaction vessel containing the reagent and the biological sample necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification. In some embodiments, the biological sample does not undergo RNA extraction when providing a reaction vessel containing biological reagents and biological samples necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification. In some embodiments, the method comprises the steps of adding a solubilizer to a reaction vessel before or during the provision of a reaction vessel containing a reagent and a biological sample necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification . In some embodiments, the solubilizer comprises a buffer. In some embodiments, the target RNA is released in the biological sample during at least one cycle of the primer extension reaction.

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

In some embodiments, DNA amplification is through a 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 step comprises detecting a detectable amount of a DNA product indicative of the presence of target RNA in the biological sample at a cycle limit (Ct) of less than 50, less than 40, less than 30, less than 20, less than 10, . In some embodiments, the amplification step yields a detectable amount of DNA product indicative of the presence of target RNA in the biological sample in a time period of less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the amplification step is non-emulsion based.

In some embodiments, the recipient is a treating physician, pharmaceutical company, or subject. In some embodiments, the step of performing a multiple cycle primer extension reaction on the reaction mixture to yield a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample may be 30 cycles or less, 20 cycles or less, or 10 Cycle. In some embodiments, the detection is optical detection, electrostatic detection, or electrochemical detection. In some embodiments, the method comprises providing a reaction container comprising a reagent and a biological sample necessary to perform reverse transcription 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 invention provides a method of amplifying a target nucleic acid present in a biological sample obtained from a subject. The method comprises the steps of: (a) providing a reaction vessel containing a biological sample and a reagent necessary to perform nucleic acid amplification to obtain a reaction mixture comprising (i) a deoxyribonucleic acid (DNA) polymerase and optionally A reverse transcriptase, and (ii) a primer set for a target nucleic acid; And (b) performing a plurality of series of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplification product indicative of the presence of a target nucleic acid in the biological sample, each series comprising (i) a denaturation temperature and a denaturation period , Followed by (ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period, wherein the individual series comprises at least one of denaturing conditions < RTI ID = 0.0 > And / or at least one other individual series at multiple times in extension conditions.

In some embodiments, the target nucleic acid is a ribonucleic acid. In some embodiments, the reagent is required to perform reverse transcription in parallel with deoxyribonucleic acid amplification. In some embodiments, the amplified product is an amplified amplified deoxyribonucleic acid product. In some embodiments, the biological sample is not purified in (a). In some embodiments, the method further comprises applying the target nucleic acid to at least one denaturing condition prior to step (b). In some embodiments, one or more denaturing conditions are selected in the denaturing temperature profile and denaturant.

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

In some embodiments, the method further comprises applying the target nucleic acid to at least one denaturing condition between a first series and a second series of a plurality of primer extension reaction series. In some embodiments, the individual series may include at least one, at least two, at least three, or at least four of the ramping rate, denaturation temperature, denaturation period, elongation temperature and elongation period between denaturation temperature and elongation temperature It is different. In some embodiments, the individual series are different for the ramping rate, denaturation temperature, denaturation period, elongation temperature and elongation period between denaturation temperature and elongation temperature.

In some embodiments, the plurality of primer extension reaction series comprises a first series and a second series, wherein the first series comprises more than 10 cycles, each cycle of the first series comprising: (i) Incubating the reaction mixture at about < RTI ID = 0.0 > 35 C-65 C < / RTI > for less than 1 minute, and the second series comprises more than 10 cycles (I) incubating the reaction mixture at about 92 ° C-95 ° C for less than 30 seconds, and then (ii) incubating the reaction mixture at about 40 ° C-60 ° C for less than 1 minute .

In some embodiments, multiple series of primer extension reactions yield a detectable amount of amplified product that is indicative of the presence of target nucleic acid in the biological sample at low cycle limits, as compared to a single series of primer extension reactions under similar denaturation and elongation conditions do. In some embodiments, the method further comprises pre-heating the biological sample prior to step (b) for a pre-heat treatment period of 10 minutes, 2 minutes, or 1 minute or less at a pre-heat treatment temperature of 90 ° C to 100 ° C do. In some embodiments, the pre-heat treatment temperature is 92 ° C-95 ° C. In some embodiments, the pre-heat treatment period is less than about 30 seconds.

In another aspect, the 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 for accepting a user request to amplify target RNA in a biological sample; (b) receiving, in response to a user request, a reaction mixture comprising a reagent and a biological sample necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification in a reaction vessel, Wherein the reagent is selected from the group consisting of (i) a reverse transcriptase, (ii) a DNA polymerase, and (3) a primer extension reaction to amplify the target RNA, And (iii) a set of primers for the target RNA, each of the cycles comprising: (i) incubating the reaction mixture at a denaturation temperature for a denaturation period of 60 seconds or less, then (ii) Lt; / RTI > and an incubation period of less than about 2 seconds; And (c) an output module operatively coupled to the amplification module, the output module including an output module that outputs information about the target RNA or DNA product to the recipient.

In another embodiment, the system comprises: (a) an input module that accepts a user request to amplify a target RNA in a biological sample; (b) a reaction mixture containing (i) a biological sample obtained from the subject, and a reagent necessary for performing reverse transcription and optionally deoxyribonucleic acid (DNA) amplification, wherein the reagent comprises ( 1) a reverse transcriptase; and (2) a primer set for the target RNA; And (ii) performing a plurality of cycles of primer extension reactions to yield a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample for the reaction mixture; (iii) an amplification module for detecting the amount of the amplified DNA product of (iii) and (iv) outputting information on the amount of amplified DNA product to the recipient, the amount of time for completing (i) - An amplification module of about 30 minutes or less; And (c) an output module operatively coupled to the amplification module, the output module including an output module for communicating information to the receiver. In some embodiments, the output module is an electronic display. In some embodiments, the electronic display includes a user interface. In some embodiments, the output module is a communications interface operatively coupled to a computer network.

In another aspect, the 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 for accepting a user request to amplify a target nucleic acid in a biological sample; (b) a reaction mixture comprising a reagent required to perform biological samples and nucleic acid amplification in response to a user request, said reagent comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) An amplification module for carrying out a plurality of series of primer extension reactions to receive a reaction mixture comprising a primer set in a reaction vessel and to generate an amplified product indicative of the presence of a target nucleic acid in the biological sample for the reaction mixture in the reaction vessel, , Each series comprising the steps of: (i) incubating the reaction mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and then (ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period , Wherein the individual series comprises at least two cycles of denaturation and / or extension conditions A plurality of amplifying modules at least one other individual series different from that; And (c) an output module operatively coupled to the amplification module, the output module outputting information about the nucleic acid or amplified product to the recipient.

In another aspect, the invention provides a computer readable medium comprising machine executable code that, when executed by one or more computer processors, comprises a machine-executable code for performing a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from the subject do. In one embodiment, the method comprises the steps of: (a) providing a reaction vessel comprising a reagent and a biological sample necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, Comprises a set of primers for (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a target RNA, and (b) performing multiple cycles of primer extension reaction on the reaction mixture in the reaction vessel (I) incubating the reaction mixture at a denaturation temperature for a denaturation period of less than or equal to 60 seconds, and then (i. E. and ii) incubating the reaction mixture at an extension temperature for an extension period of 60 seconds or less.

In another embodiment, the method comprises the steps of: (a) receiving a biological sample from a subject; (b) providing a reaction container comprising a reagent and a biological sample required to perform reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, wherein the reagent comprises (i) a reverse transcriptase and (ii) a set of primers for the target RNA; (c) performing a plurality of cycles of primer extension reaction to yield a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample for the reaction mixture; (d) detecting the amount of the DNA product of (c); And (e) outputting information on the amount of DNA product to the recipient, wherein the amount of time to complete (a) - (e) is less than about 30 minutes.

In another aspect, the invention provides a computer readable medium comprising machine executable code that, 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 the steps of (a) providing a reaction vessel comprising a biological sample and a reagent necessary to perform nucleic acid amplification to obtain a reaction mixture, wherein the reagent comprises (i) a DNA polymerase and A reverse transcriptase, and (ii) a primer set for a target nucleic acid; And (b) performing a plurality of series of primer extension reactions to produce an amplified product from the target nucleic acid for the reaction mixture in the reaction vessel, each series comprising (i) reacting the reaction mixture with Incubating under denaturing conditions, followed by (ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period, wherein the individual series comprises two or more cycles of denaturation conditions and / or elongation conditions And different from one or more other plurality of separate series.

A further aspect of the invention provides a system for amplifying a target nucleic acid in a biological sample obtained from a subject. The system includes an electronic display screen that includes a user interface for displaying graphical elements that a user can access to execute an amplification protocol for amplifying a target nucleic acid in a biological sample. The system also includes a computer processor coupled to the electronic display screen and programmed to execute an amplification protocol when selecting a graphical element by a user. The amplification protocol comprises performing a plurality of series of primer extension reactions to produce an amplified product indicative of the presence of the target nucleic acid in the biological sample for a reaction mixture comprising a reagent and a biological sample required to perform nucleic acid amplification. Each series of primer extension reactions comprises incubating the reaction mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and then incubating the reaction mixture under elongation conditions characterized by an elongation temperature and an elongation period. . ≪ / RTI > The individual series are different from at least one other individual series of the multiple series in denaturation conditions and / or elongation conditions.

In some embodiments, the amplification protocol further comprises selecting a primer set for the target nucleic acid. In some embodiments, the reagent may comprise a deoxyribonucleic acid (DNA) polymerase, an optional reverse transcriptase, and a set of primers for the target nucleic acid. In some embodiments, the user interface may display a plurality of graphical elements. Each of the graphic elements may be associated with a predetermined amplification protocol among a plurality of amplification protocols. In some embodiments, each of the graphical elements may be associated with a disease. Some amplification protocols among a plurality of amplification protocols can be specified to assess the presence of a disease in a subject. In some embodiments, the disease may be associated with a virus, such as an RNA virus or a DNA virus. In some embodiments, 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, A hepatitis B virus, a hepatitis C virus, a hepatitis C virus, a hepatitis D virus, a hepatitis E virus, a hepatitis G virus, an Epstein-Barr virus, a mononucleic virus, a cytomegalovirus, a SARS virus, a West Nile fever virus, Measles virus, herpes simplex virus, small pox 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 the 55 adenovirus (ADV55) or the 7 adenovirus (ADV7). In some embodiments, the hepatitis C virus may be armored RNA-C hepatitis virus (RNA-HCV). In some embodiments, the disease is 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 specified to assess the presence of the disease based on the presence of the amplified product. In some embodiments, the disease may be associated with a virus, such as an RNA virus or a DNA virus. In some embodiments, 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, A hepatitis B virus, a hepatitis C virus, a hepatitis C virus, a hepatitis D virus, a hepatitis E virus, a hepatitis G virus, an Epstein-Barr virus, a mononucleic virus, a cytomegalovirus, a SARS virus, a West Nile fever virus, Measles virus, herpes simplex virus, small pox 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 the 55 adenovirus (ADV55) or the 7 adenovirus (ADV7). In some embodiments, the hepatitis C virus may be armored RNA-C hepatitis virus (RNA-HCV). In some embodiments, the disease is associated with a pathogenic bacterium (e. G., Mycobacterium tuberculosis) or a pathogenic protozoan (e. G., Plasmodium).

Additional aspects and advantages of the present invention will be readily appreciated by those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the invention. As will be realized, the subject matter may be different and different embodiments, and some of the details thereof may be modified without departing from the scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Transfer 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.

The novel features of the present application will be described in detail in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS A better understanding of the features and advantages of the present invention may be obtained by reference to the following specific description in which illustrative embodiments utilizing the principles of the invention are described and in the accompanying drawings (also referred to herein as " Can be obtained.
Figure 1 is a schematic diagram illustrating an exemplary system.
FIGS. 2A and 2B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 1. FIG.
FIGS. 3A and 3B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 1. FIG.
FIGS. 4A and 4B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 2. FIG.
FIG. 5 is a graph showing the results of the exemplary nucleic acid amplification reactions described in Example 3. FIG.
6A and 6B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 4. FIG.
FIGS. 7A and 7B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 4. FIG.
Figures 8A and 8B are cross- Lt; / RTI > is a graph showing the results of the exemplary nucleic acid amplification reactions described in Example 4.
Figures 9A and 9B show Lt; / RTI > is a graph showing the results of the exemplary nucleic acid amplification reactions described in Example 4.
FIGS. 10A and 10B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 4. FIG.
11 Lt; / RTI > is a graph showing the results of the exemplary nucleic acid amplification reactions described in Example 5.
12 is a cross- Lt; / RTI > is a graph showing the results of the exemplary nucleic acid amplification reactions described in Example 5.
13 is a graph showing the results of the exemplary nucleic acid amplification reactions described in Example 7. Fig.
14 is a graph showing the results of an exemplary nucleic acid amplification reaction described in Example 9. Fig.
FIGS. 15A and 15B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 10. FIG.
FIGS. 16A and 16B are graphs showing the results of the exemplary nucleic acid amplification reactions described in Example 10. FIG.
17 is a graph showing the results of the nucleic acid amplification reaction described in Example 11. Fig.
18 is a graph showing the results of the nucleic acid amplification reaction described in Example 12. Fig.
19A and 19B are graphs showing the results of the nucleic acid amplification reaction described in Example 13. Fig.
20 is a graph showing the results of the nucleic acid amplification reaction described in Example 14. Fig.
21 is a graph showing the results of the nucleic acid amplification reaction described in Example 15. Fig.
22A and 22B are graphs showing the results of the nucleic acid amplification reaction described in Example 17. Fig.
23A, 23B and 23C are graphs showing the results of the nucleic acid amplification reaction described in Example 18. FIG.
24A and 24B are graphs showing the results of the nucleic acid amplification reaction described in Example 19. FIG.
25A and 25B are graphs showing the results of the nucleic acid amplification reaction described in Example 19. Fig.
26A and 26B are graphs showing the results of the nucleic acid amplification reaction described in Example 20. Fig.
27 is a graph showing the results of the nucleic acid amplification reaction described in Example 21. Fig.
28A is a schematic diagram of an exemplary electronic display with an exemplary user interface.
Figure 28b is a schematic diagram of an exemplary electronic display having an exemplary user interface.

While various embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, one skilled in the art can make various changes, changes, and substitutions. It should be understood that various alternatives to the embodiments described herein may be applied.

The singular forms "a "," a ", and "the ", as used in the specification and the claims, include a plurality of references unless explicitly stated otherwise. For example, the term "one cell" includes a plurality of cells, including a mixture thereof.

As used herein, the terms "amplify" and "amplification" are used interchangeably and generally refer to one or more copies of a nucleic acid or "amplified product ". The term "DNA amplification" generally refers to the generation of one or more copies of a DNA molecule or "amplified DNA product ". The term " reverse transcription amplification " generally refers to the generation of deoxyribonucleic acid (DNA) from ribonucleic acid (RNA) templates via reverse transcriptase action.

The term " cycle limit "or" Ct ", as used herein, refers to the cycle during a thermal cycle in which the increase in the detectable signal due to the amplified product has reached a statistically significant level above the reference signal.

As used herein, the terms "denature" and "denaturation" are used interchangeably and refer generally to the total or partial unwinding of the helix structure of the double-stranded nucleic acid, and in some cases the unwinding of the secondary structure of the single- stranded nucleic acid. Degeneration involves inactivation of the cell wall (s) or viral envelope of the pathogen, and inactivation of the inhibitor protein (s). The conditions under which denaturation occurs generally mean the temperatures at which denaturation occurs and the term " denaturation period " generally refers to the amount of time allotted for denaturation to occur.

The term "elongation " as used herein refers to the introduction of nucleotides into nucleic acids in a generally template-specified manner. Kidneys can occur, for example, with the aid of polymerases or reverse transcriptases. The condition under which the kidneys occur is generally the temperature at which the kidneys occur, and the "kidney period" generally refers to the amount of time allotted for kidneys to occur.

As used herein, the term "nucleic acid" refers to a polymeric form of a nucleotide of any length of deoxyribonucleotide (dNTP) or ribonucleotide (rNTP), or analog thereof. The nucleic acid has an arbitrary tertiary structure and can perform any known or unknown function. Non-limiting examples of nucleic acids include, but are not limited to, coding or non-coding regions of DNA, RNA, genes or gene fragments, loci defined by linkage analysis, loci, exons, intron messenger RNA (mRNA), transcription RNA, ribosomal RNA, (S), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant nucleic acid, branched nucleic acid, plasmid, vector, isolated DNA of any sequence, Probes, and primers. The nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the nucleic acid. The nucleotide sequence of the nucleic acid may include a non-nucleotide component. The nucleic acid may be conjugated or conjugated with, for example, a reporter agent, and further modified after polymerization.

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

The term "reaction mixture" as used herein generally refers to a composition comprising reagents necessary to complete nucleic acid amplification (e.g., DNA amplification, RNA amplification), and non-limiting examples of such reagents include, A DNA polymerase, a reverse transcriptase (for example, reversal of RNA), a suitable buffer (including a zwitterion buffer), a co-factor (for example, , Divalent and monovalent cations), dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG), etc.). In some cases, the reaction mixture may also comprise at least one reporter agent.

The term "reporter agent " as used herein refers to a composition that produces a detectable signal whose presence or absence can be used to detect the presence of amplified products.

As used herein, the term "target nucleic acid" refers to a nucleic acid molecule in the starting population of nucleic acid molecules having a nucleotide sequence that is preferably measured for its presence, quantity, and / or sequence, or one or more of these changes. The target nucleic acid can be any type of nucleic acid, including DNA, RNA, and analogs thereof. As used herein, "target ribonucleic acid (RNA)" means a target nucleic acid that is RNA. As used herein, "target deoxyribonucleic acid (DNA)" refers to a target nucleic acid that is generally DNA.

As used herein, the term "subject" means an independent entity or medium that generally has testable or detectable gene information. The subject may be a person or an entity. The subject may be a vertebrate animal, for example a mammal. Non-limiting examples of mammals include rodents and animals, apes, humans, farm animals, sports animals, and pets. Other examples of the subject 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 steps of: (a) providing a reaction container comprising a reagent and a biological sample necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification, to provide a reaction mixture, wherein the reagent comprises (i ) Reverse transcriptase, (ii) a DNA polymerase, and (iii) a set of primers for the target RNA; And (b) performing a plurality of cycles of primer extension reaction on the reaction mixture in the reaction vessel to produce an amplified DNA product indicative of the presence of target RNA, amplifying the target RNA, each cycle comprising (i) Incubating the reaction mixture at a denaturation temperature for a denaturation period of 60 seconds or less, and then (ii) incubating the reaction mixture at an elongation temperature for an elongation period of 60 seconds or less.

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 steps of: (a) receiving a biological sample from a subject; (b) providing a reaction container comprising a reagent and a biological sample required to perform reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, wherein the reagent comprises (i) a reverse transcriptase and (ii) a set of primers for the target RNA; (c) performing a plurality of cycles of primer extension reactions on the reaction mixture to yield a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample; (d) detecting the amount of the amplified DNA product of (c); And (e) outputting information about the amount of amplified DNA product to the recipient, wherein the amount of time for completing (a) - (e) is less than about 30 minutes.

In one aspect, the present invention provides a method for amplifying a target nucleic acid present in a biological sample obtained from a subject. The method comprises the steps of: (a) providing a reaction vessel comprising a biological sample and reagents necessary to perform nucleic acid amplification to obtain a reaction mixture, wherein the reagent comprises (i) a deoxyribonucleic acid (DNA) polymerase and , And (ii) a set of primers for the target nucleic acid; And (b) performing a plurality of series of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified product indicative of the presence of a target nucleic acid in the biological sample, each series comprising (i) Incubating under denaturing conditions characterized by a temperature and a denaturation period, followed by (ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period, wherein the individual series comprises And differing from at least one other individual series of multiple series in denaturation and / or elongation conditions.

In any of various aspects, the nucleic acid from the biological sample obtained from the subject is amplified. In some cases, biological samples are obtained directly from the subject. Biological samples obtained directly from the subject usually mean biological samples that have not been further processed after being obtained from the subject, except for any means used to collect the biological sample from the subject for subsequent processing. For example, blood approaches the circulatory system of the subject, removing blood from the subject (e.g., through a needle), and putting the removed blood into the container. The container may contain reagents (e. G., Anticoagulants) to aid in further analysis of the blood sample. In another example, a cotton swab can be used to access the epithelial cells on the two oral surfaces of the subject. After obtaining a biological sample from the subject, a swab containing the biological sample may be contacted with a fluid (e.g., a buffer) to recover the biological fluid from the swab.

In some embodiments, the biological sample is not purified upon presentation to the reaction vessel. In some embodiments, the nucleic acid of the biological sample is not extracted upon delivery of the biological sample to the reaction vessel. For example, RNA or DNA in a biological sample may not be extracted from the biological sample when the biological sample is provided to the reaction vessel. In addition, in some embodiments, the target nucleic acid (e. G., Target RNA or target DNA) present in the biological sample is not concentrated prior to providing the biological sample to the reaction vessel.

Any suitable biological sample, including nucleic acids, may be obtained from the subject. The biological sample can be a solid matter (e. G., A biological tissue) or a fluid (e. G., A biological fluid). In general, the biological fluid may comprise any fluid associated with a living organism. Non-limiting examples of biological samples include blood (or blood components - e.g., leukocytes, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, The pancreas, the cerebrospinal fluid, the tissue, the throat swab, the biopsy, the abdomen, the amniotic fluid, the liver, the kidney, the stomach, the bone marrow, the stool, the semen, the vaginal fluid and the tumor tissue obtained from the anatomical position , Muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, body fluids, sputum, pus, microbial gun, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, stomach and digestive juices, tears, Nasal mucosa, skin cells, plasma, nasal swabs or nasopharynx, spinal fluid, umbilical cord blood, lymphatic fluid, and / or other secretions 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 the subject include access to the circulatory system (e.g., intravenously or intraarterially with a syringe or other needle), and secreted biological samples (e.g., stool, urine, , Saliva, etc.) may be collected surgically (e.g., biopsies), swabbing (e.g., oral swabs, oral pharynx swabs), pipetting, and breathing. The biological sample may also be obtained from any anatomical portion of the subject where the desired biological sample is located.

In any of various aspects, the target nucleic acid is amplified to produce an amplified product. The target nucleic acid may be the target RNA or the target DNA. Where the target nucleic acid is a target RNA, the target RNA may be any type of RNA, including the RNA types described herein. In some embodiments, the target RNA is a viral RNA. In some embodiments, the viral RNA may be pathogenic to the subject. Non-limiting examples of pathogenic viral RNA include human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza viruses (e.g., H1N1, H3N2 , Hepatitis A virus, hepatitis B virus, hepatitis C virus (for example, armored RNA-HCV virus) virus, hepatitis D virus, hepatitis E virus, hepatitis G virus Virus, Epstein-Barr virus, mononucleic virus, cytomegalovirus, SARS virus, West Nile fever virus, poliovirus, and measles virus.

If the target nucleic acid is a target DNA, the target DNA may be any type of DNA, including the DNA types described 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, small fox, adenovirus (e. G., Type 55 adenovirus, type 7 adenovirus) and varicella virus (e. In some cases, the target DNA can be bacterial DNA. Bacterial DNA can be a hospital-acquired bacterium for a subject, a M. tuberculosis, a bacteria known to cause pneumonia. In some cases, the target DNA can be one or more protozoan-type protozoan-derived DNA capable of causing a pathogenic protozoan, e. G., Malaria.

In any of the various aspects of the invention, a biological sample from the subject is provided with reagents necessary for 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 comprises a body including an inner surface, an outer surface, an open end, and an opposite sealing end. In some embodiments, the reaction vessel may comprise a cap. The cap is configured to contact the body at its open end such that when the contact is made, the open end of the reaction vessel is closed. In some cases, the cap is permanently associated with the reaction vessel so that it remains attached to the reaction vessel in the open and sealed state. In some cases, the cap is removable, such that upon opening the reaction vessel, the cap is separated from the reaction vessel. In some embodiments, the reaction vessel may be sealed, optionally hermetically sealed.

The reaction vessel may be of various sizes, shapes, weights, and steric configurations. In some instances, the reaction vessel may be round or ovoid cylindrical. In some embodiments, the reaction vessel may be rectangular, square, diamond, round, oval, or triangular in shape. The reaction vessel may be regular or irregular. In some embodiments, the sealing end of the reaction vessel may have a sharp, rounded, or flat surface. Non-limiting examples of types of reaction vessels may include tubes, wells, capillaries, cartridges, cuvettes, centrifuge tubes, or pipette tips. The reaction vessel may be made of any suitable material of a non-limiting example of such material, including glass, metal, plastic, and combinations thereof.

In some embodiments, the reaction vessel is part of an array of reaction vessels. Reactor vessel arrays are particularly useful for automating processes and / or for processing multiple samples simultaneously. For example, the reaction vessel may be a well of a microwell plate composed of a plurality of wells. In another example, the reaction vessel may be contained in a well of a thermal block of a thermal cycler, where each block of the thermal cycler may comprise a plurality of wells each of which contains a sample vessel. The array of reaction vessels may comprise any suitable number of reaction vessels. For example, the 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 portion of the reaction vessel array can also be addressed individually by the fluid handling device so that the fluid handling device can accurately identify the reaction vessel and dispense the appropriate fluid material into the reaction vessel. The fluid handling device may be useful for automating the addition of fluid material to a reaction vessel.

In some embodiments, the reaction vessel may comprise a plurality of thermal zones. Thermal zones in the reaction vessel can be obtained by exposing different regions of the reaction vessel to different temperature cycling conditions. For example, the reaction vessel may include an upper column zone and a lower column zone. The top thermal zone can accommodate reagents and biological samples necessary to obtain a reaction mixture for nucleic acid amplification. The reaction mixture is then subjected to a thermal cycling protocol. After a desired number of cycles, for example, the reaction mixture may be slowly, but continuously, leaked from the upper heat zone to the lower heat zone. In the bottom heat zone, a desired number of cycles of the second thermal cycling protocol, which is different from that of the upper heat zone, is then performed on the reaction mixture. This strategy is particularly useful when amplifying DNA using nested PCR. In some embodiments, a thermal zone may be created in the reaction vessel with the aid of a thermally responsive lamination material in the reaction vessel. In such a case, heat treatment of the thermally sensitive laminated material can be used to release the reaction mixture from one thermal zone to another. In some embodiments, the reaction vessel may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more column zones.

In some embodiments, a reaction vessel containing a thermal zone can be used for the treatment of the biological sample prior to nucleic acid amplification. For example, a solubilizer may be added to the first column zone of the reaction vessel prior to adding the reagents and biological sample required for nucleic acid amplification. When a biological sample and a reagent are added to a reaction vessel containing a solubilizer, a reaction mixture is obtained which is capable of dissolving species (e.g., cells or virus particles) in the biological agent. Alternatively, the solubilizer may be added to the first column zone of the reaction mixture concurrently with the biological sample and the reagent. Dissolving the cells and virus particles in the biological sample in the first thermal zone using a technique that makes the thermal conditions suitable for the action of the solubilizer with respect to the first column zone are used to release the nucleic acid in the biological sample into the reaction mixture. After dissolution, the reaction mixture is then allowed to enter the second column region of the reaction vessel for amplification of the released nucleic acid using the amplification method described herein.

If a solubilizing agent is desired, any suitable solubilizing agent known in the art can be used, including commercially available solubilizing agents. Non-limiting examples of solubilizers include, but are not limited to, Tris-HCl, EDTA, a detergent (e.g., Triton X-100, SDS), lysozyme, glucolase, Proteinase E, viral endolysin, exolysin, Lyticcase, Proteinase K, endor lysine and exorhysin from bacteriophage, endoricein from bacterial phage PM2, B. subtilis Bacteriophage Bacteria derived from PBSX Endolysin, Lactobacillus prophage Lj928, Lj965, Bacteriophage 15 Endocrine derived from Phiadh, Streptococcus pneumoniae Bacteriophage Cp-I-derived endolysin, Streptococcus pneumoniae Endokinase derived from the bifunctional peptidoglycans lysine of Streptococcus agalactiae bacteriophage B30, endorizine and exolysine derived from the bacteriophage bacteria, endoricyzin derived from Listeria bacteriophage, endolin-endorysin, cell 20 lysis gene, holWMY Staphylococcus wameri M phage varphiWMY, Staphylococcus and head M phage varphiWMY, and combinations thereof. In some cases, the buffering agent may comprise a solubilizer (e. G., A lysis buffer). An example of a dissolution 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 amplified product. In addition, the amplification of the nucleic acid may be linear, exponential, or a combination thereof. Amplification may be emulsion-based or non-emulsion-based. Non-limiting examples of nucleic acid amplification 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 amplified product may be DNA. When the target RNA is amplified, the DNA is obtained through reverse transcription of the RNA and then amplification of the DNA can be used to obtain the amplified DNA product. The amplified DNA product may represent the presence of target RNA in the biological sample. When the DNA is amplified, any DNA amplification method known in the art can be adopted. Non-limiting examples of DNA amplification methods include, but are not limited to, polymerase chain reaction (PCR), PCR assays (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, , Nested PCR, nested PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, mini-primer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interglacial PCR, touchdown 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 can be achieved by nested PCR, which can improve the detection sensitivity of the amplified DNA product.

In various aspects, the nucleic acid amplification methods described herein may be performed in parallel. Generally, a parallel amplification reaction is an amplification reaction that occurs simultaneously in the same reaction vessel. The parallel nucleic acid amplification reaction can be performed, for example, by including reagents necessary for each nucleic acid amplification reaction in the reaction vessel to obtain a reaction mixture and adjusting the conditions necessary for each nucleic acid amplification reaction to the reaction mixture. For example, reverse transcription amplification and DNA amplification can be performed in parallel by tailoring the reaction mixture to conditions suitable to provide the reagents necessary for both amplification reactions in the reaction vessel to obtain a reaction mixture and perform both amplification reactions. DNA generated by reverse transcription of RNA can be amplified in parallel to produce amplified DNA products. Any suitable number of nucleic acid amplification reactions may 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, The reaction is carried out in parallel.

The advantage of performing nucleic acid amplification in parallel is that it involves rapid conversion between coupled nucleic acid amplifications. For example, the target nucleic acid (e. G., Target RNA, target DNA) may be extracted or released from the biological sample during the heating period of the parallel nucleic acid amplification. In the case of the target RNA, for example, a biological sample comprising the target RNA can be heated and the target RNA released from the biological sample. The released target RNA can immediately initiate reverse transcription (via reverse transcription) to generate complementary DNA. Complementary DNA can then be amplified immediately, often in approximately seconds, units. Release of Target RNA from Biological Samples and Complementarity of Target RNA Short time between reverse transcription to DNA can help to minimize the effects of inhibitors in biological samples that may interfere with reverse transcription and / or DNA amplification.

In any of various aspects, a nucleic acid amplification reaction can be performed using a set of primers for the target nucleic acid. Typically, the primer set comprises at least one primer. For example, a primer set includes 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 for different nucleic acid amplification reactions. For example, a primer set may comprise a first primer required to produce a first strand of nucleic acid product complementary to at least a portion of a target nucleic acid, and a second strand of nucleic acid product complementary to at least a portion of the first strand of the nucleic acid product And a second primer that is complementary to the nucleic acid strand product necessary to produce the second primer.

For example, the primer set may be for a target RNA. The primer set comprises a first primer that can be used to generate a first strand of nucleic acid product that is complementary to at least a portion of the target RNA. In the case of a reverse transcription reaction, the first strand of the nucleic acid product may be DNA. The primer set may also comprise a second primer that can be used to generate a second strand of nucleic acid product complementarity to at least a portion of the first strand of the nucleic acid product. In the case of a reverse transcription reaction performed in parallel with DNA amplification, the second strand of the nucleic acid product may be a strand of a nucleic acid (e. G., DNA) product complementary to the DNA generated from the RNA template.

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

In some embodiments, a DNA polymerase is used. Any suitable DNA polymerase may be used, including commercially available DNA polymerase. DNA polymerase refers to an enzyme that can introduce nucleotides into strands of DNA, usually in a template-binding fashion. Non-limiting examples of DNA polymerase include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA- Taq 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 I, Hi-Fi Polymerase, Tbr Polymerase, Tfl Polymerase, Pfu Turbopolymerase, Pyrobest Polymerase, Pwo Polymerase, KOD Polymerase, Bst Polymerase, Sac Polymerase, Klenow fragment and Variants, modified products and derivatives thereof. For some hot start polymerases, denaturation steps of 94 ° C-95 ° C for 2 to 10 minutes may be required, which may change the thermal profile based on different polymerases.

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

In various aspects, the primer extension reaction is used to generate the amplified product. The primer extension reaction generally involves a cycle in which the reaction mixture is incubated at the denaturation temperature for the denaturation period and the reaction mixture is incubated at the elongation temperature for the elongation period.

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

The denaturation period may vary depending upon the particular biological sample being analyzed, the particular source of the target nucleic acid in the biological sample (e.g., viral particles, bacteria), the reagent used, and / or the desired reaction conditions. For example, the denaturation period is 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, Seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second or less. For example, the denaturation period is 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 Second, or one second or less.

The elongation temperature may vary depending upon, for example, the particular biological sample being analyzed, the particular source of the target nucleic acid (e.g., viral particles, bacteria) in the biological sample, the reagent used, and / or the desired reaction conditions. For example, the elongation temperature may be from about 30 캜 to about 80 캜. In some instances, the elongation temperature may be between about 35 ° C and about 72 ° C. In some instances, the elongation temperature may be about 45 ° C to about 65 ° C. In some instances, the elongation temperature may be between about 35 ° C and about 65 ° C. In some instances, the elongation temperature may be between about 40 ° C and about 60 ° C. In some instances, the elongation temperature may be from about 50 캜 to about 60 캜. In another example, the elongation temperature is about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49 ° C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 72 ° C, 73 ° C, 74 ° C, 75 ° C, 76 ° C, 77 ° C, 78 ° C, 79 ° C, or 80 ° C .

The elongation period may vary depending upon, 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 reagent used, and / or the desired reaction conditions. For example, the stretch period may be 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, Seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, the elongation period is 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 Second, or one second or less.

In any of various 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 may be determined, for example, by the number of cycles required to obtain a detectable amplified product (e. G., A detectable amount of amplified DNA product that is indicative of the presence of target RNA in the biological sample) , Cycle limit value (Ct)). For example, the number of cycles required to obtain a detectable amplified product (e. G., A detectable amount of DNA product indicative of the presence of target RNA in the biological sample) may be 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 less than 5 cycles. Furthermore, in some embodiments, a detectable amount of the amplified product (e.g., a detectable amount of a DNA product indicative of the presence of target RNA in the biological sample) is 100, 75, 70, 65, 60, 55, 50, (Ct) of 45, 40, 35, 30, 25, 20, 15, 10 or less than 5.

The time to yield a detectable amount of amplified product indicative of the presence of the target nucleic acid for which amplification is to be amplified may vary depending on the biological sample from which the target nucleic acid is obtained, the particular nucleic acid amplification reaction being performed, and the specific number of cycles of the desired amplification reaction have. For example, amplification of the nucleic acid target may be 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 a detectable amount of amplified product that indicates the presence of the target nucleic acid in a time period of 5 minutes or less.

In some embodiments, amplification of the target RNA is performed for 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 a detectable amount of amplified DNA product that indicates the presence of target RNA in a time period of less than 5 minutes.

In some embodiments, the reaction mixture may be subjected to multiple series of primer extension reactions. Individual series of multiple series may include multiple cycles of a particular primer extension reaction characterized by, for example, specific denaturation and elongation conditions as described herein. In general, each individual series differs from at least one other individual series of multiple series, for example, in denaturation conditions and / or elongation conditions. The individual series differ from the other individual series of the multiple series, for example, for any one, two, three, or all four of the denaturation temperature, denaturation period, elongation temperature, and elongation period. Further, the plurality of series may include any number of separate series, e.g., at least about, or about 2, 3, 4, 5, 6, 7, 8, 9, 10,

For example, multiple series of primer extension reactions may include a first series and a second series. The first series may include, for example, more than 10 cycles of the primer extension reaction, wherein the individual cycles of the first series comprise (i) incubating the reaction mixture at about 92 ° C to about 95 ° C for less than 30 seconds (Ii) incubating the reaction mixture at about 35 캜 to about 65 캜 for about 1 minute or less. The second series includes, for example, more than 10 cycles of the primer extension reaction wherein each cycle of the second series comprises (i) incubating the reaction mixture at about 92 ° C to about 95 ° C for less than 30 seconds, (Ii) incubating the reaction mixture at about 40 ° C to about 60 ° C for about 1 minute or less. In this particular example, the first and second series differ in their elongation temperature conditions. However, this example is not intended to be limiting as any combination of different elongation and denaturation conditions may be used.

In some embodiments, the ramping time (i.e., the time it takes for the thermocycler to transition from one temperature to another) and / or the ramping rate may be an important factor in amplification. For example, the temperature and time at which the amplification yields a detectable amount of amplification product that indicates the presence of the target nucleic acid may vary depending on the ramping rate and / or the ramping time. The ramping rate can affect the temperature (s) and time (s) used for amplification.

In some cases, the ramping time and / or ramping rate may be different between cycles. However, in some situations, the cycle-to-cycle ramping time and / or ramping rate may be the same. The ramping time and / or ramping rate may be adjusted based on the sample (s) being processed.

In some situations, different ramp times between temperatures may be determined based on, for example, the nature of the sample and the reaction conditions. The exact temperature and incubation time may be determined based on the nature of the sample and the reaction conditions. In some embodiments, a single sample may be subjected to multiple rounds of processing (e.g., applying to amplification conditions) using multiple thermal cycles, such that each thermal cycle is different, for example, ramping time, temperature, and / or incubation time ). The best or best thermal cycle can be selected for a particular sample. This provides a robust and efficient way to cut thermal cycles for a particular sample or combination of samples being tested.

In some embodiments, the target nucleic acid may be subject to denaturation 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 subject to denaturation conditions prior to the execution of multiple series, or denaturation conditions between multiple series. For example, the target nucleic acid may be subject to denaturation conditions between the first series and the second series of multiple series. Non-limiting examples of such denaturing conditions include denaturing temperature profiles (e.g., one or more denaturing temperatures) and denaturants.

The advantage of performing multiple series of primer extension reactions is that, compared to a single series of primer extension reactions under similar denaturation and elongation conditions, a multiple series approach can detect amplified products that indicate the presence of target nucleic acids in biological samples at low cycle thresholds It is possible to calculate the amount. The use of multiple series of primer extension reactions has resulted in at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% It is possible to reduce this cycle limit value by 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In some embodiments, the biological sample may be preheated prior to performing the primer extension reaction. The temperature at which the biological sample is preheated (e.g., preheat temperature) and the duration (e.g., preheat period) may vary, for example, depending on the particular biological sample being analyzed. In some instances, the biological sample is incubated at 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, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds or less. In some instances, the biological sample may be preheated at a temperature of about 80 ° C to about 110 ° C. In some instances, the biological sample may be preheated at a temperature of about 90 ° C to about 100 ° C. In some instances, the biological sample may be preheated at about 90 ° C to about 97 ° C. In some instances, the biological sample may be preheated at a temperature of about 92 ° C to about 95 ° C. In another example, the biological sample may be stored at about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, , 94 ° C, 95 ° C, 96 ° C, 97 ° C, 98 ° C, 99 ° C, or 100 ° C.

In some embodiments, reagents necessary to perform nucleic acid amplification, including reagents required for conditions of concurrent nucleic acid amplification, also include a reporter agent that produces a detectable signal indicating the presence of the amplified product in the presence or absence of the signal . The intensity of the detectable signal may be proportional to the amount of amplified product. In some cases, if the amplified product is a nucleic acid of a different type than the initially amplified target nucleic acid, the intensity of the detectable signal may be proportional to the amount of amplified target nucleic acid initially. For example, when target RNA is amplified through amplification of DNA obtained from parallel reverse transcription and reverse transcription, the reagents required for both reactions also yield a detectable signal indicative of the presence of amplified DNA product and / or amplified target RNA Lt; RTI ID = 0.0 > reporter < / RTI > The intensity of the detectable signal may be proportional to the amount of amplified DNA product and / or amplified native target RNA. The use of reporter agents can also be used for real-time amplification methods, including real-time PCR for DNA amplification.

Reporter agents can be linked to nucleic acids, including amplified products, through shared or non-shared means. Non-limiting examples of non-covalent means may include ionic interaction, van der Waals force, hydrophobic interaction, hydrogen bonding, and combinations thereof. In some embodiments, the reporter agent binds to the initial reactant and a change in the reporter agent level can be used to detect the amplified product. In some embodiments, the reporter agent can (or may not) be detected only as the nucleic acid amplification proceeds. In some embodiments, an optically active dye (e. G., A fluorescent dye) can be used as a reporter. Non-limiting examples of dyes include but are not limited to SYBR green, SYBR blue, DAPI, propidium iodide, Hoeste, SYBR gold, ethidium proimide, acridine, proplavine, acridine orange, acrypivalin, fluorocoumarin, But are not limited to, glycine, tyrosine, daunomycin, chloroqueen, distamycin D, clomomycin, fomidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine and acridine, ethidium bromide, 1, and ethidium homodimer-2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, ACRYL, 1, YOYO-3, TOTO-1, TOTO-1, TOTO-3, TOTO-1, 1, BO-PRO-1, BO-PRO-1, BO-PRO-1, BO-PRO-1, BO- -PRO-3, TO-PRO-5, JO-P SYTO-40, SYTO-41, SY-45, RO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-42, SYTO-42, SYTO-43, SYTO-44, SYTO-45 (blue), SYTO-13, SYTO-16, SYTO-24, SYTO- SYTO-81 (SYTO-81), SYTO-80, SYTO-82, SYTO-83, SYTO-84, SYTO- 60, SYTO-63 (red), fluorescein, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (FITC), SYTO-17, SYTO-59, SYTO-61, SYTO- Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red (APC), Sybr Green I, Sybr Green II, Sybr Gold, Cell Tracker Green, 7-AAD, Ethidium homodimer I, Ethidium homodimer II, Ethidium homodimer III, Dibromide, Umbelliferone, Eosin, Green Fluorescent Protein, Those containing europium and cobium, those obtained by dissolving carboxytetra, such as trisine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein, 5 - {[2 (and 3) -5- (acetylmercapto), 5 - and / or 6-carboxyfluorescein (FAM) ) -Succinyl] amino} fluorene (SAMSA-fluorescein), lysaminrodamine B sulfonyl chloride, 5 and / or 6 carboxyhodamine (ROX) Methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophore, 8-methoxyprene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate- 653, 635, 647, 660, 680, 700, 7, 710, 710, 50, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophore.

In some embodiments, the reporter agent may be a sequence-specific oligonucleotide probe that is optically active upon hybridization with the amplified product. Owing to the sequence-specific binding of the probe to the amplified products, the use of oligonucleotide probes can increase detection sensitivity and specificity. The probe may comprise a quencher which is linked to any optically active reporter agent (e. G., A dye) described herein and is capable of blocking the optical activity of the associated dye. Using Reporter Agents Non-limiting examples of useful probes include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.

In some embodiments, the reporter agent is an RNA oligonucleotide probe comprising an optically active dye (e. G., A fluorescent dye) and a quencher is positioned adjacent to the probe. The close proximity of the quencher to the dye can block the optical activity of the dye. The probe can bind to the target sequence to be amplified. Upon destruction of the prophase due to exonuclease activity of the DNA polymerase during amplification, the quencher and the dye are separated and the free dye restores its optical activity, which can be detected later.

In some embodiments, the reporter agent may be a molecular beacon. The molecular beacon includes, for example, a quencher connected to one end of the oligonucleotide with a hairpin stereostructure. At the other end of the oligonucleotide is an optically active dye, for example a fluorescent luminescent dye. In the hairpin stereostructure, the optically active dye and quencher are close enough so that the quencher can block the optical activity of the dye. However, upon hybridization with the amplified product, the oligonucleotide is considered a linear conformation and hybridizes with the target sequence on the amplified product. The linearization of the oligonucleotide results in the separation of the optically active dye and the quencher, so that the optical activity can be stored and detected. The sequence specificity of the molecular beacons for the target sequence on the amplified product can improve detection sensitivity and specificity.

In some embodiments, the reporter agent can be a radioactive species. Non-limiting examples of radioactive species include ¹⁴ C, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, Tc 99m, ³⁵ S, or ³ H.

In some embodiments, the reporter agent may be an enzyme capable of producing a detectable signal. A detectable signal may be produced by the activity of a particular substrate and enzyme if its substrate or enzyme has multiple substrates. Non-limiting examples of enzymes that can be used as reporter agents include, but are not limited to, alkaline phosphatase, horseradish peroxidase, I ² -galactosidase, alkaline phosphatase,? -Galactosidase, acetylcholinesterase, And luciferase.

In various aspects, amplified products (e.g., amplified DNA products, amplified RNA products) can be detected. Detection of amplified products, including amplified DNA, can be performed by any suitable detection method known in the art. The particular type of detection method used may depend on, for example, whether the reporter agent is included in the reaction mixture, the specific amplification product, the type of reaction vessel used for amplification, other reagents in the reaction mixture, It may be dependent on a particular type of reporter. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, and the like. Optical detection methods include, without limitation, fluorescence measurement and UV-vis optical absorption. Spectrometry detection methods include, without limitation, mass spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, without limitation, gel based techniques such as gel electrophoresis. Electrochemical detection methods include, without limitation, electrochemical detection of amplified products after high performance liquid chromatographic separation of amplified products.

In any of its various aspects, the time required to complete the elements of the method may vary depending on the particular steps of the method. For example, the amount of time for completing the elements of the method may be from about 5 minutes to about 120 minutes. In another example, the amount of time for completing the elements of the method may be from about 5 minutes to about 60 minutes. In another example, the amount of time to complete the elements of the method may be from about 5 minutes to about 30 minutes. In another example, the amount of time for completing the elements of the method is 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, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less.

In some embodiments, information about the presence and / or amount of amplified product (e. G., Amplified DNA product) can be output to the recipient. The information about the amplified product can be output through any suitable method known in the art. In some embodiments, this information may be orally provided to the recipient. In some embodiments, this information may be provided in a report. Reports may include, but are not limited to, raw data, processing data (e.g., graphical displays (e.g., drawings, charts, data tables, data summaries, ), Any number of preferred components, including information on the measured cycle limit, the starting amount of the target polynucleotide, conclusions on the presence of the target nucleic acid, diagnostic information, prognostic information, disease information, . ≪ / RTI > The report may be provided as a printed report (e.g., hard copy) or as an electronic report. In some embodiments, this information may be provided to an electronic display (e.g., an electronic display screen), such as a monitor or television, a screen operatively connected to the unit used to obtain the amplified product, , A tablet computer screen, a mobile device screen, or the like. Both print and electronic reports can be stored in files or databases, respectively, and can be compared against future reports.

The report may also be communicated to a local or remote audience using any suitable one communication medium, including, for example, a network connection, a wireless connection, or an Internet connection. In some embodiments, the report may be sent to the recipient's device, such as a personal computer, phone, tablet, or other device. Reports can be viewed online, stored on the recipient's device, or printed. The report may also be sent to any other suitable means of transmitting information, including, by way of non-limiting example, review by the audience and / or hard copy report delivery to the recipient.

This information can also be output to a variety of audience types. Non-limiting examples of such recipients include, but are not limited to, subjects receiving biological samples, physicians treating doctors, clinical monitors for clinical trials, nurses, researchers, laboratory researchers, pharmaceutical representatives, healthcare companies, (E. G., One or more computers and / or one or more computer storage servers, e.g., a medical record of a subject), a public health worker, other health care workers, and other health care providers Equipment.

In one aspect, the present disclosure provides a system for implementing a method according to any of the methods described 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. Such a system comprises: (a) an input module for accepting a user request to amplify a target RNA in a biological sample; (b) a reagent and biological sample for carrying out reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification in response to a user request, said reagent comprising (i) a reverse transcriptase, (ii) DNA A polymerase, and (iii) a set of primers for the target RNA; An amplification module for amplifying the target RNA by performing a plurality of cycles of primer extension reaction to produce an amplified DNA product indicative of the presence of target RNA for the reaction mixture in the reaction vessel, each cycle comprising (i) denaturing the reaction mixture Incubating at a temperature for a denaturation period of 60 seconds or less, and thereafter (ii) incubating the reaction mixture at an elongation temperature for an elongation period of 60 seconds or less; And (c) an output module operatively coupled to the amplification module, the output module outputting information to the recipient about the target RNA or DNA product.

In another aspect, the 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 accepts a request for a receiver to amplify a target RNA in a biological sample; (b) a reaction mixture comprising, in response to a user request, (i) a reagent required to perform reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification, and a biological sample obtained from the subject, ) Reverse transcriptase and (2) a set of primers for the target RNA; And (ii) performing a plurality of cycles of primer extension reactions on the reaction mixture to yield a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample; (iii) detecting the amount of the amplified DNA product of (iii); And (iv) an amplification module for outputting information on the amount of amplified DNA product to the recipient, wherein the amount of time to complete (i) - (iv) is about 30 minutes or less; And (c) an output module operatively coupled to the amplification module, the output module including an output module for transmitting information to the receiver.

In another aspect, a system is provided herein for amplifying a target nucleic acid present in a biological sample obtained from a subject. The system comprises: (a) an input module for accepting a user request to amplify a target RNA in a biological sample; (b) a reaction mixture comprising a reagent required to perform biological samples and nucleic acid amplification in response to a user request, said reagent comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) An amplification module for receiving a reaction mixture comprising a primer set in a reaction vessel and performing a plurality of series of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified product indicative of the presence of a target nucleic acid in the biological sample , Each series comprising the steps of (i) incubating the reaction mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and then (ii) incubating the reaction mixture under extension conditions characterized by an elongation temperature and an elongation period , And the individual series comprises at least two of the following: denaturation conditions and / or elongation conditions An amplifier module at least one other individual series different from that of the series; And (c) an output module operatively coupled to the amplification module, the output module outputting information to the recipient about the target RNA or DNA product.

In another aspect, the invention provides a system for amplifying a target nucleic acid in a biological sample obtained from a subject. Such a system may include an electronic display system with a user interface that displays graphical elements that a user can access to execute an amplification protocol to amplify a target nucleic acid in a biological sample. The system also includes a computer processor (including any suitable device with a computer processor as described herein) programmed to execute an amplification protocol when the user selects a graphical element and is coupled to an electronic display screen. The amplification protocol involves performing a number of series of primer extension reactions to produce an amplified product for a reaction mixture comprising a reagent and a biological sample required to perform nucleic acid amplification. The amplified product may represent the presence of the target nucleic acid in the biological sample. Each series of primer extension reactions also includes incubating the reaction mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and thereafter incubating the reaction mixture under elongation conditions characterized by an elongation temperature and an elongation period. Cycle. The individual series may be different from at least one other individual series of multiple series in denaturing conditions and / or elongation conditions.

In some embodiments, the target nucleic acid can be associated with a disease. The disease may be associated with, for example, RNA viruses or DNA viruses. Examples of viruses are provided herein. In some embodiments, the disease is selected from the group consisting of pathogenic bacteria (e. G., M. tuberculosis) or pathogenic protozoa (e. G., Plasmodium of malaria) . In some embodiments, the amplification protocol may be specified to assess for the presence of the disease based on the presence of the amplified product.

In some cases, the user interface may be a graphical user interface. In addition, the user interface may include one or more graphical elements. Graphic elements may include image and / or textual information, such as pictures, icons, and characters. The graphical elements may have various sizes and orientations on the user interface. The electronic display screen may also be any suitable electronic display, including the examples described herein. Non-limiting examples of electronic display screens include a monitor, a portable device screen, a laptop computer screen, a television, a removable video game system screen, and a calculator screen. In some embodiments, the electronic display screen includes a touch screen (e.g., an electrostatic or resistive touch screen) to allow selection of the graphical elements displayed on the user interface of the electronic display screen through user touch of the electronic display screen do.

In some embodiments, the amplification protocol further comprises selecting a set of primers for the target nucleic acid. In this case, the primer set may be a set of primers specifically designed to amplify one or more sequences of the target nucleic acid molecule. In some embodiments, the amplification protocol further comprises selecting a reporter specific for one or more sequences of the target nucleic acid (for example, an oligonucleotide probe comprising an optically active species or other type of reporter agent described herein) As shown in FIG. In addition, in some embodiments, the reagents may also include any suitable reagents necessary for nucleic acid amplification, such as, for example, deoxyribonucleic acid (DNA) polymerase, a set of primers for the target nucleic acid, and ) ≪ / RTI > reverse transcriptase.

In some embodiments, the user interface may display a plurality of graphical elements. Each of the graphic elements may be associated with a predetermined amplification protocol among a plurality of amplification protocols. Each of the plurality of amplification protocols may comprise different combinations of primer extension reaction series. In some cases, the user interface may display a plurality of graphical elements associated with the same amplification protocol. An example of a user interface with multiple graphical elements associated with a given amplification protocol is shown in Figure 28A . As shown in Figure 28A , an exemplary electronic display screen 2800 associated with a computer process includes a user interface 2801. [ The user interface 2801 includes a display of graphic elements 2802 , 2803 and 2804 . Each of the graphic elements are for the "Prot. 1" for may be associated with a specific amplification protocol for a particular graphical element (e. G., Graphical elements 2802, a graphic element 2803 "Prot. 2" and "Prot. 4" for graphical element 804 ). When a user of a particular graphical element is selected (e.g., the user touches if the electronic display screen 2800 includes a touch screen that has received a user interface), the particular amplification protocol associated with the graphical element may be executed by the associated computer process have. For example, when the user selects the graphic element 2803 , the amplification "Prot. 2" is executed by the associated computer processor. Only three kinds of graphic elements When shown in the exemplary user interface 2801 of Figure 28A , the user interface may have any suitable number of graphical elements. Further, when each graphical element shown in the user interface 2801 of Fig. 28A is associated with only one amplification protocol, each graphical element of the user interface is associated with one or more amplification protocols (e. G., A series of amplification protocols) And the associated computer process executes a series of amplification protocols when interacting with the graphical element and the user.

In some embodiments, each of the graphical elements and / or disease association, and any of the multiple amplification protocols, may be designated to assess the presence of a disease in the subject. Thus, in this case, the user can select a graphical element to execute an amplification protocol (or series of amplification protocols) to assess a particular disease. In some embodiments, the disease is associated with any RNA virus or DNA virus, including viruses, e. G., Examples of such viruses described 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 , Hepatitis A virus, hepatitis B virus, hepatitis C virus (e.g., armored RNA-C hepatitis virus (RNA-HCV)), hepatitis D virus, E (hepatitis B virus or H5N1 virus) A hepatitis virus, a hepatitis virus, a hepatitis G virus, an Epstein-Barr virus, a mononucleic virus, a cytomegalovirus, a SARS virus, a West Nile fever virus, a polio virus, a measles virus, a herpes simplex virus, Such as, for example, type 55 adenovirus (ADV55), type 7 adenovirus (ADV7), and barisella virus. In some embodiments, the disease is selected from the group consisting of pathogenic bacteria (e. G., M. tuberculosis) or pathogenic protozoa (e. G., Plasmodium of malaria) .

An example of a user interface, each having a plurality of graphical elements associated with a predetermined amplification protocol, is shown in Fig. 28B . As shown in Figure 28B , an exemplary electronic display screen 2810 associated with a computer processor And a user interface 2811 . The user interface 2811 And display of graphical elements 2812 , 2813 and 2814. [ Each graphical element may be associated with a specific disease associated with the amplification protocol at least one specified for the particular disease after (for example, for the case "Ebola" graphic element (2813) of the graphical element 2812 "H1N1" And "Hep C" (hepatitis C) for graphic element 2814 ). When a user selects a particular graphic element (e.g., a user touches if the electronic display screen 2810 includes a touch-screen with a user interface), the particular amplification protocol (s) associated with the disease associated with the graphic element May be executed by an associated computer process. For example, when a user interacts with graphical element 2812 , one or more amplification protocols associated with an assay of an Ebola virus may be executed by the associated computer process. If only three types of graphical elements are shown in the exemplary user interface 2811 of Figure 28B , the user interface has any suitable number of each graphical element corresponding to the various diseases. In addition, when each graphical element shown in the user interface 2811 of Figure 28B is associated with only one disease, each graphical element of the user interface is associated with one or more diseases, (E. G., Each individual amplification protocol designated for a particular disease). For example, the graphical element allows a computer processor associated with selection of graphical elements corresponding to the Ebola virus and the H1N1 virus to execute an amplification protocol for both the Ebola virus and the H1N1 virus.

In various aspects, the system includes an input module that accepts a user request to amplify a target nucleic acid (e. G., Target RNA, target DNA) present in a biological sample obtained directly from the subject. Any suitable module capable of accepting such a user request may be used. The input module may include, for example, an apparatus comprising 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., Apple® iPad, Samsung® Galaxy Tab), mobile phones, smart phones A personal digital assistant (PDA), a video game console, a television, a music playback device (e.g., Apple® iPod), a video playback device, a pager, and a calculator. A processor may be associated with one or more controllers, computing units, and other computer system units, or may be injected into firmware if desired. When executed in software, the routine (or program) is stored in any computer readable memory, such as RAM, ROM, flash memory, magnetic disk, laser disk, or other storage medium. Similarly, such software may be communicated to the device via any known delivery method including, for example, a communication line such as a telephone line, the Internet, a local intranet, a wireless connection, or the like, or via a removable medium such as a computer readable disc, . The various steps may be performed with various blocks, operations, means, modules or techniques that may be implemented in hardware, firmware, software, or any combination thereof. Some or all of the blocks, operations, techniques, etc., when executed in hardware, may be implemented in a computer system, such as, for example, a customer integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array ), And the like.

In some embodiments, the input module is configured to accept a user request to perform amplification of the target nucleic acid. The input module may send a user request directly (e.g., via an input device, such as a keyboard, a mouse or a touch screen, operated by the user) or indirectly (e.g., via a wired or wireless connection, including the Internet) Lt; / RTI > Through the output electronics, the input module can provide a user request to the amplification module. In some embodiments, the input module includes a user interface (UI), e.g., a graphical user interface (GUI), configured to allow the user to provide a request to amplify the target nucleic acid. The GUI may include text, graphics, and / or speech components. The GUI may be provided on an electronic display, including a display of an apparatus including a computer processor. Such a display may include a resistive or capacitive touch screen.

Non-limiting examples of users include, but are not limited to, subjects from which biological samples are obtained, health care workers, clinicians (e.g., physicians, nurses, laboratory researchers), laboratory workers (e.g., hospital laboratory researchers, research scientists, pharmaceutical scientists) , Clinical monitors for clinical trials, or health industry workers.

In various aspects, the system includes an amplification module for performing a nucleic acid amplification reaction on a target nucleic acid or a portion thereof, corresponding to a user request accepted by the input module. The amplification module may be any of the methods described herein and may include any fluid handling device, at least one thermocycler, means for receiving at least one reaction vessel (e.g., a well of a thermal block of a thermocycler), an amplified Information on the presence and / or amount of the amplified product (e.g., amplified DNA product) in the detector (e.g., optical detector, spectroscopic detector, electrochemical detector) capable of detecting the product For example, raw data, processed data, or any other type of information described herein). In some cases, the amplification module includes an apparatus having a computer processor as described herein and may also analyze the detected raw data, with the help of appropriate software. Further, in some embodiments, the amplification module may include input electronics necessary to receive instructions from the input module and output electronics needed to communicate with the output module.

In some embodiments, one or more of the steps of providing material to the reaction vessel, amplifying the nucleic acid, detecting the amplified product, and outputting information may be automated by an amplification module. In some embodiments, automation involves the use of one or more fluid handlers and associated software. The automation of such processes can be carried out using some commercially available fluid handling systems. Non-limiting examples of such fluid handling machines include fluid handlers from Perkin-Elmer, Caliper Life Sciences, Tecan, Eppendorf, Apricot Design, and Velocity 11.

In some embodiments, the amplification module includes real-time detection equipment. Non-limiting examples of such devices include real-time PCR thermocycler, ABI PRISM® 7000 sequence detection system, ABI PRISM® 7700 sequence detection system, Applied Biosystems 7300 real-time PCR system, Applied Biosystems 7500 real-time PCR system, Applied Biosystems 7900 HT Fast Real- System (all from Applied Biosystems); LightCycler ™ system (Roche Diagnostics GmbH); Mx3000P ™ real-time PCR system, Mx3005P ™ real-time PCR system, and Mx4000® Multiplex Quantitative PCR system (Stratagene, La Jolla, Calif.); And Smart Cycler System (Cepheid, distributed by Fisher Scientific). In some embodiments, the amplification module may be integrated with other automation equipment such as, for example, COBAS (R) AmpliPrep / COBAS (R) TaqMan (Roche Molecular Systems), TIGRIS DTS (Hologic Gen-Probe, San Diego, CA) (Becton Dickinson), GeneXpert System (Cepheid), Filmarray® (BioFire Diagnostics) System, iCubate System, IDBox System (Luminex), EncompassMDx ™ (Rheonix) System, The Enigma® ML system (Enigma Diagnostics), the T2Dx® system (T2 Biosystems), the Verigene® system (NanoSphere), the Great Basin diagnostic system, the Unyvero ™ system (Curetis ™), the Liat ™ analyzer (IQuum) system, Biocartis' ), PanNAT system (Micronics), or Spartan (R) system (Spartan Bioscience).

In various aspects, the system includes an output module operatively connected to the amplification module. In some embodiments, the output module includes an apparatus having a process as described above for the input module. The output module may include an input electronics for communicating with the amplification module and / or including the output device described herein. In some embodiments, the output module may be an electronic display, in some cases the electronic display includes a UI. In some embodiments, the output module is a communication interface operatively coupled to a computer network, e.g., the Internet. In some embodiments, the output module may transmit information to a listener at a local or remote location using any suitable communication medium, including a computer network, a wireless network, a local intranet, or the Internet. In some embodiments, the output module may analyze the data received from the amplification module. In some cases, the output module includes a report generator capable of generating a report and sending the report to the recipient, wherein the report includes any information as described herein. In some embodiments, the output module can automatically transmit information in response to information received from the amplification module, e.g., in the form of data analysis or raw data performed by software included in the amplification module. Alternatively, the output module may receive the instruction from the user and then transmit the information. The information sent by the output module can be viewed electronically or printed by a printer.

One or more of the input module, the amplification module, and the output module may be included in the same device or may include one or more of the same components. For example, the amplification module may also include an input module, an output module, or both. In another example, an apparatus comprising a processor may be included in both the input module and the output module. The user can use the device to request amplification of the target nucleic acid and can also use it as a means to send information about the amplified product to the recipient. In some cases, an apparatus including a processor may be included in all three modules so that the apparatus, including the process, also controls, provides instructions, and communicates with a device included in the amplification module or any other module (e.g., A thermal cycler, a detector, a fluid handling device).

An exemplary system for amplifying a target nucleic acid according to the method described herein is shown in FIG. The system includes a computer 101 that may be provided as part of both the input and output modules. The user may insert a reaction vessel 102 containing the reaction mixture ready for nucleic acid amplification Into the amplification module 104 . The amplification module includes a thermal cycler ( 105 ) and a detector ( 106 ). Input module 107 includes a computer 101 and an associated input device 103 (e.g., keyboard, mouse, etc.) capable of receiving a request from a user to amplify a target nucleic acid in a reaction mixture. The input module 107 communicates the user's request to the amplification module 104 and the nucleic acid amplification begins at the thermocycler 105 . As the amplification proceeds, the detector 106 of the amplification module detects the amplified product. Information about the amplified product (e.g., raw data obtained by the detector) is transmitted from the detector 106 to the computer 101 , which is also provided as a component of the output module 108 . The computer 101 receives information from the amplification module 104 , performs any additional operations on the information, and then generates a report containing the processed information. When the report is generated, the computer 101 is a report that the final recipient 109 is a computer network over a computer network interface 110 to the via (e.g., an intranet, the Internet), via a printer (111) a high copy Or via an electronic display 112 operatively connected to the computer 101 . In some cases, the electronic display (112)

In one aspect, the present disclosure provides a computer-readable medium comprising machine executable code that, when executed by one or more processors, embodies a method according to any of the methods described herein. In another aspect, the present invention provides a computer readable medium comprising machine executable code that, when executed by one or more computer processors, embodies a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject Said method comprising the steps of: (a) providing a reaction container comprising a reagent and a biological sample necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, wherein said reagent comprises (i ) A reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for the target RNA; And (b) performing a plurality of cycles of primer extension reaction on the reaction mixture in the reaction vessel to produce an amplified DNA product indicative of the presence of target RNA, amplifying the target RNA, wherein each cycle comprises (i) Incubating the mixture at a denaturation temperature for a denaturation period of 60 seconds or less, and then (ii) incubating the reaction mixture at an elongation temperature for an elongation period of 60 seconds or less.

In another aspect, the present invention provides a computer readable medium comprising machine executable code that, when executed by one or more computer processors, embodies 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 from the subject; (b) providing a reaction container comprising a reagent and a biological sample required to perform reverse transcription amplification and optionally deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, wherein the reagent comprises (i) a reverse transcriptase and (ii) a set of primers for the target RNA; (c) performing multiple cycles of the primer extension reaction on the reaction mixture to yield a detectable amount of amplified DNA product indicative of the presence of target RNA in the biological sample; (d) detecting the amount of the DNA product of (c); And (e) outputting information on the amount of DNA product to the recipient, wherein the amount of time to complete (a) - (e) is less than about 30 minutes.

In one aspect, the present invention provides a computer readable medium comprising machine executable code that, when executed by one or more computer processors, embodies a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained from a subject , Said method comprising the steps of: (a) providing a reaction vessel comprising a biological sample and a reagent necessary to perform nucleic acid amplification to obtain a reaction mixture, said reagent comprising (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a primer set for the target nucleic acid; And (b) performing a plurality of series of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified product from the target nucleic acid, each series comprising (i) reacting the reaction mixture with a (Ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period, wherein the individual series are incubated under denaturing conditions and / or elongation conditions Of the plurality of series being different from at least one other individual series of the plurality of series.

The computer-readable medium may take various forms, including, without limitation, a type (or non-volatile) storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer (s), such as those that may be used to carry out, for example, computational steps, processing steps, Volatile storage media include dynamic memory, e.g., main memory of a computer. The type of transmission medium is coaxial cable; Copper wires and optical fibers, including wires including buses within computer systems. The carrier-wave transmission medium may take the form of electronic or electromagnetic signals, or those generated during acoustic or optical wave, such as radio frequency (FR) and infrared (IR) data communications. Common forms of computer-readable media therefore include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or a DVD- Tape, any other physical storage medium having a hole pattern, a RAM, a PROM and an EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, Or any other medium from which a computer can read program code and / or data. Many of these forms of computer readable media may be involved in carrying one or more of the instructions of one or more instructions to a processor for execution.

Example

Example  1: Virus stock  Amplification and detection of nucleic acids in samples and biological samples

Amplification and detection experiments were performed to compare the results obtained from virus standard and biological samples. Standard samples of biological samples containing RNA virus pathogens and viral pathogens were applied to the amplification conditions to amplify the RNA of the pathogen. Experimental sets were performed for each of the H3N2 and H1N1 (2007) influenza viruses. Each biological sample was obtained directly from the subject through an oral pharyngeal swab. Each virus standard sample was obtained as a serial dilution of the stock solution containing the virus. The concentration of H3N2 and H1N1 (2007) was 10 ⁶ IU / mL. For H5N1 and H1N1 (2007), amplification was performed for dilutions of 1/2, 1/20, 1/200, 1/2000, and 1/20000. In each experimental set, negative controls (e. G. Samples without viral RNA) were also amplified.

Five microliters of each sample were combined in a 25 [mu] l reaction tube with the reagents needed to perform the reverse transcription of the viral RNA and the reagents necessary to complete the amplification (e.g., parallel nucleic acid amplification) of the complementary DNA from reverse transcription. Reagents necessary for performing reverse transcription and DNA amplification are commercially available pre-mixtures containing reverse transcriptase (for example, Sensiscript and Omniscript transcriptase), DNA polymerase (for example, HotStarTaq DNA polymerase), and dNTP For example, Qiagen one-step RT-PCR or one-step RT-qPCR kit). The reaction tube also contained a TaqMan probe containing a FAM dye for detection of the amplified DNA. To generate amplified DNA products, each reaction mixture was incubated in a real-time PCR thermocycler for 5 min at 95 ° C, then at 45 ° C for 20 min, then at 95 ° C for 2 min, then at 95 ° C for 5 sec and at 55 ° C Lt; RTI ID = 0.0 > 40 < / RTI > cycles for 30 seconds. Detection of the amplified product occurred during incubation.

Amplification results for the H3N2 2 was shown in (FIG. 2A corresponds to a different viral standard samples, and Figure 2B is equivalent to the biological sample) 3 is also the amplification results of the H1N1 (2007) (Fig. 3A is a different virus standard Corresponding to the sample, and Figure 3B corresponds to the biological sample). The recorded fluorescence of the FAM dye was plotted against the number of cycles.

As shown in Figure 2A , each of the H3N2 virus standard samples showed a detectable signal above the negative control, with a Ct value of 18-32. As shown in Figure 2B , each of the viral H3N2 biological samples showed a detectable signal above the negative control, with a Ct value in the range of 29-35.

As shown in Figure 3A , except for 1/20000 dilution, each of the H1N1 (2007) virus standard samples displayed a detectable signal with a Ct value in the range of 24-35, compared to the negative control. As shown in FIG. 3B , each of the H1N1 (2007) biological samples showed a detectable signal with a Ct value in the range of 28-35, as compared to the negative control.

In general, the data shown in FIGS. 2 and 3 show that the tested viruses are amplified through the amplified DNA product, with good sensitivity, at a low concentration of 50 IU / mL, at a 4-log concentration It can be detected. In addition, the data suggests that the detection of viral RNA obtained from a biological sample from the subject can be detected in a similar manner.

Example  2: different Buffer  Amplification and detection of viral nucleic acids in the system

Amplification and detection experiments were performed to compare the results obtained using different buffers for amplification. The experimental set was performed on two different buffer systems, S1 and S2. The S1 buffer included a twitter ion buffer and BSA, and the S2 buffer contained a twitter ion buffer and sodium hydroxide. Experiments on each buffer were performed using the H5N1 influenza virus standard sample set obtained as serial dilutions of the stock solution containing the virus. The H5N1 concentration was 10 ⁶ IU / mL. Amplification was performed on dilutions of 1/2, 1/20, 1/200, 1/2000, 1/20000, 1/200000 and negative control.

Five microliters of each sample was combined in a 25 μl reaction tube with the reagents necessary to perform the reverse transcription of the viral RNA and the reagents needed to complete the amplification (eg, parallel nucleic acid amplification) of complementary DNA from reverse transcription. Reagents necessary for performing reverse transcription and DNA amplification include reverse transcriptase, DNA polymerase, dNTPs, and appropriate S1 or S2 buffers. The reaction tube also contains a TaqMan probe containing a FAM dye for detection of the amplified DNA product. To generate the amplified DNA product, each reaction mixture contains 5 cycles at 95 ° C for 5 minutes, followed by 20 cycles at 45 ° C, 2 minutes at 95 ° C, and 40 cycles of 5 seconds at 95 ° C and 30 seconds at 55 ° C Lt; RTI ID = 0.0 > RT < / RTI > thermocycler according to the protocol of denaturation and elongation conditions. Detection of the amplified product occurred during incubation.

The amplification results for Buffer System S1 are shown in Figure 4A and the results for Buffer System S2 are shown in Figure 4B . The recorded fluorescence of the FAM dye was plotted against the number of cycles.

As shown in Fig. 4A , each of the virus standard samples amplified in the buffer system S1 showed detectable signals at a Ct value in the range of 25 to 36 relative to the negative control. As shown in Figure 4B , each of the virus standard samples amplified in Buffer S2 showed a detectable signal relative to the negative control at a Ct value in the range of 25-35.

In general, the data shown in Figure 4 can be detected at a cycle limit of about 40 or less in the 5-log concentration range at a low concentration of 50 IU / mL, with good sensitivity through the amplified DNA product . In addition, the data suggests that similar amplification results can be obtained with different buffer systems.

Example  3: Among the plasma samples, hepatitis B virus ( HBV ) Amplification and detection

Amplification experiments were performed to determine the robustness of the amplification method for detecting target nucleic acids in biological samples. In a diluted human plasma sample containing hepatitis B virus (HBV) at various concentrations (e.g., 50 infectious units per milliliter (IU / mL), 200 IU / mL, 2000 IU / mL, 20000 IU / mL) Respectively. HBV is a DNA virus that replicates through RNA intermediates. HBV can be detected by direct PCR of DNA viruses. Multiple samples (n = 2-4) were added to multiple samples of negative control (e. G., HBV-free plasma) and tested at each concentration.

Add 2.5 μl of each sample and the reagents required to perform the reverse transcription of RNA and the reagents required to perform amplification (eg, parallel nucleic acid amplification) of the complementary DNA obtained by reverse transcription and each sample reagent into a 50 μl reaction tube A mixture was obtained. Reagents necessary for performing reverse transcription and DNA amplification can be obtained from commercially available pre-mixtures containing reverse transcriptase (e.g., Sensiscript and Omniscript transcriptase), DNA polymerase (e.g., HotStarTaq DNA polymerase), and dNTP For example, Qiagen one-step RT-PCR or One-Step RT-qPCR kit). The mixture also contained a TaqMan probe containing a FAM dye for detection of the amplified DNA product. In addition, the reaction mixture contained uracil-DNA glycosylase (UNG) enzyme and zwitterion buffer to prevent the inhibitory effect of the amplification inhibitor present in the plasma. Each reaction mixture was incubated at 94 ° C for 1 minute, at 50 ° C for 10 minutes, then at 94 ° C for 2 minutes, and at 94 ° C for 5 seconds and at 58 ° C for 35 seconds for 50 cycles of denaturation and elongation conditions Followed by incubation in a real-time PCR thermocycler. Detection of the amplified product occurred during incubation.

The amplification results are shown in FIG. 5 and the measured Ct values are summarized in Table 1 . The relative fluorescence unit (RFU) of the recorded dye FAM was graphed against the number of cycles in FIG. As shown in Figure 5 and Table 1 , HBV was detected at each concentration tested with a cycle limit value ranging from 28.99 to 39.39. In general, high concentrations of samples corresponded to low cycle limits.

In general, the results shown in FIG. 5 and Table 1 can be detected with a sensitivity limit of about 40 or less at a concentration as low as 50 IU / mL (tested lowest), through HBV amplified DNA products Respectively. The highest concentration tested (20000 IU / mL) was enriched approximately 400-fold over the lowest concentration tested (50 IU / mL), but the cycle limit was only about 25% higher for low concentrations suggesting that the amplification scheme is largely firm do.

Table 1: Ct results of the experiment of Example 3 Sample # IU / mL Ct One 2000 33.09 2 50 39.39 3 2.00E + 04 29 4 2000 32.97 5 200 35.51 6 2000 33.07 7 2.00E + 04 30.03 8 200 35.78 9 50 37.91 10 2.00E + 04 29.37 11 200 35.73 12 2.00E + 04 28.99

Example  4: Preincubation of biological samples before series of amplification and amplification reactions of nucleic acids in biological samples

Amplification experiments were performed to confirm the preheating effect of the biological samples on the detection sensitivity and the effect of multiple series of amplification reactions on the detection sensitivity was confirmed.

Twenty 25 [mu] l reaction mixtures were prepared, each reaction mixture containing 1 [mu] l of the pathogen species, reagent necessary for appropriate nucleic acid amplification reactions (e.g., reverse transcription for RNA species and DNA amplification, and DNA amplification for DNA species) , And a TaqMan probe containing a FAM dye. Four reaction mixtures contained H1N1 (2007) (ie RNA virus), four reaction mixtures contained H3N2 (ie RNA virus), four reaction mixtures contained H1N1 (2009) The reaction mixture contained Tuberculosis (TB) (i.e., a bacterial sample), and the four reaction mixtures contained an Alcian disease virus (ADV) (i.e., a DNA virus). The H1N1 (2007), H1N1 (2009), H3N2, and ADV pathogenic species were derived from oral pharyngeal swabs obtained from the subject. TB was obtained from stock.

Various combinations of preheat and amplification protocols were used and summarized in Table 2. For the first reaction mixture for each pathogen species, the pathogen species was preheated at 95 占 폚 for 10 minutes before addition to the reaction mixture. After addition of the pathogen species to the reaction mixture, the reaction mixture was incubated in real-time PCR thermocycler according to the protocol of denaturation and elongation conditions, including 2 cycles at 95 ° C for 2 minutes, 5 seconds at 95 ° C for 5 seconds, and 55 ° C for 30 seconds for 40 cycles Lt; / RTI > Detection of the amplified product occurred during incubation. These reaction mixtures were called PH-1 mixtures.

For the second reaction mixture for each pathogen species, the pathogen species was preheated at 50 占 폚 for 30 minutes before being added to the reaction mixture. After addition of the pathogen species to the reaction mixture, the reaction mixture was incubated in a real-time PCR thermocycler following 95 ° C for 2 minutes, 95 ° C for 5 seconds and 40 cycles of 55 ° C for 30 seconds according to the protocol of denaturation and elongation conditions. Detection of the amplified product occurred during incubation. These reaction mixtures are called PH-2 mixtures.

For the third reaction mixture for each pathogen species, the pathogen species was not preheated prior to addition to the reaction mixture. These reaction mixtures were incubated in a real-time PCR thermocycler according to the protocol of denaturation and elongation conditions including 95 ° C for 1 min, 55 ° C for 10 min, 95 ° C for 2 min, 95 ° C for 5 sec and 40 ° C for 30 sec Lt; / RTI > Detection of the amplified product occurred during incubation. These reaction mixtures were called PTC-1 mixtures.

For the fourth reaction mixture for each pathogen species, the pathogen species was not preheated prior to addition to the reaction mixture. For these reaction mixtures, a protocol was implemented that included multiple series of amplification reactions, each containing multiple cycles of denaturation and elongation conditions. After 10 cycles of 95 ° C for 1 minute and then 10 cycles of the first series (95 ° C for 5 seconds, 60-50 ° C for 20 seconds, stepwise decrease at 1 ° C / cycle for 10 seconds) and 95 ° C for 2 minutes, Real-time PCR was performed in a thermocycler according to a protocol including a second series of cycles (5 sec at 95 [deg.] C, 30 sec at 55 [deg.] C). Series 1 and Series 2 differ in their elongation temperature and elongation period. Detection of the amplified product occurred during incubation. These reaction mixtures were called PTC-2 mixtures.

Table 2: Experimental conditions of Example 4 Reaction mixture type protocol PH-1 After the pre-addition of the pathogen species to the reaction mixture at 95 캜 for 10 minutes, the reaction mixture was incubated at 95 캜 for 2 minutes, at 95 캜 for 5 seconds, at 55 캜 for 30 seconds, PH-2 After pre-heating the pathogen species before addition to the reaction mixture at 50 캜 for 30 minutes, the reaction mixture was incubated at 95 캜 for 2 minutes, at 95 캜 for 5 seconds, at 55 캜 for 30 seconds, PTC-1 95 ° C for 1 minute, 55 ° C for 10 minutes, then 95 ° C for 2 minutes, (95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles PTC-2 Followed by 1 hour at 95 ° C for 1 minute, 10 cycles of 10 cycles (95 ° C for 5 seconds, 60-50 ° C, 1 ° C / cycle stepwise, 20 seconds, 60 ° C for 10 seconds) followed by 2 minutes at 95 ° C Sec, 55 < 0 > C for 30 seconds) x 40 cycles

The results for each pathogen species is shown in Figure 6 (H1N1 (2007), Figure 7 (H3N2), Figure 8 (H1N1 (2009)), Figure 9 (TB), and 10 (ADV). Fig. 6-10 Each item A The results obtained for the reaction mixtures PH-1 and PH-2 are shown, whereas the items B in FIGS. 6-10 show the results obtained for the reaction mixtures PTC-1 and PTC-2. The Ct values determined in each experiment are summarized in Table 3. The Ct values for the PH-1 and PH-2 ADV reaction mixtures were not confirmed but correspond to the data shown in FIG. 10A .

According to the data shown in Table 3, the Ct values are fairly similar between the PH-1 and PH-2 reaction mixtures so that the pathogen species (or biological sample containing the pathogen species) can be preheated . In addition, the PTC-1 reaction mixture was similar to the Ct values for the PH-1 and PH-2 reaction mixture. The PTC-1 and PH-1 / PH-2 protocols were similar, except that PTC-1 did not include a preheating step. Thus, a comparison of the PTC-1 data with the PH-1 / PH-2 data suggested that the pre-heating of the pathogen species is not essential to obtain good susceptibility results before being provided to the reaction mixture. However, in the case of TB and ADV samples in some cases, preheating could be worse than without preheating.

However, for all pathogen species tested, PTC-2 Ct values were lower than either PH-1, PH-2, or PTC-1. Comparison of PTC-1 and PTC-2 data suggested that for the reaction mixture, performing multiple series of amplification reactions, each series involving multiple cycles of denaturation and elongation conditions could improve detection sensitivity .

Table 3: Ct results of Example 4 experiment type Sample PH-1 (Ct) PH-2 (Ct) PTC-1 (Ct) PTC-2 (Ct) RNA virus H1N1 (2007) 27 30 28 22 RNA virus H3N2 34 33 32 23 RNA virus H1N1 (2009) 32 32 32 24 DNA bacteria TB 34 32 26 20 DNA virus ADV - - 36 30

Example  5: Multiplexed sample

Amplification and detection experiments were performed to verify whether various amplification protocols could be benchmarked and multiplexed. (Eg, H1N1 (2007), H1N1 (2009), H3N2) or DNA (eg, ADV, human bovavirus (HBoV)) viral pathogens or DNA bacterial pathogens Biological samples were applied to various amplification conditions. Each biological sample was obtained directly from the subject via the oral pharyngeal swab, except for the TB sample obtained from the bacterial stock. One microliter of each sample was subjected to nucleic acid amplification as described herein and blended in a 25 [mu] l reaction tube with the reagents necessary to detect the amplified product to obtain a reaction mixture.

To evaluate the multiplexing ability of the amplification protocol, three reaction mixtures each containing one of H3N2, ADV, or a mixture of H3N2 and ADV were incubated for 2 minutes at 94 DEG C for 20 minutes, 45 DEG C for 1 minute, 94 DEG C for 5 minutes, And incubated in a real-time PCR thermocycler according to an amplification protocol comprising 50 cycles of 94 [deg.] C and 55 [deg.] C for 35 seconds. Detection of the amplified product occurred during incubation.

The results of the experiment are shown in FIG. 11 and summarized in Table 4 below. As shown in Fig. 11 , both H3N2 and TB could similarly be detected in combination or in the absence of the other. In the absence of ADV, a Ct value of 26.03 was recorded for the H3N2 reaction mixture, and in the absence of H3N2, a Ct value of 30.5 was recorded for the ADV reaction mixture. When both H3N2 and ADV were combined in a single reaction mixture, the Ct values of 26 (H3N2) and 30 (ADV) were obtained. The Ct values were approximately the same for the combined reaction mixture as compared to the single component reaction mixture. The results suggested that multiplexing could be obtained with good sensitivity and that both RNA and DNA species could be detected.

Table 4: Experimental results of H3N2 and ADV multiplexing in Example 5 type Sample Ct RNA virus H3N2 26.03 DNA virus ADV 30.5 RNA & DNA virus H3N2 & ADV 26 (H3N2) & 30 (ADV)

In another experiment to evaluate the multiplexing ability of the amplification protocol, the three reaction mixtures containing either H3N2, TB, or a mixture of H3N2 and TB, respectively, were incubated at 95 DEG C for 2 minutes, at 95 DEG C for 5 seconds, and at 55 DEG C for 30 seconds Real-time PCR thermocycler according to an amplification protocol comprising 40 cycles. Detection of the amplified product occurred during incubation.

The results of the experiment are shown in FIG. 12 and summarized in Table 5. As shown in Fig. 12 , both H3N2 and TB were detected in combination if they were combined or not. In the absence of TB, a Ct value of 32 was recorded for the H3N2 reaction mixture, and in the absence of H3N2, a Ct value of 32 was recorded for the TB reaction mixture. When both H3N2 and TB were combined in a single reaction mixture, Ct values of 29 (H3N2) and 30 (TB) were obtained. The Ct values were similar for the combined reaction mixtures as compared to the single component reaction mixture. The results suggested that multiplexing could be obtained with good sensitivity and that both RNA and DNA species could be detected in the multiplexing scheme.

Table 5: Results of H3N2 and TB multiplexing experiments of Example 5 type Sample Ct RNA virus H3N2 32 DNA virus TB 32 RNA & DNA virus H3N2 & TB 29 (H3N2) & 30 (TB)

Example  6: Benchmarking multiple series of amplification reactions

Amplification and detection experiments were performed to benchmark various amplification protocols including multiple series of amplification reactions. Including biological (including, for example, H1N1 (2007), H1N1 (2009), H3N2) or DNA (such as ADV, human bovirus (HBoV) viral pathogens or DNA bacterial pathogens Each biological sample was obtained directly from the subject via an oral pharyngeal swab, except that the TB sample was obtained from a bacterial stock. [0060] One microliter of each sample was subjected to nucleic acid amplification And combined in a 25 [mu] l reaction tube with the reagents necessary to detect the amplified product to give a reaction mixture.

In one set of experiments, an amplification protocol was performed that included two series of amplification reactions, each series containing different denaturation and elongation conditions for the amplification mixture. Six cycles of reaction mixture (two kinds including H3N2, two kinds including ADV and two types including HBoV) were incubated at 94 ° C for 1 second, followed by 11 cycles of series 1 (94 ° C for 1 second and 45 ° C for 10 seconds) Real-time PCR was carried out in a thermocycler according to an amplification protocol comprising 95 cycles of 1 minute and 95 cycles of series 2 (95 ° C for 5 seconds and 55 ° C for 30 seconds). Detection of the amplified product occurred during incubation.

The experimental results are shown in Table 6. The Ct values measured as shown in Table 6 were in the range of 8.35 to 23. The results suggest that a protocol containing multiple series of amplification reactions may be useful for obtaining good sensitivity. In addition, the results suggest that both RNA and DNA species can be detected with a protocol that includes multiple series of amplification reactions.

Table 6: H3N2, ADV, and HBoV experimental results type Sample Ct RNA virus H3N2-1 17 RNA virus H3N2-2 20 DNA virus ADV-1 18.8 DNA virus ADV-2 23 DNA virus HBoV-1 8.35 DNA virus HBoV-2 18.37

In another set of experiments, the amplification mixture was subjected to an amplification protocol comprising three series of amplification reactions, each series differing in their denaturation and / or elongation conditions. Five reaction mixtures (one including sH1N1 (2007), one including H3N2, one including pH1N1 (2009), one including ADV, and one including TB) were incubated for one minute at 94 ° C, followed by series 1 5 cycles of series 2 (94 ° C for 5 seconds and 50 ° C for 30 seconds), followed by 95 ° C for 2 minutes, followed by 5 cycles of 94 ° C for 30 seconds and 60 ° C for 30 seconds, Lt; RTI ID = 0.0 > 95 C < / RTI > for 30 seconds and 55 C for 30 seconds) in a real-time PCR thermocycler. Detection of the amplified product occurred during incubation.

The experimental results are shown in Table 7 below. As shown in Table 7, the measured Ct values ranged from 20 to 30. The results indicate that a protocol containing multiple series of amplification reactions is useful for obtaining good sensitivity. In addition, the results suggest that both RNA and DNA species can be detected with a protocol that includes multiple series of amplification reactions.

Table 7: Results of sH1N1 (2007), H3N2, pH1N1 (2009), ADV, and TB experiments of Example 6 type Sample Ct RNA virus sH1N1 (2007) 22 RNA virus H3N2 23 RNA virus pH1N1 (2009) 24 DNA virus ADV 30 DNA bacteria TB 20

Example  7: Benchmarking multiple series of amplification reactions

Amplification and detection experiments were performed to benchmark multiple amplification protocols including multiple series of amplification reactions. Biological samples containing H3N2 were applied to various amplification conditions. Each biological sample was taken directly from the subject via an oral pharyngeal swab. One microliter of each sample was subjected to nucleic acid amplification as described herein and combined in a 25 [mu] l reaction tube with reagents necessary to detect the amplified product to obtain a reaction mixture.

An amplification protocol was performed on the amplification mixture, some of which included one of the three different first amplification reactions and a second series, and the three first series comprised different denaturation and elongation conditions than the second series. Each of the first series and the second series includes a plurality of cycles. Other experiments were performed with only the second series, without the first series. In a real-time PCR thermocycler, each of the four reaction mixtures containing H3N2 was incubated according to one of the amplification protocols shown in Table 8 below.

Table 8: Experimental Protocol of Example 7 reaction
mixture protocol One X 5 cycles at 94 占 폚 for 1 minute (series 1A-94 占 폚 for 1 second, 45 占 폚 for 2 minutes) x 5 cycles at 95 占 폚 for 1 minute (series 2 - 95 占 폚 for 5 seconds, 55 占 폚 for 30 seconds) cycle 2 (5 seconds at series 2 - 95 ° C, 30 seconds at 55 ° C) for 2 minutes at 80 ° C (1 second at series 1B - 80 ° C and 2 minutes at 45 ° C) x 50 cycles 3 At 50 ° C for 2 minutes, at 45 ° C for 30 minutes, at 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 50 cycles 4 X 50 cycles (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 50 cycles (1 second at 94 ° C, 1 second at 45 ° C for 30 seconds)

The results of the experiment are shown in Fig. 13 and shown in Table 9 below. As shown in Fig. 13 , Reaction mixture 3 had a maximum Ct value (28.59). Others, including the multiple series, had low values in the range of 8.5 to 26.5. The results suggest that a protocol containing multiple series of amplification reactions is useful for obtaining good sensitivity. In addition, the results suggest that a protocol containing multiple series of amplification reactions can achieve better sensitivity compared to protocols with only a single series.

Table 9: Test results of Example 7 Reaction mixture Ct One 22.97 2 26.5 3 28.59 4 8.5

Example  8: Benchmarking multiple series of amplification reactions

Amplification and detection experiments were performed to benchmark various amplification protocols including multiple series of amplification reactions. Biological samples containing H3N2 were applied to various amplification conditions. Each biological sample was obtained directly from the subject through an oral pharyngeal swab. One microliter of each sample was subjected to nucleic acid amplification as described herein and combined in a 25 [mu] l reaction tube with the reagents necessary to detect the amplified product to obtain a reaction mixture.

An amplification protocol was performed on the amplification mixture, some of which included one of the six first series of amplification reactions and the second series, and the six first series comprised different denaturation and extension conditions than the second series. Six other experiments were performed without the first series. In a real-time PCR thermocycler, each of the 12 reaction mixtures containing H3N2 was incubated according to one of the amplification protocols shown in Table 10 below.

Table 10: Experimental Protocol of Example 8 reaction
mixture protocol One 95 ° C for 3 minutes, 45 ° C for 5 minutes, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles 2 95 ° C for 10 minutes, 45 ° C for 5 minutes, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles 3 95 ° C for 3 minutes, 45 ° C for 20 minutes, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles 4 95 ° C for 10 minutes, 45 ° C for 20 minutes, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles 5 95 ° C for 10 minutes, 45 ° C for 3 minutes, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles 6 45 ° C for 20 minutes, 95 ° C for 1 minute, (Series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 40 cycles 7 (Series 1A - 94 ° C for 1 second, 45 ° C for 10 seconds) x 10 cycles, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) x 50 cycles 8 95 ° C for 5 seconds, and 55 ° C for 30 seconds) at 94 ° C for 10 seconds (series 1B to 94 ° C for 1 second, 45 ° C for 10 seconds) x 10 cycles, 50 cycles 9 (Series 2C - 94C for 10 seconds, 45C for 20 seconds) x 10 cycles, 95C for 1 minute, (series 2 - 95C for 5 seconds, 55C for 30 seconds) x 50 cycles 10 (Series 1D - 94 ° C for 10 seconds, 45 ° C for 20 seconds) x 10 cycles, 95 ° C for 1 minute, (series 2 - 95 ° C for 5 seconds, 55 ° C for 30 seconds) 50 cycles 11 (Series 2E - 95 ° C for 5 seconds, 55 ° C for 30 seconds) at 94 ° C for 2 minutes (series 1E - 94 ° C for 30 seconds, 45 ° C for 60 seconds) x 10 cycles, x 50 cycles 12 95 ° C for 5 seconds, and 55 ° C for 30 seconds) at 94 ° C for 10 seconds (series 1F-94 ° C for 30 seconds, 45 ° C for 60 seconds) x 10 cycles, 50 cycles

The experimental results are shown in Table 11 below. The Ct values ranged from 14.53 to 27.28 and the reaction mixture 2-5 had no detectable product. In general, the reaction mixtures that did not carry out multiple series of amplification reactions had higher detectable products or higher Ct values than the reaction mixtures that performed multiple series of amplification reactions. The results indicate that a protocol containing multiple series of amplification reactions is useful for obtaining good sensitivity. In addition, the results suggest that a protocol containing multiple series of amplification reactions can achieve better sensitivity compared to protocols containing only a single series. In some cases, multiple series of amplification reactions are required to generate a detectable amount of the amplified product.

Table 11: Test results of Example 8 Reaction mixture Ct One 26.03 2 - 3 - 4 - 5 - 6 27.28 7 21.64 8 19.56 9 17.2 10 14.53 11 19.2 12 -

Example  9: Comparative results of tablets and crude samples

Amplification and detection experiments were performed to compare the results obtained from the purified and crude samples. Amplification protocols were performed on purified and crude biological samples containing HlNl. Each biological sample was obtained directly from the subject through an oral pharyngeal swab. One microliter of each sample was subjected to nucleic acid amplification as described herein and combined in a 25 [mu] l reaction tube with reagents necessary to detect the amplified product to obtain a reaction mixture. Three reaction mixtures were produced, two of which contained a sample purified by either column or magnetic purification. The third reaction mixture comprises a crude sample.

The reaction mixture was incubated in a real-time PCR thermocycler according to an amplification protocol consisting of 94 ° C for 2 minutes, 45 ° C for 20 minutes, 94 ° C for 1 minute, followed by 50 cycles of 94 ° C for 5 seconds and 55 ° C for 35 seconds. Detection of the amplified product occurred during incubation.

The results of the experiment are shown in FIG. 14 and shown in Table 12 below. table As shown in FIG. 12, the Ct values measured in the range of 27 to 31 were similar between the purified and untreated samples by various means. The results suggest that the sample tablet is not essential for obtaining similar detection sensitivities.

Table 12: Example 9 Sample Type Ct Column purification 31 Magnetic bead purification 27 Crude 28

Example  10: Analysis of specimens and saliva samples

Amplification and detection experiments were performed on H3N2 virus-containing blood and saliva samples. Four different samples were tested. The two samples contain whole blood or saliva samples and the two samples contain 10-fold dilutions (in PBS) of whole blood or saliva samples. Each of the four samples was combined with the reagents needed to perform reverse transcription of the viral RNA and the reagents needed to complete the amplification of the complementary DNA obtained by reverse transcription. Reagents necessary for performing reverse transcription and DNA amplification can be obtained from commercially available pre-mixtures containing reverse transcriptase (e.g., Sensiscript and Omniscript transcriptase), DNA polymerase (e.g., HotStarTaq DNA polymerase), and dNTP For example, Takara one-step RT-PCR or one-step RT-qPCR kit). The reaction tube also contained a TaqMan probe containing a FAM dye for detection of the amplified DNA product. To generate the amplified DNA product, each reaction mixture was subjected to a denaturation and extension protocol at 45 ° C for 20 minutes followed by 94 ° C for 2 minutes, followed by 94 ° C for 5 seconds and 42 ° C for 35 seconds at 55 ° C And incubated in a real-time PCR thermocycler. Detection of the amplified product occurred during incubation.

The amplification result for H3N2 is shown in FIG. 15 (FIG. 15A corresponds to the undiluted blood , FIG. 15B corresponds to the diluted blood) and FIG. 16 (FIG. 16A corresponds to the undiluted saliva, and FIG. 16B corresponds to the diluted saliva box). The recorded fluorescence of the FAM dye was graphed according to the number of cycles.

As shown in FIGS. 15 and 16 , both the undiluted and diluted blood and saliva reaction mixtures exhibited detectable signals in the Ct value range of 24-33. Thus, the results shown in Figures 15 and 16 suggest that the undiluted biological sample can be analyzed with good sensitivity, with a Ct value of about 40 or less. The data also suggests that if the dilution of the sample is necessary for the analysis, the amplified product can be detected in a similar manner. In some cases, if too much of the inhibitor from the sample is present, the dilution may be another way to remove the inhibition in the sample, e.g. whole blood.

Example  11: Nested PCR

Amplification and detection experiments were performed on H1N1 virus-containing samples. Eight species samples were tested. Each sample contains a H1N1 (2007) virus stock. Samples were each diluted in PBS and the dilution ratios were as shown in Table 13 below. The virus stock concentration is 1x10 ⁶ IU / mL. To generate the amplified DNA product, the reaction mixture containing the predetermined sample was incubated according to a protocol of denaturation and elongation conditions. (I) in a first run, heating the mixture in a thermocycler at 94 DEG C for 5 minutes at 94 DEG C for 1 minute and then 10 or 15 cycles at 57 DEG C for 10 seconds (as shown in Table 13 below); And (ii) in a second run, heating the mixture in a thermocycler at 94 ° C for 1 minute followed by 94 ° C for 5 seconds and 35 ° C for 30 seconds at 57 ° C. 1 시리즈 series diluted organisms were added to a Takara one-step qPCR premix of 25 uL reaction volume. After the first run for a given cycle, 1 [mu] l from the reaction was added to the second running reaction mixture. Amplification of the H1N1 and the results are shown in Fig. This figure shows a relative fluorescent light-emitting unit (RFU) recorded according to a function of the number of cycles. The graphs for each of the eight samples (1-8) are shown in the figure. The sample with the detectable signal has the Ct value shown in Table 13.

Table 13: Test results of Example 11 # One 2 3 4 5 6 7 8 Sample dilution 1/10 1/100 1/1000 0 1/10 1/100 1/1000 0 Ct 18 21 27 - 11 17 24 - At the first execution time
cycle 10 cycles 15 cycles

Example  12: Amplification and Detection of Ebola Recombinant Plasmids

Amplification and detection experiments were carried out on human whole blood samples containing various copies of recombinant plasmids corresponding to Zyer Ebola virus (ZyR-EBOV). 8 samples were tested. Six of the samples contained recombinant plasmids of a specific copy number (250000, 25000, 2500, 250, 25 and 2.5 copies) and two of the samples (one blood only, one water only) served as control samples. Whole blood samples were analyzed without sample purification.

Each sample is combined with a reagent (for example, a DNA polymerase, a dNTP, a primer, a cofactor, a suitable buffer, a primer, etc.) required for nucleic acid amplification and a reporter (for example, an oligonucleotide probe including a FAM dye) To obtain a reaction mixture. A summary of the various reaction mixtures by sample number, including the number of copies of the recombinant plasmids, is shown in Table 14. To generate the amplified product, two series of denaturation and set conditions were performed for each reaction mixture. 2 series are as follows: (i) 95 ° C for 1 minute after 15 cycles of 95 ° C for 1 second and 45 ° C for 1 second in the first series; And (ii) 45 cycles of 95 ° C for 5 seconds and 55 ° C for 10 seconds in the second series. During the second series, a reporter derived signal was recorded to generate an amplification graph and a Ct value was obtained. The amplification graph for the experiment is shown in Figure 18 , and each is labeled with a sample number corresponding to the sample number shown in Table 14. The result shown in Fig. 18 represents a relative fluorescent light-emitting unit (RFU) recorded according to the function of the number of cycles. The Ct values obtained from the graph shown in Fig. 18 are summarized in Table 15. < tb >< TABLE >

As shown in Fig. 18 , the recombinant plasmid was detected through the amplified product for all the samples containing the recombinant plasmid except for the sample 6. In addition, the recombinant plasmids were not detected in the control samples (Samples 7 and 8). Thus, the results shown in Fig. 18 suggest that, in some cases, the detection sensitivity of 25 copies of plasmid / rxn can be obtained without sample purification using multiple series of denaturation and elongation conditions.

Table 14: Experimental reaction mixture of Example 12 Sample Plasmids (copies / rxn) One 250000 2 25000 3 2500 4 250 5 25 6 2.5 7 0 (blood only) 8 0 (water only)

Table 15: Ct values measured in the experiment of Example 12 Sample Copies / rxn Ct One 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 various copies of the Zygos ebola virus (ZyR-EBOV) pseudovirus. 8 samples were tested in duplicate (Dual Set # 1 and Double Set # 2) for a total of 16 samples. Six of the samples included pseudorabies with a certain number of copies (2500000, 250000, 25000, 2500, 250 and 25 copies) and two samples (one with blood and one with water) served as control samples . Whole blood samples were analyzed without sample purification.

Each sample is treated with a reagent (e.g., a reverse transcriptase, a DNA polymerase, a dNTP, a cofactor, a primer, a suitable buffer, etc.) and a reporter (an oligonucleotide probe including a FAM dye) necessary for reverse transcription and nucleic acid amplification To give a 30 [mu] l reaction mixture. A summary of the reaction mixture by sample number, including the number of copies of pseudovirus, is shown in Table 16. To generate amplified products from pseudovirus, two series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) 95 ° C for 1 minute after 15 cycles of 95 ° C for 1 second and 45 ° C for 1 second in the first series; And (ii) 45 cycles of 95 ° C for 5 seconds and 55 ° C for 10 seconds in the second series. During the second series, a reporter derived signal was recorded to generate an amplification graph and a Ct value was obtained. Amplification graphs for the experiments are shown in Figures 19a (Double Set # 1) and Figure 19b (Dual Set # 2), each labeled with a sample number corresponding to that shown in Table 16. The results shown in Figs. 19A and 19B show the relative fluorescent light emitting units (RFU) recorded according to the function of the number of cycles. The Ct values obtained in the graphs shown in FIGS. 19A and 19B are summarized in Table 17, where "Ct 1" corresponds to double set # 1 and "Ct 2" corresponds to double set # 2.

As shown in Figs. 19A and 19B , pseudorabies were detected in both double sets through amplified products for all samples (samples 1-6) containing pseudovirus. In addition, pseudovirus was not detected in any of the control samples (Samples 7 and 8). Thus, the results shown in Figs. 19A and 19B suggest that, in some cases, detection sensitivity of 25 copies of virus / rxn can be obtained without sample purification using multiple series of denaturation and elongation conditions.

Table 16: Experimental reaction mixture of Example 13 Sample Pseudovirus (copies / rxn) One 2500000 2 250000 3 25000 4 2500 5 250 6 25 7 0 (blood only) 8 0 (water only)

Table 17: Ct value measured in the experiment of Example 13 Sample Copies / rxn Ct 1 Ct 2 One 2500000 8.57 8.44 2 250000 12.09 11.27 3 25000 15.03 14.99 4 2500 18.90 18.87 5 250 21.71 21.71 6 25 27.86 39.42 7 0 (blood only) --- --- 8 0 (water only) --- ---

Example  14: Amplification and detection of Ebola virus

Amplification and detection experiments were performed on human whole blood samples containing various copies of the Zygos ebola virus (ZyR-EBOV) pseudovirus. 8 samples were tested. 6 samples contained pseudoravirus with a specific copy number (2500000, 250000, 25000, 2500, 250 and 25) and 2 samples (with 20000 copies of pseudovirus positive control, only water) served as control samples . Whole blood samples were analyzed without sample purification.

Each sample is subjected to reverse transcription and amplification using reagents necessary for nucleic acid amplification (e.g., reverse transcriptase, DNA polymerase, primer, dNTP, cofactor, suitable buffer, etc.) and a reporter (e.g., oligonucleotide probe ) To give a 30 [mu] l reaction mixture. A summary of the various reaction mixtures by sample number, including the number of pseudovircie copies, is shown in Table 18. < tb >< TABLE > To generate amplified products from pseudovirus, two series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) 95 ° C for 1 minute after 15 cycles of 95 ° C for 1 second and 45 ° C for 1 second in the first series; And (ii) 35 cycles of 95 ° C for 5 seconds and 55 ° C for 10 seconds in the second series. In the second series, a reporter-derived signal was recorded to generate an amplification graph and a Ct value was obtained. An amplification graph for the experiment is shown in Figure 20 , each labeled with a sample number corresponding to that shown in Table 18. The result shown in Fig. 20 shows a relative fluorescent light-emitting unit (RFU) recorded according to the function of the number of cycles. The Ct values obtained from the graph shown in Fig. 20 are summarized in Table 19. < tb >< TABLE >

As shown in Figure 20 , pseudorabies were detected through amplified products for all samples (samples 1-6) containing pseudovirus, including samples containing positive control pseudovirus (sample 7). Pseudovirus was also not detected in the water alone control sample (Sample 8). Thus, the results shown in Figure 20 , in some cases, Suggesting that the detection sensitivity of the virus / rxn of 25 copies can be obtained using multiple series of denaturation and elongation conditions and without sample purification.

Table 18: Experimental reaction mixture of Example 14 Sample Pseudovirus (copies / rxn) One 2500000 2 250000 3 25000 4 2500 5 250 6 25 7 20000 (positive control pseudovirus) 8 0 (water only)

Table 19: Ct value measured in the experiment of Example 14 Sample Copies / rxn Ct One 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 group) 15.94 8 0 (water only) ---

Example  15: Amplification and detection of Ebola virus

Amplification and detection experiments were performed on a human whole blood sample containing one of the two copies of the Zygos ebola virus (ZyR-EBOV) pseudovirus (250 copies / rxn or 25 copies / rxn). Each whole blood sample was tested for a total of 8 samples using one of four reagent systems. Each of the reagent systems (B-1, B-2, B-3 and B-4) contains reagents (for example, reverse transcriptase, DNA polymerase, primer, dNTP, co- Etc.) and reporter agents (e.g., oligonucleotide probes comprising FAM dyes). Each different reagent system contains different concentrations of various components in the reagent system. Each whole blood sample was combined with its appropriate reagent system to yield a 30 [mu] l reaction mixture. A summary of the various reaction mixtures by sample number is shown in Table 20, including the number of copies of pseudovirus and the reagent system. To generate amplified products from pseudovirus, two reaction series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) 95 ° C for 1 minute after 15 cycles of 95 ° C for 1 second and 45 ° C for 1 second in the first series; And (ii) in the second series, 40 cycles of 95 ° C for 5 seconds and 55 ° C for 10 seconds. During the second series, a reporter derived signal was recorded to generate an amplification graph and a Ct value was obtained. An amplification graph for the experiment is shown in Figure 21 , each labeled with a sample number corresponding to that shown in Table 20. The result shown in Fig. 21 represents a relative fluorescent light-emitting unit (RFU) recorded according to the function of the number of cycles. The Ct values obtained in the graph shown in FIG. 21 are summarized in Table 21. < tb >< TABLE >

As shown in Fig. 21 , pseudorhavirus was detected through amplified products for all samples, including samples with 25 copies / rxn. Thus, the results shown in FIG. 21 suggest that, in some cases, the detection sensitivity of 25 copies of virus / rxn can be achieved using a number of series of denaturation and elongation conditions, without sample purification, with different reagent systems.

Table 20: Experimental reaction mixture of Example 15 Sample Pseudovirus (copies / rxn) Reagent system One 250 B-1 2 25 B-1 3 250 B-2 4 25 B-2 5 250 B-3 6 25 B-3 7 250 B-4 8 25 B-4

Table 21: Ct measured in the experiment of Example 15 Sample Copies / rxn Ct One 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: Zaire  Real time for Ebola virus PCR  detection

The patient's serum samples for the Zygoszvola virus were analyzed using the one-step qPCR method of the present application. The sample was not purified. The sample includes a 9-Zie Ebola virus positive sample and a 7-Zie Ebola virus negative sample. Roche LC96 real-time PCR system was used.

The programs adopted in this example to analyze the samples are shown in Table 22 below.

Table 22: Thermal cycling programs step Temperature time Number of cycles One 42 ° C 1 minute 1 cycle 2 95 ℃ 5 seconds 10 cycles 45 ° C 10 seconds 3 95 ℃ 1 minute 1 cycle 4 95 ℃ 5 seconds 40 cycles 55 ° C 10 seconds (reading)

The results of the first-step qPCR method are shown in Table 23. The one-step qPCR method that was tested for the Zygos-Ebola virus showed 100% consistency compared to the proven reagents and methods.

Table 23: Results Sample # Coyote 1st Step QPCR Method (Cq) Proven reagents and methods (Cq)  consistency One N / A N / A has exist 2 26.53 29.73 has exist 3 17.68 19.53 has exist 4 N / A N / A has exist 5 N / A N / A has exist 6 N / A N / A has exist 7 N / A N / A has exist 8 21.52 20.98 has exist 9 18.97 18.88 has exist 10 24.97 24.44 has exist 11 18.92 18.91 has exist 12 26.32 25.22 has exist 13 20.48 20.85 has exist 14 18.5 20.45 has exist 15 N / A N / A has exist

Example  17: Amplification and detection of malaria

Amplification and detection experiments were performed on a human whole blood sample containing an unknown concentration of malaria pathogen. Two sets of experiments were performed. In a first set of experiments, a double experiment was performed on a 1: 4 dilution (in 1X PBS) of a human whole blood sample, where experiments involving samples containing plasmids and whole blood corresponding to malaria pathogens were performed, Were carried out. In a second set of experiments, various dilutions (1: 4, 1:40, 1: 400, 1: 4000, 1: 40000 and 1: 400000) of human whole blood samples in 1X PBX with blood alone and water- Were carried out. Whole blood samples were analyzed without sample purification.

Each sample is combined with a reagent (e.g., DNA polymerase, primer, dNTP, cofactor, suitable buffer, etc.) necessary for nucleic acid amplification and a reporter (e.g., oligonucleotide probe including FAM dye) Mu] l of reaction mixture. A summary of the reaction mixture according to sample number, including dilution, for the first set of experiments is shown in Table 24. < tb >< TABLE > A summary of the reaction mixture according to sample number, including dilution in the second set of experiments, is shown in Table 25. < tb >< TABLE > To generate amplified products from malaria pathogens, two series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) in the first series, 13 cycles of 95 占 폚 for 1 second and 45 占 폚 for 1 second, then 95 占 폚 for 1 minute; And (ii) 45 cycles of 95 ° C for 5 seconds and 55 ° C for 10 seconds in the second series. During the second series, a signal by the reporter agent was recorded to generate an amplification graph. The amplification graph for the first set of experiments is shown in Figure 22A , and the amplification graph for the second set of experiments is shown in Figure 22B . Each graph was labeled with its corresponding sample number in Tables 24 and 25, respectively. The results shown in Figs. 22A and 22B were expressed as a relative fluorescent light-emitting unit (RFU) recorded according to the function of the number of cycles.

As shown in Figure 22A , the malaria pathogen was detected through the amplified products for the two reaction mixtures (Samples 1 and 2) and the positive control (Sample 3) containing the recombinant plasmids. In addition, malaria pathogens were not detected in water alone samples (Sample 4). Thus, the results shown in Figure 22B suggest that malaria pathogens can, in some cases, be detected without sample purification using multiple series of elongation and denaturation conditions.

As shown in FIG. 22B , malaria pathogens were detected through amplified products for all reaction mixtures (samples 1-6) containing whole blood samples. In addition, malaria pathogens were not detected in water alone and blood alone samples (Samples 7 and 8). Thus, the results shown in FIG. 22B suggest that the pathogen (s), including malaria pathogens, can be detected in dilutions of up to 1: 400000 without sample purification using multiple series of denaturation and elongation conditions in some cases .

Table 24: Experimental reaction mixture for the first experimental set of Example 17 Sample Dilution One 1: 4 2 1: 4 3 1: 2 (Plasma of whole blood control group) 4 None (water only)

Table 25: Experimental reaction mixture for the second experimental set of Example 17 Sample Dilution One 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: Dengue  Amplification and detection of viruses

Amplification and detection experiments were performed on samples obtained from cultures containing unknown concentrations of dengue virus. Three sets of experiments were performed. In the first set of experiments, a duplicate experiment was performed on the undiluted culture, the experiment was performed on a 1:10 dilution of the culture, and an experiment was performed on a control with only water. In the second set of experiments, experiments were performed on various dilutions of the culture (no dilution, 1: 10, 1: 100, 1: 1000, 1: 10000, 1: 100000 and 1: 1000000) Respectively. In the third set of experiments, experiments were performed with various dilutions of the culture (no dilution, 1:10, 1: 100, 1: 1000, and 1: 10000) with a control sample only with water. Cultured samples were analyzed without sample purification.

2 μl of each sample was subjected to reverse transcription and amplification using a reagent (for example, a reverse transcriptase, a DNA polymerase, a primer, a dNTP, a cofactor, a suitable buffer and the like) necessary for nucleic acid amplification and a reporter Nucleotide probe) to obtain a 30 [mu] l reaction mixture. A summary of the reaction mixture, including dilution, for 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 the amplified product from the virus, two series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) 10 cycles of 42 ° C for 1 minute, 95 ° C for 5 seconds and 45 ° C for 10 seconds, then 95 ° C for 1 minute, in the first series; And (ii) 45 cycles of 95 ° C for 5 seconds and 55 ° C for 10 seconds in the second series. During the second series, a reporter originated signal was recorded and an amplification graph was generated. The amplification graph for the first experimental set is shown in Figure 23A , the amplification graph for the second experiment set is shown in Figure 23B , the amplification graph for the third experiment is 23C . Each graph was labeled with the corresponding sample number in Tables 26, 27 and 28, respectively. The results shown in Figs. 23A , 23B, and 23C were expressed as a relative fluorescent light-emitting unit (RFU) recorded according to the function of the number of cycles. Ct values obtained in the graphs shown in Figs. 23A , 23B and 23C are shown in Tables 26, 27 and 28, respectively.

As shown in FIG. 23A , viruses were detected through amplified products for three reaction mixtures (samples 1-3) containing viruses. In addition, the virus was not detected in the water-only control sample (Sample 4). Thus, the results shown in Figure 23A suggest that Dengue virus can be detected using multiple series of elongation and denaturation conditions in some cases.

As shown in Figure 23 (b), the virus was detected through the amplified product for the reaction mixture containing Dengue virus and not diluted (Sample 1) or diluted 1: 1000 (Samples 2, 3 and 4). However, the Ct value was not determined for the 1: 1000 reaction mixture (Sample 4). Viruses were not detected in high dilutions (Samples 5, 6 and 7) or water alone control samples (Sample 8). Thus, the results shown in FIG. 23B can be detected in dilutions of up to 1: 1000 in some cases, without sample purification, using multiple series of denaturation and elongation conditions, with Ct values of up to 1: 100 ≪ / RTI >

As shown in Figure 23C , viruses were detected via amplified products for reaction mixtures containing Dengue virus, un-diluted (Sample 1) or diluted 1: 1000 (Samples 2, 3 and 4). However, the Ct value was not determined for the 1: 1000 reaction mixture. The virus was not detected in the higher dilutions (Sample 5) or water alone control sample (Sample 6). Thus, the results shown in Figure 23C can be detected in dilutions of up to 1: 1000 without sample purification using a number of series of denaturation and elongation conditions, in some cases, where the Ct value is up to 1: 100 Suggesting that it may be generated in dilution.

Table 26: Experimental reaction mixture and measured Ct values for the first experimental set of Example 18 Sample Dilution Ct value One Do not 19.32 2 Do not 20.40 3 1:10 23.23 4 Virus free (water only) ---

≪ tb > < tb > < tb > < tb > Sample Dilution Ct value One Do not 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 Virus free (water only) ---

Table 28: Experimental reaction mixture and measured Ct values for the third set of experiments of Example 18 Sample Dilution Ct value One Do not 19.22 2 1:10 22.43 2 1: 100 26.55 4 1: 1000 --- 5 1: 10000 --- 6 Virus free (water only) ---

Example  19: single nucleotide polymorphism ( SNP )

Amplification and detection experiments were performed on blood samples and human oral pharyngeal swabs containing specific genotypes of chromosomes P450 2C19, CYP2C19 * 2 (with the "GA" genotype) or CYP2C19 * 3 (with the "GG" genotype). One set of samples for human oral pharyngeal swabs and one set of two sets of experiments for samples obtained from blood were performed. In the first set of experiments, seven different samples obtained from the human oral pharynx swab were analyzed without sample purification. In a second set of experiments, five different blood samples were analyzed without sample purification.

Each sample is incubated with a reagent (e.g., a DNA polymerase, a primer, a dNTP, a cofactor, a suitable buffer, etc.) necessary for nucleic acid amplification and two reporter agents (for example, FAM dye for detecting amplification of nucleic acid Oligonucleotide probes, including a Texas red dye to detect "GA " genotypes). To generate the amplified product, a thermocycling protocol was carried out for 5 minutes at 95 ° C for 5 seconds, and 50 cycles at 49 ° C for 10 seconds for 5 seconds for each reaction mixture. During thermal cycling, a signal from the reporter was recorded to generate an amplification graph. An amplification graph for the first set of experiments (human oral pharyngeal swab) is shown in Figure 24a (signal corresponding to the FAM oligonucleotide probe) and Figure 24b (signal corresponding to the Texas red oligonucleotide probe). The graph for the second set of experiments (blood sample) is shown in Figure 25a (signal corresponding to FAM oligonucleotide probe) and Figure 25b (signal corresponding to Texas Red Oligonucleotide probe). The results shown in Figs. 24A , 24B , 25A and 25B show the relative fluorescent light emitting units (RFU) recorded according to the function of the number of cycles. Each graph is labeled with the corresponding reaction mixture number in Table 29 (oral pharyngeal swab test) or Table 30 (blood test). The recorded Ct values for the amplification graph are shown in Table 29 or Table 30 together with the measured genotypes. 24B or In the amplification graph in which the Texas Red derived signal was observed in Figure 25B , the corresponding reaction mixture was found to have the "GA" genotype. Also, in the amplification graph in which no TX red-derived signal was observed in FIG. 24B or 25B , the corresponding reaction mixture was confirmed to have the "GG" genotype.

As shown in Figure 24A , the amplified product was observed in each reaction mixture with samples taken from oral pharyngeal swabs, suggesting that nucleic acid amplification occurred. However, as shown in FIG. 24B , the amplified products were observed in only a portion of the reaction mixture (sample 1, 4, 6 and 7) having the sample obtained from oral pharyngeal cotton swab, ≪ / RTI > In the other reaction mixtures (reaction mixtures 2, 3 and 5) no amplified products were observed and these reaction mixtures correspond to the "GG" genotype. The results shown in Figures 24A and 24B were verified by amplification and detection experiments using DNA extracted from oral swab samples (data not shown). Thus, the results shown in FIGS. 24A and 24B suggest that, in some cases, the SNP can be detected by performing amplification in samples obtained from oral pharyngeal swabs without sample purification.

As shown in Fig. 25A , the amplified product was observed for each reaction mixture having a sample obtained from blood, suggesting that nucleic acid amplification occurred. However, as shown in FIG. 25B , the amplified products were observed in only a portion of the reaction mixture (samples 1, 2 and 5) having samples obtained from the blood, and these reaction mixtures corresponded to the "GA" genotype. In the other reaction mixtures (reaction mixtures 3 and 4), no amplified product was observed, and these reaction mixtures corresponded to the "GG" genotype. The results in Figures 25a and 25b were verified by nucleic acid sequencing. Thus, the results of FIGS. 25A and 25B suggest that, in some cases, SNPs can be detected through real-time amplification of samples obtained from blood without sample purification.

Table 29: Ct values and genotypes measured for the oral pharyngeal swab of Example 19 Reaction mixture Ct-FAM Reporter Ct-Texas Red Reporter genotype One 38.70 40.25 GA 2 38.28 --- GG 3 34.16 --- GG 4 33.18 33.75 GA 5 35.20 --- GG 6 33.08 33.59 GA 7 36.45 37.01 GA

Table 30: Ct values and genotypes measured for the blood experiment of Example 19 Reaction mixture Ct-FAM Reporter Ct-Texas Red Reporter genotype One 38.36 36.24 GA 2 39.97 39.67 GA 3 41.25 --- GG 4 33.96 --- GG 5 35.68 34.12 GA

Example  20: 55 Adenovirus ( ADV55 ) And 7 type Of the adenovirus (ADV7)  Amplification and detection

Amplification and detection experiments were performed on samples obtained from oral pharyngeal swabs containing various copies of the 55 adenovirus (ADV55) or the unknown concentration of type 7 adenovirus type 7 (ADV7). One set for the sample with ADV55 and two sets of experiments for the experiment with ADV7 were performed. In the first set of experiments, six different experiments with samples containing different copies of ADV55 (1, 10, 100, 1000, 10000, and 100000 copies) were performed without sample purification with experiments on negative controls. In a second set of experiments, eight different experiments with samples containing an unknown copy number of ADV7 were performed without sample purification.

Each sample is mixed with a reagent (for example, a DNA polymerase, a primer, a dNTP, a cofactor, a suitable buffer, etc.) required for nucleic acid amplification and a reporter (for example, an oligonucleotide probe including a FAM dye) . A summary of the reaction mixture, including the ADV55 copy number for the first set of experiments, is shown in Table 31. To generate the amplified product from the virus, two series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) 95 ° C for 1 minute after 20 cycles of 95 ° C for 1 second and 45 ° C for 1 second in the first series; And (ii) 35 cycles of 95 ° C for 5 seconds and 60 ° C for 34 seconds in the second series. During the second series, a signal from the reporter was recorded to generate an amplification graph and to obtain a Ct value. An amplification graph for the first set of experiments is shown in Fig. 26A , and each labeled with a reaction mixture number corresponds to that shown in Table 31. The amplification graph for the second set of experiments is shown in Figure 26B and the corresponding Ct values are shown in Table 32. [ 26b was labeled to correspond to the reaction mixture number shown in Table 32. [ The results shown in Figs. 26A and 26B are represented by a relative fluorescent light-emitting unit (RFU) recorded according to the function of the number of cycles.

As shown in Figure 26A , ADV55 was detected through amplified products 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 in FIG. 26A suggested that the ADV55 virus could be detected in several cases using multiple series of elongation and denaturation conditions at various dilution levels without sample purification.

As shown in Figure 26B , ADV7 was detected through amplified products for all reaction mixtures. Thus, the results shown in Figure 26B suggest that the ADV7 virus may, in some cases, be detected using multiple series of elongation and denaturation conditions without sample purification.

Table 31: Experimental reaction mixture for ADV55 experiment of Example 20 Reaction mixture ADV55 Copies / Rxn One One 2 10 3 100 4 1000 5 10000 6 100000 7 0 (negative control group)

Table 32: Ct values for the ADV7 experiment of Example 20 Reaction mixture Ct value One 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: Armored RNA Hepatitis C virus (RNA- HCV ) Amplification and detection

Amplification and detection experiments were performed on plasma samples containing various copies of armored RNA hepatitis C virus (RNA-HCV). Three different experiments with samples containing different copies of RNA-HCV (10, 100, and 500 copies) were performed without sample purification with experiments performed on negative controls.

Each sample is subjected to reverse transcription in a reaction mixture and a reagent (for example, an oligosaccharide such as a reverse transcriptase, a DNA polymerase, a primer, a dNTP, a coenzyme, a suitable buffer, etc.) necessary for nucleic acid amplification and a reporter Nucleotide probe). A summary of the reaction mixture containing the RNA-HCV copy number is shown in Table 33. To generate the amplified DNA product from the virus, two series of denaturation and elongation conditions were performed for each reaction mixture. 2 series are as follows: (i) in the first series, 20 cycles of 1 minute at 95 ° C, 1 second at 95 ° C and 1 second at 45 ° C; And (ii) 55 cycles of 5 seconds at 95 占 폚 and 34 seconds at 60 占 폚 in the second series. During the second series, a signal from the reporter agent was recorded to generate an amplification graph. The amplification graph for the first set of experiments is shown in Figure 27 , and each of the numbers labeled by the number corresponding to the number of reaction mixtures is shown in Table 33. The result shown in Fig. 27 represents a relative fluorescent light-emitting unit (RFU) recorded according to the cycle number function.

As shown in Fig. 27 , RNA-HCV was detected through amplified products for all reaction mixtures (reaction mixture 1-3) containing samples containing virus. RNA-HCV was not detected in the negative control reaction mixture (reaction mixture 4). Thus, the results shown in Figure 27 suggest that, in some cases, RNA-HCV can be detected using multiple series of elongation and denaturation conditions without sample purification. The detection sensitivity of 10 copies / rxn could also be achieved.

Table 33: Experimental reaction mixture for RNA-HCV experiment of Example 21 Reaction mixture Number of RNA-HCV copies / Rxn One 10 2 100 3 500 4 0 (negative control group)

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can be effected 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 applied to practice the invention. The following claims define the scope of the invention and are intended to encompass such methods and compositions within the claims and their equivalents.

Claims (84)

A method for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject,
(a) providing a reaction container comprising the biological sample and a reagent necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, wherein the reagent comprises (i) a reverse transcriptase , (ii) a DNA polymerase, and (iii) a primer set for the target RNA. And
(b) performing a plurality of cycles of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified DNA product indicative of the presence of the target RNA and amplifying the target RNA, i) incubating the reaction mixture at a denaturation temperature for a denaturation period of 60 seconds or less, and thereafter (ii) incubating the reaction mixture at an elongation temperature for an elongation period of 60 seconds or less
≪ / RTI > 2. The method of claim 1, wherein the reagent further comprises a reporter agent that produces a detectable signal indicative of the presence of the amplified DNA product. 3. The amplification method according to claim 2, wherein the intensity of the detectable signal is proportional to the amount of amplified DNA product or target RNA. 3. The method according to claim 2, wherein the reporter agent is a dye. 2. The amplification method according to claim 1, wherein the primer set comprises at least one primer. 6. The amplification method of claim 5, wherein the set comprises a first primer to generate strands complementary to the target RNA. 2. The amplification method according to claim 1, wherein the target RNA is a viral RNA. 2. The method of claim 1, wherein the reaction vessel comprises a body and a cap. The amplifying method according to claim 1, wherein the reaction vessel adopts a shape of a pipette tip. 2. The amplifying method according to claim 1, wherein the reaction vessel is part of an array of reaction vessels. 11. The method of claim 10, wherein the reaction vessel is individually addressable by a fluid handling device. 2. The method of claim 1, wherein the reaction vessel comprises at least two heat zones. The amplification method according to claim 1, wherein the denaturation temperature is about 90 ° C to 100 ° C. 14. The amplifying method according to claim 13, wherein the denaturation temperature is about 92 [deg.] C to 95 [deg.] C. The amplification method of claim 1, wherein the extension temperature is about 35 ° C to 72 ° C. 16. The amplifying method according to claim 15, wherein the extension temperature is about 45 DEG C to 65 DEG C. 2. The amplifying method according to claim 1, wherein the denaturation period is 30 seconds or less. 2. The method of claim 1, wherein the stretching period is 30 seconds or less. 3. The amplification method according to claim 1, wherein the target RNA is not concentrated before step (a). 2. The method of claim 1, wherein in step (a), the biological sample is not subjected to RNA extraction. The amplification method according to claim 1, further comprising the step of adding a solubilizer to the reaction vessel before or during step (a). 2. The method of claim 1, wherein the biological sample is a biological fluid from the subject. 2. The amplification method according to claim 1, wherein the amplification yields a detectable amount of a DNA product indicative of the presence of the target RNA in the biological sample at a cycle limit (Ct) of less than 40. 2. The amplification method according to claim 1, wherein the amplification yields a detectable amount of a DNA product indicative of the presence of the target RNA in the biological sample for a period of 10 minutes or less. The amplifying method according to claim 1, wherein the reaction vessel is sealed. The amplification method according to claim 1, wherein the DNA amplification is a polymerase chain reaction. 27. The amplification method according to claim 26, wherein the polymerase chain reaction is a nested polymerase chain reaction. 2. The method of claim 1, wherein the target RNA is released from the biological sample during at least one cycle of the primer extension reaction. A method for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject,
(a) receiving the biological sample obtained from the subject;
(b) providing a reaction container comprising the biological sample and a reagent necessary to perform reverse transcription and optionally deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, the reagent comprising (i) An enzyme and (ii) a set of primers for the target RNA;
(c) performing a plurality of cycles of primer extension reactions on the reaction mixture to yield a detectable amount of amplified DNA product indicative of the presence of the target RNA in the biological sample;
(d) detecting the amount of said amplified DNA product of (c); And
(e) outputting information on the amount of the amplified DNA product to a receiver
Lt; / RTI >
wherein the amount of time for completing (a) - (e) is about 30 minutes or less. 30. The method of claim 29, wherein the recipient is a treating physician, a pharmaceutical company, or the subject. 30. The amplification method according to claim 29, wherein (b) comprises DNA amplification. 30. The amplification method according to claim 29, wherein (c) is performed at 30 cycles or less. 30. The method of claim 29, wherein the information is output as a report. 30. The method of claim 29, wherein the information is output to an electronic display. 30. The method of claim 29, wherein the amount of time is 10 minutes or less. A method for amplifying a target nucleic acid present in a biological sample obtained from an experimental subject,
(a) providing a reaction container comprising the biological sample and a reagent necessary to perform nucleic acid amplification to obtain a reaction mixture, the reagent comprising (i) a deoxyribonucleic acid (DNA) polymerase, and optionally a reverse transcriptase An enzyme, and (ii) a set of primers for the target nucleic acid; And
(b) performing a plurality of series of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified product indicative of the presence of the target nucleic acid in the biological sample, each series comprising (i) Incubating the mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and then (ii) incubating the reaction mixture under extensional conditions characterized by an elongation temperature and an elongation period, Wherein the individual series is different from the one or more other individual series of the multiple series in the denaturing condition and / or the elongating condition
/ RTI > 37. The method of claim 36, wherein the target nucleic acid is a ribonucleic acid. 37. The method of claim 36, wherein the reagent is required to perform reverse transcription in parallel with deoxyribonucleic acid amplification. 37. The method of claim 36, wherein in (b), the amplified product is an amplified deoxyribonucleic acid product. 37. The method of claim 36, wherein (a) said biological sample is not purified. 37. The method of claim 36, wherein (a) said biological sample is concentrated. 37. The method of claim 36, wherein in (a), the biological sample is diluted. 37. The method of claim 36, further comprising applying the target nucleic acid to at least one denaturation condition before (b). 44. The amplifying method according to claim 43, wherein the at least one denaturing condition is selected from a denaturing temperature profile and a denaturing agent. 37. The amplification method of claim 36, further comprising applying the target nucleic acid to at least one denaturation condition between a first series and a second series of multiple series of primer extension reactions. 37. The method of claim 36, wherein the individual series is different in at least one of a ramping rate, a denaturation temperature, a denaturation period, an elongation temperature and an elongation period between denaturation temperature and elongation temperature. 47. The method of claim 46, wherein the individual series is different in at least either of a lambda ratio between denaturation temperature and elongation temperature, denaturation temperature, denaturation period, elongation temperature, and elongation period. 37. The method of claim 36, wherein the plurality of series of primer extension reactions comprises a first series and a second series, wherein the first series comprises more than 10 cycles, each cycle of the first series comprising: (i) Incubating the reaction mixture at about 92 DEG C to 95 DEG C for less than 30 seconds, and then (ii) incubating the reaction mixture at about 35 DEG C to 65 DEG C for less than 1 minute, Wherein each cycle of the second series comprises: (i) incubating the reaction mixture at about 92 DEG C to 95 DEG C for less than 30 seconds, then (ii) incubating the reaction mixture at about 40 DEG C RTI ID = 0.0 > 60 C < / RTI > for less than 1 minute. 37. The method of claim 36, wherein a number of the series of primer extension reactions comprise amplified products that exhibit the presence of the target nucleic acid in the biological sample at a lower cycle limit value as compared to a single series of primer extension reactions under similar denaturation and elongation conditions And calculating a detectable amount. 37. The method of claim 36, further comprising preheating the biological sample (b) prior to a preheat period of less than 10 minutes at a preheat temperature of 90 < 0 > C to 100 < 0 > C. 51. The method of claim 50, wherein the preheating period is one minute or less. A system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject,
(a) an input module for accepting a user request to amplify the target RNA from the biological sample;
(b) in response to the user request,
Said reagent comprising (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a DNA polymerase. ) A reaction mixture containing a set of primers for the target RNA;
Performing a plurality of cycles of primer extension reaction on the reaction mixture in the reaction vessel to generate an amplified DNA product indicative of the presence of the target RNA to amplify the target RNA, each cycle comprising (i) Incubating the reaction mixture at a denaturation temperature for a denaturation period of 60 seconds or less, and then (ii) incubating the reaction mixture at an elongation temperature for an elongation period of 60 seconds or less
Amplification module; And
(c) an output module operatively coupled to the amplification module, the output module outputting information about the target RNA or the DNA product to a receiver,
≪ / RTI > A system for amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject,
(a) an input module for accepting a user request to amplify the target RNA from the biological sample;
(b) in response to the user request,
(i) a reaction mixture comprising said biological sample obtained from said subject and a reagent necessary for performing reverse transcription and optionally deoxyribonucleic acid (DNA) amplification, said reagent comprising (1) a reverse transcriptase and (2) ) A reaction mixture containing a set of primers for the target RNA;
(ii) performing a plurality of cycles of primer extension reactions on the reaction mixture to yield a detectable amount of amplified DNA product indicative of the presence of the target RNA in the biological sample;
(iii) detecting the amount of said amplified DNA product of (ii);
(iv) outputting information on the amount of the amplified DNA product to the recipient,
The amount of time for completing (i) - (iv) is about 30 minutes or less
Amplification module; And
(c) an output module operatively coupled to the amplification module, the output module transmitting the information to a receiver,
≪ / RTI > A system for amplifying a target nucleic acid present in a biological sample obtained from a subject,
(a) an input module for accepting a user request to amplify the target nucleic acid from the biological sample;
(b) in response to the user request,
Wherein said reagent comprises (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) a set of primers for said target nucleic acid ≪ / RTI > in a reaction vessel;
Wherein a plurality of series of primer extension reactions are performed on the reaction mixture in the reaction vessel to produce an amplified product indicative of the presence of the target nucleic acid in the biological sample, each series comprising (i) (Ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period, wherein each series comprises two or more cycles of denaturing conditions And / or differing from the other series of one or more of the multiple series in the elongation condition
Amplification module; And
(c) an output module operatively coupled to the amplification module, the output module outputting information about the target nucleic acid or the amplified product to a receiver,
≪ / RTI > A computer readable medium comprising machine executable code embodying a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject when executed by one or more computer processors, the method comprising:
(a) providing a reaction container comprising the biological sample and a reagent necessary to perform reverse transcription in parallel with deoxyribonucleic acid (DNA) amplification to obtain a reaction mixture, wherein the reagent comprises (i) a reverse transcriptase , (ii) a DNA polymerase, and (iii) a primer set for the target RNA. And
(b) performing a plurality of cycles of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified DNA product indicative of the presence of the target RNA, and amplifying the target RNA, i) incubating the reaction mixture at a denaturation temperature for a denaturation period of 60 seconds or less, and thereafter (ii) incubating the reaction mixture at an elongation temperature for an elongation period of 60 seconds or less
Readable medium. A computer readable medium comprising machine executable code embodying a method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained directly from a subject when executed by one or more computer processors, the method comprising:
(a) receiving the biological sample obtained from the subject;
(b) obtaining a reaction mixture comprising the biological sample and a reagent necessary for performing reverse transcription and optionally deoxyribonucleic acid (DNA) amplification, the reagent comprising (i) a reverse transcriptase And (ii) a set of primers for the target RNA;
(c) performing a plurality of cycles of primer extension reactions on the reaction mixture to yield a detectable amount of amplified DNA indicative of the presence of the target RNA in the biological sample;
(d) detecting the amount of said DNA product of (c); And
(e) outputting information on the amount of the DNA product to the recipient
Lt; / RTI >
wherein the amount of time for completing (a) - (e) is less than or equal to about 30 minutes. A computer readable medium comprising machine executable code embodying a method of amplifying a target nucleic acid present in a biological sample obtained from a subject when executed by one or more computer processors,
(a) providing a reaction container comprising the biological sample and a reagent necessary to perform nucleic acid amplification to obtain a reaction mixture, wherein the reagent comprises (i) a DNA polymerase and optionally a reverse transcriptase, and (ii) Comprising a primer set for the target nucleic acid; And
(b) performing a plurality of series of primer extension reactions on the reaction mixture in the reaction vessel to produce an amplified product indicative of the presence of the target nucleic acid in the biological sample, each series comprising (i) Incubating the mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and then (ii) incubating the reaction mixture under extensional conditions characterized by an elongation temperature and an elongation period, Wherein the individual series is different from the one or more other individual series of the multiple series in the denaturing condition and / or the elongating condition
Readable medium. A system for amplifying a target nucleic acid in a biological sample obtained from a subject,
An electronic display screen including a user interface for displaying graphical elements that a user can access to execute 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 amplification protocol when the user selects the graphical element;
Wherein the amplification protocol comprises:
Performing a plurality of series of primer extension reactions on a reaction mixture comprising the biological sample and a reagent necessary to perform nucleic acid amplification to produce an amplified product indicative of the presence of the target nucleic acid in the biological sample, (I) incubating the reaction mixture under denaturing conditions characterized by denaturation temperature and denaturation period, and thereafter (ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation period Wherein the individual series is different from the one or more other individual series of the multiple series in the denaturing condition and / or the elongating condition
. ≪ / RTI > 59. The system of claim 58, wherein the amplification protocol further comprises selecting a primer set for the target nucleic acid. 59. The system of claim 58, wherein the reagent comprises (i) a deoxyribonucleic acid (DNA) polymerase and optionally a reverse transcriptase, and (ii) a set of primers for the target nucleic acid. 59. The system of claim 58, wherein the user interface displays a plurality of graphical elements, each graphical element being associated with a predetermined one of a plurality of amplification protocols. 62. The system of claim 61, wherein each of the graphical elements is associated with a disease, and wherein a predetermined amplification protocol of the plurality of amplification protocols is specified to assess the presence of the disease in the subject. 63. The system of claim 62, wherein the disease is associated with a virus. 64. The system of claim 63, wherein the virus is an RNA virus. 64. The system of claim 63, wherein said virus is a DNA virus. 63. The method of claim 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 B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile fever virus, A virus, a measles virus, a herpes simplex virus, a small pox virus, an adenovirus, and a varicella virus. 67. The system of claim 66, wherein said influenza virus is selected from the group consisting of H1N1 virus, H3N2 virus, H7N9 virus and H5N1 virus. 67. The system of claim 66, wherein the adenovirus is a 55 adenovirus (ADV55) or a 7 adenovirus (ADV7). 67. The system of claim 66, wherein the hepatitis C virus is armored RNA-hepatitis C virus (RNA-HCV). 62. The system of claim 61, wherein the disease is associated with a pathogenic bacterium or a pathogenic protozoan. 69. The system of claim 68, wherein said pathogenic bacteria is Mycobacterium tuberculosis . 69. The system of claim 68, wherein said pathogenic protozoan is Plasmodium . 59. The system of claim 58, wherein the target nucleic acid is associated with a disease. 73. The system of claim 73, wherein the amplification protocol is designated to assess the presence of the disease based on the presence of the amplified product. 73. The system of claim 73, wherein said disease is associated with a virus. 78. The system of claim 75, wherein the virus is an RNA virus. 77. The system of claim 75, wherein the virus is a DNA virus. 78. The method of claim 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 B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile fever virus, A virus, a measles virus, a herpes simplex virus, a small pox virus, an adenovirus, and a varicella virus. 79. The system of claim 78, wherein said influenza virus is selected from the group consisting of H1N1 virus, H3N2 virus, H7N9 virus and H5N1 virus. 79. The system of claim 78, wherein said adenovirus is a 55 adenovirus (ADV55) or a 7 adenovirus (ADV7). 79. The system of claim 78, wherein the hepatitis C virus is armored RNA-C hepatitis virus (RNA-HCV). 73. The system of claim 73, wherein the disease is associated with a pathogenic bacterium or a pathogenic protozoan. 83. The system of claim 82, wherein the pathogenic bacteria is M. tuberculosis. 83. The system of claim 82, wherein the pathogenic protozoan is plasmodium.

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