USOO9546,389 B2

(12) United States Patent (10) Patent No.: US 9,546,389 B2 Li (45) Date of Patent: Jan. 17, 2017

(54) METHODS AND SYSTEMS FOR NUCLEIC (56) References Cited ACIDAMPLIFICATION U.S. PATENT DOCUMENTS (71) Applicant: Coyote Bioscience Co., Ltd., Beijing 5,759,821. A * 6/1998 Teasdale ...... C12O 1/6806 (CN) 435,912 2004/0209331 A1* 10, 2004 Ririe ...... BO1L 3,505 435,912 (72) Inventor: Xiang Li, Beijing (CN) 2008/0085541 A1 4/2008 Spangler 2012/0308990 A1* 12/2012 TeMaat ...... BO1L, 7,52 (73) Assignee: COYOTE BIOSCIENCE CO.,LTD., 435/3 Beijing (CN) 2013/0022963 A1 1/2013 Exner et al. FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 CN 101597652. A 12/2009 U.S.C. 154(b) by 0 days. CN 103074349 A 5, 2013 JP EP 1069 190 A2 * 1 2001 ...... C12O 1/686 WO WO-2012109604 A1 8, 2012 (21) Appl. No.: 14/963,986 OTHER PUBLICATIONS (22) Filed: Dec. 9, 2015 Callahan et al. Use of a portable real-time reverse transcriptase (65) Prior Publication Data polymerase chain reaction assay for rapid detection of foot-and mouth disease virus. JAVMA 220: 1636-1642 (2002).* US 2016/O115513 A1 Apr. 28, 2016 Gilbert et al. Typing of bovine viral diarrhea viruses directly from blood of persistently infected cattle by multiplex PCR. Journal of Clinical Microbiology 37:2020-2023 (1999).* International search report and written opinion dated Feb. 26, 2015 Related U.S. Application Data for PCT Application No. CN2014/094914. International search report and written opinion dated Nov. 26, 2014 (63) Continuation of application No. for PCT Application No. CN2013/090425. PCT/CN2014/094914, filed on Dec. 25, 2014, which is a continuation-in-part of application No. * cited by examiner PCT/CN2013/090425, filed on Dec. 25, 2013. Primary Examiner — Samuel Woolwine (74) Attorney, Agent, or Firm — Wilson, Sonsini, (51) Int. Cl. Goodrich & Rosati CI2O I/68 (2006.01) (57) ABSTRACT CI2P 19/34 (2006.01) The present disclosure provides methods and systems for CI2O 1/70 (2006.01) amplifying nucleic acid samples. In some aspects, the meth GOIN 2L/64 (2006.01) ods and systems described provided can be useful in con BOIL 7/00 (2006.01) ducting multiple nucleic acid amplification reactions in (52) U.S. Cl. parallel. In some embodiments, methods and systems pro CPC ...... CI2P 19/34 (2013.01); B0IL 7/52 vided herein can be useful in conducting reverse transcrip (2013.01); C12O 1/68 (2013.01): CI2O I/686 tion and DNA amplification in parallel. Moreover, in some (2013.01); C12O 1/6862 (2013.01); C12O aspects, the methods and systems described herein can be I/70 (2013.01); G0IN 2 1/64 (2013.01); BOIL useful in analysis of nucleic acid samples. In some embodi 2200/14 (2013.01); B0IL 2300/027 (2013.01) ments, methods and systems provided herein can be useful (58) Field of Classification Search for conducting multiple series of primer extension reactions, None which can aid in analysis of a nucleic acid sample. See application file for complete search history. 29 Claims, 46 Drawing Sheets U.S. Patent Jan. 17, 2017 Sheet 1 of 46 US 9,546,389 B2

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Fig. 28B US 9,546,389 B2 1. 2 METHODS AND SYSTEMIS FOR NUCLEC polymerase, and (iii) a primer set for the target RNA, to ACIDAMPLIFICATION obtain a reaction mixture; and (b) Subjecting the reaction mixture in the reaction vessel to multiple cycles of a primer CROSS-REFERENCE extension reaction to generate amplified DNA product that is indicative of the presence of the target RNA, each cycle This application is a continuation of Patent Cooperation comprising (i) incubating the reaction mixture at a denatur Treaty Application No. PCT/CN2014/094914, filed on Dec. ing temperature for a denaturing duration that is less than or 25, 2014, which is a continuation-in-part of Patent Coop equal to 60 seconds, followed by (ii) incubating the reaction eration Treaty Application No. PCT/CN2013/090425, filed mixture at an elongation temperature for an elongation on Dec. 25, 2013, said applications are incorporated herein 10 duration that is less than or equal to 60 seconds, thereby by reference in their entireties for all purposes. amplifying the target RNA. In another embodiment, the method comprises: (a) receiving the biological sample that BACKGROUND has been obtained from the subject; (b) providing a reaction vessel comprising the biological sample and reagents nec Nucleic acid amplification methods permit selected 15 essary for conducting reverse transcription amplification and amplification and identification of nucleic acids of interest optionally deoxyribonucleic acid (DNA) amplification, the from a complex mixture. Such as a biological sample. To reagents comprising (i) a reverse transcriptase and (ii) a detect a nucleic acid in a biological sample, the biological primer set for the target RNA, to obtain a reaction mixture; sample is typically processed to isolate nucleic acids from (c) Subjecting the reaction mixture to multiple cycles of a other components of the biological sample and other agents primer extension reaction to yield a detectable amount of that may interfere with the nucleic acid and/or amplification. amplified DNA product that is indicative of the presence of Following isolation of the nucleic acid of interest from the the target RNA in the biological sample; (d) detecting the biological sample, the nucleic acid of interest can be ampli amount of amplified DNA product of (c); and outputting fied, via, for example, amplification methods known in the information regarding the amount of amplified DNA product art, Such as thermal cycling based approaches (e.g., poly 25 to a recipient, wherein an amount of time for completing merase chain reaction (PCR)). Following amplification of (a)-(e) is less than or equal to about 30 minutes. In some the nucleic acid of interest, the products of amplification can embodiments, the amount of time is less than or equal to 20 be detected and the results of detection interpreted by an minutes, less than or equal to 10 minutes, or less than or end-user. The extraction of nucleic acid from a biological equal to 5 minutes. sample prior to amplification of the nucleic acid, however, 30 In some embodiments, the reagents further comprise a can be time consuming, resulting in a reduced time effi reporter agent that yields a detectable signal indicative of the ciency for the process as a whole. presence of the amplified DNA product. In some embodi Point-of-care (POC) testing has the potential to improve ments, the intensity of the detectable signal is proportional the detection and management of infectious diseases in to the amount of the amplified DNA product or target RNA. resource-limited settings with poor laboratory infrastructure, 35 In some embodiments, the reporter agent is a dye. In some or in remote areas where there are delays in the receipt of embodiments, the primer set comprises one or more primers. laboratory results and potential complications to following In Some embodiments, the primer set comprises a first up with patients. POC testing also could render state of the primer to generate a strand that is complementary to the art health care facilities more capable of delivering sample target RNA. In some embodiments, the primer set comprises to-answer results to patients during a single visit. Inefficien 40 a second primer to generate a strand that is complementary cies in POC methods and devices, however, limit what can to a DNA product that is complementary to at least a portion be achieved. For example, preparation of nucleic acids (e.g., of the target RNA. In some embodiments, the target RNA is of a pathogen) from complex sample types (e.g., biological viral RNA. In some embodiments, the viral RNA is patho samples) entails highly skilled personnel, in a dedicated genic to the subject. In some embodiments, the viral RNA is laboratory space, to manually perform multiple processing 45 selected from the group consisting of HIV I, HIV II, Ebola steps and Subsequent testing, with reporting of results often virus, Dengue virus, orthomyxoviruses, hepevirus, and/or occurring hours or even days later. hepatitis A, B, C (e.g., Armored RNA-HCV virus), D, and E Thus, there exists a need for rapid, accurate methods and viruses. devices for analyzing nucleic acids from complex sample In some embodiments, the reaction vessel comprises a types. Such methods and devices may be useful, for 50 body and a cap. In some embodiments, the cap is removable. example, in realizing fast sample-to-answer detection and In some embodiments, the reaction vessel adopts a format of management of diseases detectable via their nucleic acid. a pipette tip. In some embodiments, the reaction vessel is part of an array of reaction vessels. In some embodiments, SUMMARY the reaction vessel part of an array of reaction vessels is 55 individually addressable by a fluid handling device. In some The present disclosure provides methods and systems for embodiments, the reaction vessel comprises two or more efficient amplification of nucleic acids, such as RNA and thermal Zones. In some embodiments, the reaction vessel is DNA molecules. Amplified nucleic acid product can be sealed, optionally hermetically sealed. detected rapidly and with good sensitivity. In some embodiments, the denaturing temperature is from In one aspect, the disclosure provides a method of ampli 60 about 90° C. to 100° C., or from about 92° C. to 95°C. In fying a target ribonucleic acid (RNA) present in a biological Some embodiments, the elongation temperature is from sample obtained directly from a Subject. In one embodiment, about 35° C. to 72° C., or from about 45° C. to 65° C. In the method comprises: (a) providing a reaction vessel com Some embodiments, the denaturing duration is less than or prising the biological sample and reagents necessary for equal to 30 seconds. In some embodiments, the elongation conducting reverse transcription amplification in parallel 65 duration is less than or equal to 30 seconds. with deoxyribonucleic acid (DNA) amplification, the In some embodiments, the target RNA has not undergone reagents comprising (i) a reverse transcriptase, (ii) a DNA concentration prior to providing a reaction vessel compris US 9,546,389 B2 3 4 ing the biological sample and reagents necessary for con incubating the reaction mixture under a denaturing condition ducting reverse transcription amplification in parallel with characterized by a denaturing temperature and a denaturing deoxyribonucleic acid (DNA) amplification. In some duration, followed by (ii) incubating the reaction mixture embodiments, the biological sample has not undergone under an elongation condition characterized by an elonga RNA extraction when providing a reaction vessel compris tion temperature and an elongation duration, wherein an ing the biological sample and reagents necessary for con individual series differs from at least one other individual ducting reverse transcription amplification in parallel with series of the plurality with respect to the denaturing condi deoxyribonucleic acid (DNA) amplification. In some tion and/or the elongation condition. embodiments, the method further comprises the step of In some embodiments, the target nucleic acid is a ribo adding a lysis agent to the reaction vessel prior to or during 10 nucleic acid. In some embodiments, the reagents are neces providing a reaction vessel comprising the biological sample sary for conducting reverse transcription amplification in and reagents necessary for conducting reverse transcription parallel with deoxyribonucleic acid amplification. In some amplification in parallel with deoxyribonucleic acid (DNA) embodiments, the amplified product is amplified deoxyri amplification. In some embodiments, the lysis agent com bonucleic acid product. In some embodiments, the biologi prises a buffer. In some embodiments, the target RNA is 15 cal sample is not purified in (a). In some embodiments, the released from the biological sample during one or more method further comprises subjecting the target nucleic acid cycles of the primer extension reaction. to one or more denaturing conditions prior to (b). In some In some embodiments, the biological sample is a biologi embodiments, the one or more denaturing conditions are cal fluid from the subject. In some embodiments, the bio selected from a denaturing temperature profile and a dena logical sample is selected from the group consisting of turing agent. breath, blood, urine, feces, saliva, cerebrospinal fluid and In some embodiments, the biological sample is diluted. SWeat. This may aid in minimizing inhibitions. In some embodi In some embodiments, DNA amplification is via the ments, the biological sample is concentrated. This may aid polymerase chain reaction. In some embodiments, the poly in increasing or otherwise improving sensitivity. merase chain reaction is nested polymerase chain reaction. 25 In some embodiments, the method further comprises In some embodiments, DNA amplification is linear ampli Subjecting the target nucleic acid to one or more denaturing fication. In some embodiments, the amplifying yields a conditions between a first series and a second series of the detectable amount of DNA product indicative of the pres plurality of series of primer extension reactions. In some ence of the target RNA in the biological sample at a cycle embodiments, the individual series differ with respect to at threshold value (Ct) of less than 50, less than 40, less than 30 least any one, at least any two, at least any three, or at least 30, less than 20, less than 10, or less than 5. In some any four of ramping rate between denaturing temperature embodiments, the amplifying yields a detectable amount of and elongation temperature, denaturing temperature, dena DNA product indicative to the presence of the target RNA turing duration, elongation temperature and elongation dura in the biological sample at a time period of 30 minutes or tion. In some embodiments, the individual series differ with less, 20 minutes or less, or 10 minutes or less. In some 35 respect to ramping rate between denaturing temperature and embodiments, the amplifying is non-emulsion based. elongation temperature, denaturing temperature, denaturing In some embodiments, the recipient is a treating physi duration, elongation temperature and elongation duration. cian, a pharmaceutical company, or the Subject. In some In some embodiments, the plurality of series of primer embodiments, Subjecting the reaction mixture to multiple extension reactions comprises a first series and a second cycles of a primer extension reaction to yield a detectable 40 series, the first series comprising more than ten cycles, each amount of amplified DNA product that is indicative of the cycle of the first series comprising (i) incubating the reaction presence of the target RNA in the biological sample is mixture at about 92°C.-95°C. for no more than 30 seconds, conducted in 30 cycles or less, 20 cycles or less, or 10 cycles followed by (ii) incubating the reaction mixture at about 35° or less. In some embodiments, detecting is optically detect C.-65° C. for no more than 1 minute, the second series ing, electrostatically detecting, or electrochemically detect 45 comprising more than ten cycles, each cycle of the second ing. In some embodiments, the method comprises providing series comprising (i) incubating the reaction mixture at a reaction vessel comprising the biological sample and about 92° C.-95°C. for no more than 30 seconds, followed reagents necessary for conducting reverse transcription by (ii) incubating the reaction mixture at about 40°C.-60° C. amplification and deoxyribonucleic acid (DNA) amplifica for no more than 1 minute. tion. 50 In some embodiments, the plurality of series of primer In some embodiments, the information is outputted as a extension reactions yields a detectable amount of amplified report. In some embodiments, the report is an electronic product that is indicative of the presence of the target nucleic report. In some embodiments, the information is outputted to acid in the biological sample with a lower cycle threshold an electronic display. value as compared to a single series of primer extension In another aspect, the disclosure provides a method of 55 reactions under comparable denaturing and elongation con amplifying a target nucleic acid present in a biological ditions. In some embodiments, the method further com sample obtained from a Subject. The method comprises: (a) prises, prior to (b), pre-heating the biological sample at a providing a reaction vessel comprising the biological sample pre-heating temperature from 90° C. to 100° C. for a and reagents necessary for conducting nucleic acid ampli pre-heating duration of no more than 10 minutes, 2 minutes, fication, the reagents comprising (i) a deoxyribonucleic acid 60 or 1 minute. In some embodiments, the pre-heating tem (DNA) polymerase and optionally a reverse transcriptase, perature is from 92 C. to 95°C. In some embodiments, the and (ii) a primer set for the target nucleic acid, to obtain a pre-heating duration is no more than about 30 seconds. reaction mixture; and (b) Subjecting the reaction mixture in In another aspect, the disclosure provides a system for the reaction vessel to a plurality of series of primer extension amplifying a target ribonucleic acid (RNA) present in a reactions to generate amplified product that is indicative of 65 biological sample obtained directly from a subject. In one the presence of the target nucleic acid in the biological embodiment, the systems comprises: (a) an input module sample, each series comprising two or more cycles of (i) that receives a user request to amplify the target RNA in the US 9,546,389 B2 5 6 biological sample; (b) an amplification module that, in (c) an output module operatively coupled to the amplifica response to the user request: receives, in a reaction vessel, a tion module, wherein the output module outputs information reaction mixture comprising the biological sample and regarding the nucleic acid or the amplified product to a reagents necessary for conducting reverse transcription recipient. amplification in parallel with deoxyribonucleic acid (DNA) In another aspect, the disclosure provides a computer amplification, the reagents comprising (i) a reverse tran readable medium comprising machine executable code that, Scriptase, (ii) a DNA polymerase, and (iii) a primer set for upon execution by one or more computer processors, imple the target RNA; and subjects the reaction mixture in the ments a method of amplifying a target ribonucleic acid reaction vessel to multiple cycles of a primer extension (RNA) present in a biological sample obtained directly from reaction to generate amplified DNA product that is indica 10 a Subject. In one embodiment, the method comprises: (a) tive of the presence of the target RNA, each cycle compris providing a reaction vessel comprising the biological sample ing (i) incubating the reaction mixture at a denaturing and reagents necessary for conducting reverse transcription temperature for a denaturing duration that is less than or amplification in parallel with deoxyribonucleic acid (DNA) equal to 60 seconds, followed by (ii) incubating the reaction amplification, the reagents comprising (i) a reverse tran mixture at an elongation temperature for an elongation 15 Scriptase, (ii) a DNA polymerase, and (iii) a primer set for duration that is less than or equal to 60 seconds, thereby the target RNA, to obtain a reaction mixture; and (b) amplifying the target RNA; and (c) an output module Subjecting the reaction mixture in the reaction vessel to operatively coupled to the amplification module, wherein multiple cycles of a primer extension reaction to generate the output module outputs information regarding the target amplified DNA product that is indicative of the presence of RNA or the DNA product to a recipient. the target RNA, each cycle comprising (i) incubating the In another embodiment, the system comprises (a) an input reaction mixture at a denaturing temperature for a denatur module that receives a user request to amplify the target ing duration that is less than or equal to 60 seconds, followed RNA in the biological sample; (b) an amplification module by (ii) incubating the reaction mixture at an elongation that, in response to the user request: (i) receives, in a reaction temperature for an elongation duration that is less than or vessel, a reaction mixture comprising the biological sample 25 equal to 60 seconds, thereby amplifying the target RNA. that has been obtained from the Subject and reagents nec In another embodiment, the method comprises: (a) receiv essary for conducting reverse transcription amplification and ing the biological sample that has been obtained from the optionally deoxyribonucleic acid (DNA) amplification, the Subject; (b) providing a reaction vessel comprising the reagents comprising (1) a reverse transcriptase and (2) a biological sample and reagents necessary for conducting primer set for the target RNA; and (ii) subjects the reaction 30 reverse transcription amplification and optionally deoxyri mixture to multiple cycles of a primer extension reaction to bonucleic acid (DNA) amplification, the reagents compris yield a detectable amount of amplified DNA product that is ing (i) a reverse transcriptase and (ii) a primer set for the indicative of the presence of the target RNA in the biological target RNA, to obtain a reaction mixture; (c) Subjecting the sample; (iii) detects the amount of amplified DNA product reaction mixture to multiple cycles of a primer extension of (iii); and (iv) outputs information regarding the amount of 35 reaction to yield a detectable amount of amplified DNA amplified DNA product to a recipient, wherein an amount of product that is indicative of the presence of the target RNA time for completing (i)-(iv) is less than or equal to about 30 in the biological sample; (d) detecting the amount of DNA minutes; and (c) an output module operatively coupled to the product of (c); and (e) outputting information regarding the amplification module, wherein the output module transmits amount of DNA product to a recipient, wherein an amount the information to a recipient. In some embodiments, the 40 of time for completing (a)-(e) is less than or equal to about output module is an electronic display. In some embodi 30 minutes. ments, the electronic display comprises a user interface. In In another aspect, the disclosure provides a computer Some embodiments, the output module is a communication readable medium comprising machine executable code that, interface operatively coupled to a computer network. upon execution by one or more computer processors, imple In another aspect, the disclosure provides a system for 45 ments a method of amplifying a target nucleic acid present amplifying a target nucleic acid present in a biological in a biological sample obtained from a subject. In one sample obtained from a subject. The system comprises: (a) embodiment, the method comprises (a) providing a reaction an input module that receives a user request to amplify the vessel comprising the biological sample and reagents nec target nucleic acid in the biological sample; (b) an amplifi essary for conducting nucleic acid amplification, the cation module that, in response to the user request: receives, 50 reagents comprising (i) a DNA polymerase and optionally a in a reaction vessel, a reaction mixture comprising the reverse transcriptase, and (ii) a primer set for the target biological sample and reagents necessary for conducting nucleic acid, to obtain a reaction mixture; and (b) Subjecting nucleic acid amplification, the reagents comprising (i) a the reaction mixture in the reaction vessel to a plurality of DNA polymerase and optionally a reverse transcriptase, and series of primer extension reactions to generate amplified (ii) a primer set for the target nucleic acid; and Subjects the 55 product from the target nucleic acid, each series comprising reaction mixture in the reaction vessel to a plurality of series two or more cycles of (i) incubating the reaction mixture of primer extension reactions to generate amplified product under a denaturing condition characterized by a denaturing that is indicative of the presence of the target nucleic acid in temperature and a denaturing duration, followed by (ii) the biological sample, each series comprising two or more incubating the reaction mixture under an elongation condi cycles of (i) incubating the reaction mixture under a dena 60 tion characterized by an elongation temperature and an turing condition characterized by a denaturing temperature elongation duration, wherein an individual series differs and a denaturing duration, followed by (ii) incubating the from at least one other individual series of the plurality with reaction mixture under an elongation condition character respect to the denaturing condition and/or the elongation ized by an elongation temperature and an elongation dura condition. tion, wherein an individual series differs from at least one 65 An additional aspect of the disclosure provides a system other individual series of the plurality with respect to the for amplifying a target nucleic acid in a biological sample denaturing condition and/or the elongation condition; and obtained from a subject. The system can comprise an US 9,546,389 B2 7 8 electronic display screen that comprises a user interface that titis C virus, hepatitis D virus, hepatitis E virus, hepatitis G displays a graphical element that is accessible by a user to virus, Epstein-Barr virus, mononucleosis virus, cytomega execute an amplification protocol to amplify the target lovirus, SARS virus, West Nile Fever virus, polio virus, nucleic acid in the biological sample. The system can also measles virus, herpes simplex virus, Smallpox virus, adeno comprise a computer processor coupled to the electronic 5 virus, and Varicella virus. In some embodiments, the influ display screen and programmed to execute the amplification enza virus can be selected from the group consisting of protocol upon selection of the graphical element by the user. H1N1 virus, H3N2 virus, H7N9 virus and H5N1 virus. In The amplification protocol can comprise Subjecting a reac Some embodiments, the adenovirus may be adenovirus type tion mixture comprising the biological sample and reagents 55 (ADV55) or adenovirus type 7 (ADV7). In some embodi necessary for conducting nucleic acid amplification to a 10 plurality of series of primer extension reactions to generate ments, the hepatitis C virus may be armored RNA-hepatitis amplified product that is indicative of the presence of the C virus (RNA-HCV). In some embodiments, the disease target nucleic acid in the biological sample. Each series of may be associated with a pathogenic bacterium (e.g., Myco primer extension reactions can include two or more cycles of bacterium tuberculosis) or a pathogenic protozoan (e.g., incubating the reaction mixture under a denaturing condition 15 Plasmodium). characterized by a denaturing temperature and a denaturing Additional aspects and advantages of the present disclo duration, followed by incubating the reaction mixture under sure will become readily apparent to those skilled in this art an elongation condition characterized by an elongation from the following detailed description, wherein only illus temperature and an elongation duration. An individual series trative embodiments of the present disclosure are shown and may differ from at least one other individual series of the described. As will be realized, the present disclosure is plurality with respect to the denaturing condition and/or the capable of other and different embodiments, and its several elongation condition. details are capable of modifications in various obvious In some embodiments, the amplification protocol can respects, all without departing from the disclosure. Accord further comprise selecting a primer set for the target nucleic ingly, the drawings and description are to be regarded as acid. In some embodiments, the reagents may comprise a 25 illustrative in nature, and not as restrictive. deoxyribonucleic acid (DNA) polymerase, an optional reverse transcriptase, and a primer set for the target nucleic INCORPORATION BY REFERENCE acid. In some embodiments, the user interface can display a plurality of graphical elements. Each of the graphical ele All publications, patents, and patent applications men ments can be associated with a given amplification protocol 30 tioned in this specification are herein incorporated by ref among a plurality of amplification protocols. In some erence to the same extent as if each individual publication, embodiments, each of the graphical elements may be asso patent, or patent application was specifically and individu ciated with a disease. A given amplification protocol among ally indicated to be incorporated by reference. the plurality of amplification protocols can be directed to assaying a presence of the disease in the Subject. In some 35 BRIEF DESCRIPTION OF THE DRAWINGS embodiments, the disease may be associated with a virus such as for example an RNA virus or a DNA virus. In some The novel features of the invention are set forth with embodiments, the virus can be selected from the group particularity in the appended claims. A better understanding consisting of human immunodeficiency virus I (HIV I), of the features and advantages of the present invention will human immunodeficiency virus II (HIV II), an orthomyxo 40 be obtained by reference to the following detailed descrip virus, Ebola virus, Dengue virus, influenza viruses, hepevi tion that sets forth illustrative embodiments, in which the rus, hepatitis A virus, hepatitis B virus, hepatitis C virus, principles of the invention are utilized, and the accompany hepatitis D virus, hepatitis E virus, hepatitis G. virus, ing drawings (also “Figure' and “Fig.” herein), of which: Epstein-Barr virus, mononucleosis virus, cytomegalovirus, FIG. 1 is schematic depicting an example system. SARS virus, West Nile Fever virus, polio virus, measles 45 FIGS. 2A and 2B are graphs depicting results of example virus, herpes simplex virus, Smallpox virus, adenovirus, and nucleic acid amplification reactions described in Example 1. Varicella virus. In some embodiments, the influenza virus FIGS. 3A and 3B are graphs depicting results of example can be selected from the group consisting of H1N1 virus, nucleic acid amplification reactions described in Example 1. H3N2 virus, H7N9 virus and H5N1 virus. In some embodi FIGS. 4A and 4B are graphs depicting results of example ments, the adenovirus may be adenovirus type 55 (ADV55) 50 nucleic acid amplification reactions described in Example 2. or adenovirus type 7 (ADV7). In some embodiments, the FIG. 5 is a graph depicting results of example nucleic acid hepatitis C virus may be armored RNA-hepatitis C virus amplification reactions described in Example 3. (RNA-HCV). In some embodiments, the disease may be FIGS. 6A and 6B are graphs depicting results of example associated with a pathogenic bacterium (e.g., Mycobacte nucleic acid amplification reactions described in Example 4. rium tuberculosis) or a pathogenic protozoan (e.g., Plasmo 55 FIGS. 7A and 7B are graphs depicting results of example dium). nucleic acid amplification reactions described in Example 4. In some embodiments, the target nucleic acid may be FIGS. 8A and 8B are graphs depicting results of example associated with a disease. In some embodiments, the ampli nucleic acid amplification reactions described in Example 4. fication protocol can be directed to assaying a presence of FIGS. 9A and 9B are graphs depicting results of example the disease based on a presence of the amplified product. In 60 nucleic acid amplification reactions described in Example 4. Some embodiments, the disease may be associated with a FIGS. 10A and 10B are graphs depicting results of virus such as, for example, an RNA virus or a DNA virus. example nucleic acid amplification reactions described in In some embodiments, the virus can be selected from the Example 4. group consisting of human immunodeficiency virus I (HIV FIG. 11 is a graph depicting results of example nucleic I), human immunodeficiency virus II (HIV II), an 65 acid amplification reactions described in Example 5. orthomyxovirus, Ebola virus, Dengue virus, influenza FIG. 12 is a graph depicting results of example nucleic viruses, hepevirus, hepatitis. A virus, hepatitis B virus, hepa acid amplification reactions described in Example 5. US 9,546,389 B2 10 FIG. 13 is a graph depicting results of example nucleic As used herein, the term “cycle threshold' or “Ct' gen acid amplification reactions described in Example 7. erally refers to the cycle during thermocycling in which an FIG. 14 is a graph depicting results of example nucleic increase in a detectable signal due to amplified product acid amplification reactions described in Example 9. reaches a statistically significant level above background FIGS. 15A and 15B are graphs depicting results of 5 signal. example nucleic acid amplification reactions described in As used herein, the terms “denaturing and “denatur Example 10. ation' are used interchangeably and generally refer to the FIGS. 16A and 16B are graphs depicting results of full or partial unwinding of the helical structure of a double example nucleic acid amplification reactions described in Stranded nucleic acid, and in Some cases the unwinding of Example 10. 10 the secondary structure of a single Stranded nucleic acid. FIG. 17 is a graph depicting results of nucleic acid Denaturation may include the inactivation of the cell wall(s) amplification reactions described in Example 11. of a pathogen or the shell of a virus, and the inactivation of FIG. 18 is a graph depicting results of nucleic acid the protein(s) of inhibitors. Conditions at which denaturation amplification reactions described in Example 12. 15 may occur include a “denaturation temperature' that gener FIG. 19A and FIG. 19B are graphs depicting results of ally refers to a temperature at which denaturation is permit nucleic acid amplification reactions described in Example ted to occur and a “denaturation duration” that generally 13. refers to an amount of time allotted for denaturation to occur. FIG. 20 is a graph depicting results of nucleic acid As used herein, the term “elongation' generally refers to amplification reactions described in Example 14. the incorporation of nucleotides to a nucleic acid in a FIG. 21 is a graph depicting results of nucleic acid template directed fashion. Elongation may occur via the aid amplification reactions described in Example 15. of an enzyme. Such as, for example, a polymerase or reverse FIG. 22A and FIG. 22B are graphs depicting results of transcriptase. Conditions at which elongation may occur nucleic acid amplification reactions described in Example include an “elongation temperature' that generally refers to 17. 25 a temperature at which elongation is permitted to occur and FIG. 23A, FIG. 23B and FIG. 23C are graphs depicting an “elongation duration” that generally refers to an amount results of nucleic acid amplification reactions described in of time allotted for elongation to occur. Example 18. As used herein, the term “nucleic acid' generally refers to FIG. 24A and FIG. 24B are graphs depicting results of a polymeric form of nucleotides of any length, either deoxy nucleic acid amplification reactions described in Example 30 ribonucleotides (dNTPs) or ribonucleotides (rNTPs), or ana 19. logs thereof. Nucleic acids may have any three dimensional FIG. 25A and FIG. 25B are graphs depicting results of structure, and may perform any function, known or nucleic acid amplification reactions described in Example unknown. Non-limiting examples of nucleic acids include 19. DNA, RNA, coding or non-coding regions of a gene or gene FIG. 26A and FIG. 26B are graphs depicting results of 35 fragment, loci (locus) defined from linkage analysis, exons, nucleic acid amplification reactions described in Example introns, messenger RNA (mRNA), transfer RNA, ribosomal 2O. RNA, short interfering RNA (siRNA), short-hairpin RNA FIG. 27 is a graph depicting results of nucleic acid (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recom amplification reactions described in Example 21. binant nucleic acids, branched nucleic acids, plasmids, Vec FIG. 28A is a schematic of an example electronic display 40 tors, isolated DNA of any sequence, isolated RNA of any having an example user interface. sequence, nucleic acid probes, and primers. A nucleic acid FIG. 28B is a schematic of an example electronic display may comprise one or more modified nucleotides, such as having an example user interface. methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made DETAILED DESCRIPTION 45 before or after assembly of the nucleic acid. The sequence of nucleotides of a nucleic acid may be interrupted by non While various embodiments of the invention have been nucleotide components. A nucleic acid may be further modi shown and described herein, it will be obvious to those fied after polymerization, Such as by conjugation or binding skilled in the art that such embodiments are provided by way with a reporter agent. of example only. Numerous variations, changes, and Substi 50 As used herein, the term “primer extension reaction s tutions may occur to those skilled in the art without depart generally refers to the denaturing of a double-stranded ing from the invention. It should be understood that various nucleic acid, binding of a primer to one or both strands of the alternatives to the embodiments of the invention described denatured nucleic acid, followed by elongation of the herein may be employed. primer(s). As used in the specification and claims, the singular form 55 As used herein, the term “reaction mixture' generally “a”, “an and “the include plural references unless the refers to a composition comprising reagents necessary to context clearly dictates otherwise. For example, the term “a complete nucleic acid amplification (e.g., DNA amplifica cell includes a plurality of cells, including mixtures thereof. tion, RNA amplification), with non-limiting examples of As used herein, the terms “amplifying and “amplifica Such reagents that include primer sets having specificity for tion” are used interchangeably and generally refer to gen 60 target RNA or target DNA, DNA produced from reverse erating one or more copies or “amplified product of a transcription of RNA, a DNA polymerase, a reverse tran nucleic acid. The term “DNA amplification' generally refers scriptase (e.g., for reverse transcription of RNA), suitable to generating one or more copies of a DNA molecule or buffers (including Zwitterionic buffers), co-factors (e.g., “amplified DNA product. The term “reverse transcription divalent and monovalent cations), dNTPs, and other amplification' generally refers to the generation of deoxy 65 enzymes (e.g., uracil-DNA glycosylase (UNG)), etc). In ribonucleic acid (DNA) from a ribonucleic acid (RNA) Some cases, reaction mixtures can also comprise one or more template via the action of a reverse transcriptase. reporter agents. US 9,546,389 B2 11 12 As used herein, a “reporter agent' generally refers to a the reaction vessel to a plurality of series of primer extension composition that yields a detectable signal, the presence or reactions to generate amplified product that is indicative of absence of which can be used to detect the presence of the presence of the target nucleic acid in the biological amplified product. sample, each series comprising two or more cycles of (i) As used herein, the term “target nucleic acid' generally 5 incubating the reaction mixture under a denaturing condition refers to a nucleic acid molecule in a starting population of characterized by a denaturing temperature and a denaturing nucleic acid molecules having a nucleotide sequence whose duration, followed by (ii) incubating the reaction mixture presence, amount, and/or sequence, or changes in one or under an elongation condition characterized by an elonga more of these, are desired to be determined. A target nucleic tion temperature and an elongation duration, wherein an acid may be any type of nucleic acid, including DNA, RNA, 10 individual series differs from at least one other individual and analogues thereof. As used herein, a “target ribonucleic series of the plurality with respect to the denaturing condi acid (RNA) generally refers to a target nucleic acid that is tion and/or the elongation condition. RNA. As used herein, a “target deoxyribonucleic acid In any of the various aspects, nucleic acid from a bio (DNA) generally refers to a target nucleic acid that is DNA. logical sample obtained from a Subject is amplified. In some As used herein, the term “subject,” generally refers to an 15 cases, the biological sample is obtained directly from the entity or a medium that has testable or detectable genetic Subject. A biological sample obtained directly from a subject information. A subject can be a person or individual. A generally refers to a biological sample that has not been Subject can be a vertebrate. Such as, for example, a mammal. further processed after being obtained from the subject, with Non-limiting examples of mammals include murines, sim the exception of any means used to collect the biological ians, humans, farm animals, sport animals, and pets. Other sample from the Subject for further processing. For example, examples of Subjects include food, plant, soil, and water. blood is obtained directly from a subject by accessing the In one aspect, the disclosure provides a method of ampli Subject's circulatory system, removing the blood from the fying a target ribonucleic acid (RNA) present in a biological Subject (e.g., via a needle), and entering the removed blood sample obtained directly from a subject. The method com into a receptacle. The receptacle may comprise reagents prises: (a) providing a reaction vessel comprising the bio 25 (e.g., anti-coagulants) Such that the blood sample is useful logical sample and reagents necessary for conducting for further analysis. In another example, a Swab may be used reverse transcription amplification in parallel with deoxyri to access epithelial cells on an oropharyngeal Surface of the bonucleic acid (DNA) amplification, the reagents compris Subject. After obtaining the biological sample from the ing (i) a reverse transcriptase, (ii) a DNA polymerase, and Subject, the Swab containing the biological sample can be (iii) a primer set for the target RNA, to obtain a reaction 30 contacted with a fluid (e.g., a buffer) to collect the biological mixture; and (b) Subjecting the reaction mixture in the fluid from the Swab. reaction vessel to multiple cycles of a primer extension In some embodiments, a biological sample has not been reaction to generate amplified DNA product that is indica purified when provided in a reaction vessel. In some tive of the presence of the target RNA, each cycle compris embodiments, the nucleic acid of a biological sample has not ing (i) incubating the reaction mixture at a denaturing 35 been extracted when the biological sample is provided to a temperature for a denaturing duration that is less than or reaction vessel. For example, the RNA or DNA in a bio equal to 60 seconds, followed by (ii) incubating the reaction logical sample may not be extracted from the biological mixture at an elongation temperature for an elongation sample when providing the biological sample to a reaction duration that is less than or equal to 60 seconds, thereby vessel. Moreover, in some embodiments, a target nucleic amplifying the target RNA. 40 acid (e.g., a target RNA or target DNA) present in a In another aspect, the disclosure provides a method of biological sample may not be concentrated prior to provid amplifying a target ribonucleic acid (RNA) present in a ing the biological sample to a reaction vessel. biological sample obtained directly from a subject. The Any Suitable biological sample that comprises nucleic method comprises: (a) receiving the biological sample that acid may be obtained from a Subject. A biological sample has been obtained from the subject; (b) providing a reaction 45 may be solid matter (e.g., biological tissue) or may be a fluid vessel comprising the biological sample and reagents nec (e.g., a biological fluid). In general, a biological fluid can essary for conducting reverse transcription amplification and include any fluid associated with living organisms. Non optionally deoxyribonucleic acid (DNA) amplification, the limiting examples of a biological sample include blood (or reagents comprising (i) a reverse transcriptase and (ii) a components of blood—e.g., white blood cells, red blood primer set for the target RNA, to obtain a reaction mixture; 50 cells, platelets) obtained from any anatomical location (e.g., (c) Subjecting the reaction mixture to multiple cycles of a tissue, circulatory system, bone marrow) of a subject, cells primer extension reaction to yield a detectable amount of obtained from any anatomical location of a Subject, skin, amplified DNA product that is indicative of the presence of heart, lung, kidney, breath, bone marrow, stool, semen, the target RNA in the biological sample; (d) detecting the vaginal fluid, interstitial fluids derived from tumorous tissue, amount of amplified DNA product of (c); and (e) outputting 55 breast, pancreas, cerebral spinal fluid, tissue, throat Swab, information regarding the amount of amplified DNA product biopsy, placental fluid, amniotic fluid, liver, muscle, Smooth to a recipient, wherein an amount of time for completing muscle, bladder, gallbladder, colon, intestine, brain, cavity (a)-(e) is less than or equal to about 30 minutes. fluids, sputum, pus, micropiota, meconium, breast milk, In one aspect, the disclosure provides a method of ampli prostate, esophagus, thyroid, serum, saliva, urine, gastric fying a target nucleic acid present in a biological sample 60 and digestive fluid, tears, ocular fluids, Sweat, mucus, ear obtained from a subject. The method comprises: (a) provid wax, oil, glandular secretions, spinal fluid, hair, fingernails, ing a reaction vessel comprising the biological sample and skin cells, plasma, nasal Swab or nasopharyngeal wash, reagents necessary for conducting nucleic acid amplifica spinal fluid, cord blood, emphatic fluids, and/or other excre tion, the reagents comprising (i) a deoxyribonucleic acid tions or body tissues. (DNA) polymerase and optionally a reverse transcriptase, 65 A biological sample may be obtained from a Subject by and (ii) a primer set for the target nucleic acid, to obtain a any means known in the art. Non-limiting examples of reaction mixture; and (b) Subjecting the reaction mixture in means to obtain a biological sample directly from a subject US 9,546,389 B2 13 14 include accessing the circulatory system (e.g., intravenously with non-limiting examples of Such materials that include or intra-arterially via a syringe or other needle), collecting a glasses, metals, plastics, and combinations thereof. secreted biological sample (e.g., feces, urine, sputum, saliva, In some embodiments, a reaction vessel is part of an array etc.), Surgically (e.g., biopsy), Swabbing (e.g., buccal Swab, of reaction vessels. An array of reaction vessels may be oropharyngeal Swab), pipetting, and breathing. Moreover, a particularly useful for automating methods and/or simulta biological sample may be obtained from any anatomical part neously processing multiple samples. For example, a reac of a Subject where a desired biological sample is located. tion vessel may be a well of a microwell plate comprised of In any of the various aspects, a target nucleic acid is a number of wells. In another example, a reaction vessel may amplified to generate an amplified product. A target nucleic be held in a well of a thermal block of a thermocycler, acid may be a target RNA or a target DNA. In cases where 10 wherein the block of the thermal cycle comprises multiple the target nucleic acid is a target RNA, the target RNA may wells each capable of receiving a sample vessel. An array be any type of RNA, including types of RNA described comprised of reaction vessels may comprise any appropriate elsewhere herein. In some embodiments, the target RNA is number of reaction vessels. For example, an array may viral RNA. In some embodiments, the viral RNA may be comprise at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 35, 48, pathogenic to the Subject. Non-limiting examples of patho 15 96, 144, 384, or more reaction vessels. A reaction vessel part genic viral RNA include human immunodeficiency virus I of an array of reaction vessels may also be individually (HIV I), human immunodeficiency virus II (HIV II), addressable by a fluid handling device, such that the fluid orthomyxoviruses, Ebola virus, Dengue virus, influenza handling device can correctly identify a reaction vessel and viruses (e.g., H1N1, H3N2, H7N9, or H5N1), hepesvirus, dispense appropriate fluid materials into the reaction vessel. hepatitis A virus, hepatitis B virus, hepatitis C (e.g., armored Fluid handling devices may be useful in automating the RNA-HCV virus) virus, hepatitis D virus, hepatitis E virus, addition of fluid materials to reaction vessels. hepatitis G. virus, Epstein-Barr virus, mononucleosis virus, In some embodiments, a reaction vessel may comprise cytomegalovirus, SARS virus, West Nile Fever virus, polio multiple thermal Zones. Thermal Zones within a reaction virus, and measles virus. vessel may be achieved by exposing different regions of the In cases where the target nucleic acid is a target DNA, the 25 reaction vessel to different temperature cycling conditions. target DNA may be any type of DNA, including types of For example, a reaction vessel may comprise an upper DNA described elsewhere herein. In some embodiments, the thermal Zone and a lower thermal Zone. The upper thermal target DNA is viral DNA. In some embodiments, the viral Zone may be capable of a receiving a biological sample and DNA may be pathogenic to the subject. Non-limiting reagents necessary to obtain a reaction mixture for nucleic examples of DNA viruses include herpes simplex virus, 30 acid amplification. The reaction mixture can then be Sub Smallpox, adenovirus (e.g., Adenovirus Type 55, Adenovirus jected to a first thermocycling protocol. After a desired Type 7) and Varicella virus (e.g., chickenpox). In some number of cycles, for example, the reaction mixture can cases, a target DNA may be a bacterial DNA. The bacterial slowly, but continuously leak from the upper thermal Zone DNA may be from a bacterium pathogenic to the subject to the lower thermal Zone. In the lower thermal Zone, the Such as, for example, Mycobacterium tuberculosis—a bac 35 reaction mixture is then subjected to a desired number of terium known to cause tuberculosis. In some cases, a target cycles of a second thermocycling protocol different from DNA may be a DNA from a pathogenic protozoan, Such as, that in the upper thermal Zone. Such a strategy may be for example one or more protozoans of the Plasmodium type particularly useful when nested PCR is used to amplify that can cause Malaria. DNA. In some embodiments, thermal Zones may be created In any of the various aspects of the present disclosure, a 40 within a reaction vessel with the aid of thermal sensitive biological sample obtained from a subject is provided with layering materials within the reaction vessels. In Such cases, reagents necessary for nucleic acid amplification in a reac heating of the thermal sensitive layering materials may be tion vessel to obtain a reaction mixture. Any suitable reac used to release reaction mixtures from one thermal Zone to tion vessel may be used. In some embodiments, a reaction the next. In some embodiments, the reaction vessel com vessel comprises a body that can include an interior Surface, 45 prises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more an exterior Surface, an open end, and an opposing closed thermal Zones. end. In some embodiments, a reaction vessel may comprise In some embodiments, a reaction vessel comprising ther a cap. The cap may be configured to contact the body at its mal Zones may be used for processing of a biological sample open end. Such that when contact is made the open end of the prior to nucleic acid amplification. For example, a lysis reaction vessel is closed. In some cases, the cap is perma 50 agent may be added to a first thermal Zone of a reaction nently associated with the reaction vessel such that it vessel prior to adding a biological sample and reagents remains attached to the reaction vessel in open and closed necessary for nucleic acid amplification. When the biologi configurations. In some cases, the cap is removable. Such cal sample and reagents are added to the reaction vessel that when the reaction vessel is open, the cap is separated comprising the lysis agent, a reaction mixture capable of from the reaction vessel. In some embodiments, a reaction 55 lysing species (e.g., cells or viral particles) within the vessel may be sealed, optionally hermetically sealed. biological is obtained. Alternatively, a lysis agent can be A reaction vessel may be of varied size, shape, weight, added to the first thermal Zone of the reaction mixture and configuration. In some examples, a reaction vessel may concurrently with the biological sample and reagents. Sub be round or oval tubular shaped. In some embodiments, a jecting the first thermal Zone to temperature conditions reaction vessel may be rectangular, square, diamond, circu 60 Suitable for action of the lysis agent may be used to lyse cells lar, elliptical, or triangular shaped. A reaction vessel may be and viral particles in the biological sample in the first regularly shaped or irregularly shaped. In some embodi thermal Zone, Such that nucleic acids in the biological ments, the closed end of a reaction vessel may have a sample are released into the reaction mixture. After lysis, the tapered, rounded, or flat Surface. Non-limiting examples of reaction mixture can then be permitted to enter a second types of a reaction vessel include a tube, a well, a capillary 65 thermal Zone of the reaction vessel for amplification of the tube, a cartridge, a cuvette, a centrifuge tube, or a pipette tip. released nucleic acid, using amplification methods described Reaction vessels may be constructed of any suitable material herein. US 9,546,389 B2 15 16 In cases where a lysis agent is desired, any suitable lysis parallel to generate an amplified DNA product. Any suitable agent known in the art may be used, including commercially number of nucleic acid amplification reactions may be available lysis agents. Non-limiting examples of lysis agents conducted in parallel. In some cases, at least 1, 2, 3, 4, 5, 6, include Tris-HCl, EDTA, detergents (e.g., Triton X-100, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more SDS), lysozyme, glucolase, proteinase E, viral endolysins, nucleic acid amplification reactions are conducted in paral exolysins Zymolose, lyticase, proteinase K, endolysins and lel. exolysins from bacteriophages, endolysins from bacterio An advantage of conducting nucleic acid amplification phage PM2, endolysins from the B. subtilis bacteriophage reactions in parallel can include fast transitions between PBSX, endolysins from Lactobacillus prophages Lj928, coupled nucleic acid amplification reactions. For example, a Lj965, bacteriophage 15 Phiadh, endolysin from the Strep 10 target nucleic acid (e.g., target RNA, target DNA) may be tococcus pneumoniae bacteriophage Cp-I, bifunctional pep extracted or released from a biological sample during heat tidoglycan lysin of Streptococcus agalactiae bacteriophage ing phases of parallel nucleic acid amplification. In the case B30, endolysins and exolysins from prophage bacteria, of a target RNA, for example, the biological sample com endolysins from Listeria bacteriophages, holin-endolysin, prising the target RNA can be heated and the target RNA cell 20 lysis genes, holWMY Staphylococcus waneri M 15 released from the biological sample. The released target phage varphi WMY. IySWMY of the Staphylococcus waneri RNA can immediately begin reverse transcription (via M phage varphiWMY. and combinations thereof. In some reverse transcription amplification) to produce complemen cases a buffer may comprise a lysis agent (e.g., a lysis tary DNA. The complementary DNA can then be immedi buffer). An example of a lysis buffer is sodium hydroxide ately amplified, often on the order of seconds. Short times (NaOH). between release of a target RNA from a biological sample Any type of nucleic acid amplification reaction known in and reverse transcription of the target RNA to complemen the art may be used to amplify a target nucleic acid and tary DNA may help minimize the effects of inhibitors in the generate an amplified product. Moreover, amplification of a biological sample that may impede reverse transcription nucleic acid may linear, exponential, or a combination and/or DNA amplification. thereof. Amplification may be emulsion based or may be 25 In any of the various aspects, primer sets directed to a non-emulsion based. Non-limiting examples of nucleic acid target nucleic acid may be utilized to conduct nucleic acid amplification methods include reverse transcription, primer amplification reaction. Primer sets generally comprise one extension, polymerase chain reaction, ligase chain reaction, or more primers. For example, a primer set may comprise helicase-dependent amplification, asymmetric amplification, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primers. In some rolling circle amplification, and multiple displacement 30 cases, a primer set or may comprise primers directed to amplification (MDA). In some embodiments, the amplified different amplified products or different nucleic acid ampli product may be DNA. In cases where a target RNA is fication reactions. For example, a primer set may comprise amplified, DNA can be obtained by reverse transcription of a first primer necessary to generate a first strand of nucleic the RNA and subsequent amplification of the DNA can be acid product that is complementary to at least a portion of used to generate an amplified DNA product. The amplified 35 the target nucleic acid and a second primer complementary DNA product may be indicative of the presence of the target to the nucleic acid strand product necessary to generate a RNA in the biological sample. In cases where DNA is second strand of nucleic acid product that is complementary amplified, any DNA amplification method known in the art to at least a portion of the first strand of nucleic acid product. may be employed. Non-limiting examples of DNA ampli For example, a primer set may be directed to a target fication methods include polymerase chain reaction (PCR), 40 RNA. The primer set may comprise a first primer that can be variants of PCR (e.g., real-time PCR, allele-specific PCR, used to generate a first strand of nucleic acid product that is assembly PCR, asymmetric PCR, digital PCR, emulsion complementary to at least a portion the target RNA. In the PCR, dial-out PCR, helicase-dependent PCR, nested PCR, case of a reverse transcription reaction, the first strand of hot start PCR, inverse PCR, methylation-specific PCR, nucleic acid product may be DNA. The primer set may also miniprimer PCR, multiplex PCR, nested PCR, overlap 45 comprise a second primer that can be used to generate a extension PCR, thermal asymmetric interlaced PCR, touch second strand of nucleic acid product that is complementary down PCR), and ligase chain reaction (LCR). In some cases, to at least a portion of the first strand of nucleic acid product. DNA amplification is linear. In some cases, DNA amplifi In the case of a reverse transcription reaction conducted in cation is exponential. In some cases, DNA amplification is parallel with DNA amplification, the second strand of achieved with nested PCR, which can improve sensitivity of 50 nucleic acid product may be a strand of nucleic acid (e.g., detecting amplified DNA products. DNA) product that is complementary to a strand of DNA In various aspects, nucleic acid amplification reactions generated from an RNA template. described herein may be conducted in parallel. In general, Where desired, any suitable number of primer sets may be parallel amplification reactions are amplification reactions used. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more that occur in the same reaction vessel and at the same time. 55 primer sets may be used. Where multiple primer sets are Parallel nucleic acid amplification reactions may be con used, one or more primer sets may each correspond to a ducted, for example, by including reagents necessary for particular nucleic acid amplification reaction or amplified each nucleic acid amplification reaction in a reaction vessel product. to obtain a reaction mixture and Subjecting the reaction In some embodiments, a DNA polymerase is used. Any mixture to conditions necessary for each nucleic amplifica 60 Suitable DNA polymerase may be used, including commer tion reaction. For example, reverse transcription amplifica cially available DNA polymerases. A DNA polymerase tion and DNA amplification may be conducted in parallel, by generally refers to an enzyme that is capable of incorporat providing reagents necessary for both amplification methods ing nucleotides to a strand of DNA in a template bound in a reaction vessel to form to obtain a reaction mixture and fashion. Non-limiting examples of DNA polymerases Subjecting the reaction mixture to conditions Suitable for 65 include Taq polymerase. Tth polymerase, Tli polymerase, conducting both amplification reactions. DNA generated Pfu polymerase, VENT polymerase, DEEPVENT poly from reverse transcription of the RNA may be amplified in merase, EX-Taq polymerase, LA-Taq polymerase, Expand US 9,546,389 B2 17 18 polymerases, Sso polymerase, Poc polymerase, Pab poly tion temperature may be from about 40° C. to about 60° C. merase, Mth polymerase, Pho polymerase, ES4 polymerase, In some examples, an elongation temperature may be from Tru polymerase, Tac polymerase, Tne polymerase, Tma about 50° C. to about 60° C. In still other examples, an polymerase, Tih polymerase, Tifi polymerase, Platinum Taq elongation temperature may be about 35°, 36° C., 37° C. polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl poly 5 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° merase, Pfutubo polymerase, Pyrobest polymerase, Pwo C., 46° C. 470 C., 48° C., 499 C., 50° C. 510 C., 52° C., 53° polymerase, KOD polymerase, Bst polymerase, Sac poly C., 540 C., 550 C., 56° C. 57°C., 58° C., 59° C., 60° C. 610 merase, Klenow fragment, and variants, modified products C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C, 69° and derivatives thereof. For certain Hot Start Polymerase, a C., 70° C. 710 C., 720 C., 73° C. 740 C., 75° C., 76° C., 770 denaturation step at 94° C.-95° C. for 2 minutes to 10 10 minutes may be required, which may change the thermal C., 78° C. 79° C., or 80° C. profile based on different polymerases. Elongation durations may vary depending upon, for In some embodiments, a reverse transcriptase is used. Any example, the particular biological sample analyzed, the Suitable reverse transcriptase may be used. A reverse tran particular source of target nucleic acid (e.g., viral particle, Scriptase generally refers to an enzyme that is capable of 15 bacteria) in the biological sample, the reagents used, and/or incorporating nucleotides to a strand of DNA, when bound the desired reaction conditions. For example, an elongation to an RNA template. Non-limiting examples of reverse duration may be less than or equal to 300 seconds, 240 transcriptases include HIV-1 reverse transcriptase, M-MLV seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, reverse transcriptase, AMV reverse transcriptase, telomerase 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, reverse transcriptase, and variants, modified products and 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, derivatives thereof. 5 seconds, 2 seconds, or 1 second. For example, an elon In various aspects, primer extension reactions are utilized gation duration may be no more than 120 seconds, 90 to generate amplified product. Primer extension reactions seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 generally comprise a cycle of incubating a reaction mixture seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 at a denaturation temperature for a denaturation duration and 25 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. incubating a reaction mixture at an elongation temperature In any of the various aspects, multiple cycles of a primer for an elongation duration. extension reaction can be conducted. Any suitable number Denaturation temperatures may vary depending upon, for of cycles may be conducted. For example, the number of example, the particular biological sample analyzed, the cycles conducted may be less than about 100, 90, 80, 70, 60. particular source of target nucleic acid (e.g., viral particle, 30 bacteria) in the biological sample, the reagents used, and/or 50, 40, 30, 20, 10, or 5 cycles. The number of cycles the desired reaction conditions. For example, a denaturation conducted may depend upon, for example, the number of temperature may be from about 80° C. to about 110°C. In cycles (e.g., cycle threshold value (Ct)) necessary to obtain Some examples, a denaturation temperature may be from a detectable amplified product (e.g., a detectable amount of about 90° C. to about 100° C. In some examples, a dena 35 amplified DNA product that is indicative of the presence of turation temperature may be from about 90° C. to about 97 a target RNA in a biological sample). For example, the C. In some examples, a denaturation temperature may be number of cycles necessary to obtain a detectable amplified from about 92° C. to about 95°C. In still other examples, a product (e.g., a detectable amount of DNA product that is denaturation temperature may be about 80°, 81° C., 82°C., indicative of the presence of a target RNA in a biological 83° C., 840 C., 850 C., 86° C., 87° C., 88° C., 89° C., 90° 40 sample) may be less than about or about 100 cycles, 75 C., 910 C., 920 C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° cycles, 70 cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, C., 99 C., or 100° C. 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 Denaturation durations may vary depending upon, for cycles, 10 cycles, or 5 cycles. Moreover, in some embodi example, the particular biological sample analyzed, the ments, a detectable amount of an amplifiable product (e.g., particular source of target nucleic acid (e.g., viral particle, 45 a detectable amount of DNA product that is indicative of the bacteria) in the biological sample, the reagents used, and/or presence of a target RNA in a biological sample) may be the desired reaction conditions. For example, a denaturation obtained at a cycle threshold value (Ct) of less than 100, 75, duration may be less than or equal to 300 seconds, 240 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5. seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, The time for which amplification yields a detectable 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 50 amount of amplified product indicative of the presence of a 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, target nucleic acid amplified can vary depending upon the 5 seconds, 2 seconds, or 1 second. For example, a denatur biological sample from which the target nucleic acid was ation duration may be no more than 120 seconds, 90 obtained, the particular nucleic acid amplification reactions seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 to be conducted, and the particular number of cycles of seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 55 amplification reaction desired. For example, amplification of seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. a target nucleic acid may yield a detectable amount of Elongation temperatures may vary depending upon, for amplified product indicative to the presence of the target example, the particular biological sample analyzed, the nucleic acid at time period of 120 minutes or less; 90 particular source of target nucleic acid (e.g., viral particle, minutes or less; 60 minutes or less; 50 minutes or less; 45 bacteria) in the biological sample, the reagents used, and/or 60 minutes or less; 40 minutes or less; 35 minutes or less; 30 the desired reaction conditions. For example, an elongation minutes or less; 25 minutes or less; 20 minutes or less; 15 temperature may be from about 30° C. to about 80°C. In minutes or less; 10 minutes or less; or 5 minutes or less. Some examples, an elongation temperature may be from In some embodiments, amplification of a target RNA may about 35° C. to about 72°C. In some examples, an elonga yield a detectable amount of amplified DNA product indica tion temperature may be from about 45° C. to about 65° C. 65 tive to the presence of the target RNA at time period of 120 In some examples, an elongation temperature may be from minutes or less; 90 minutes or less; 60 minutes or less; 50 about 35° C. to about 65° C. In some examples, an elonga minutes or less; 45 minutes or less; 40 minutes or less; 35 US 9,546,389 B2 19 20 minutes or less; 30 minutes or less; 25 minutes or less: 20 primer extension reaction. In the case of a plurality of series minutes or less; 15 minutes or less; 10 minutes or less; or 5 of primer extension reactions, the target nucleic acid may be minutes or less. Subjected to a denaturing condition prior to executing the In some embodiments, a reaction mixture may be Sub plurality of series or may be subjected to a denaturing jected to a plurality of series of primer extension reactions. condition between series of the plurality. For example, the An individual series of the plurality may comprise multiple target nucleic acid may be subjected to a denaturing condi cycles of a particular primer extension reaction, character tion between a first series and a second series of a plurality ized, for example, by particular denaturation and elongation of series. Non-limiting examples of Such denaturing condi conditions as described elsewhere herein. Generally, each tions include a denaturing temperature profile (e.g., one or individual series differs from at least one other individual 10 more denaturing temperatures) and a denaturing agent. series in the plurality with respect to, for example, a dena An advantage of conducting a plurality of series of primer turation condition and/or elongation condition. An indi extension reaction may be that, when compared to a single vidual series may differ from another individual series in a series of primer extension reactions under comparable dena plurality of series, for example, with respect to any one, two, turing and elongation conditions, the plurality of series three, or all four of denaturing temperature, denaturing 15 approach yields a detectable amount of amplified product duration, elongation temperature, and elongation duration. that is indicative of the presence of a target nucleic acid in Moreover, a plurality of series may comprise any number of a biological sample with a lower cycle threshold value. Use individual series such as, for example, at least about or about of a plurality of series of primer extension reactions may 2, 3, 4, 5, 6, 7, 8, 9, 10, or more individual series. reduce such cycle threshold values by at least about or about For example, a plurality of series of primer extension 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, reactions may comprise a first series and a second series. The 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% when first series, for example, may comprise more than ten cycles compared to a single series under comparable denaturing of a primer extension reaction, where each cycle of the first and elongation conditions. series comprises (i) incubating a reaction mixture at about In some embodiments, a biological sample may be pre 92° C. to about 95°C. for no more than 30 seconds followed 25 heated prior to conducting a primer extension reaction. The by (ii) incubating the reaction mixture at about 35° C. to temperature (e.g., a preheating temperature) at which and about 65° C. for no more than about one minute. The second duration (e.g., a preheating duration) for which a biological series, for example, may comprise more than ten cycles of sample is preheated may vary depending upon, for example, a primer extension reaction, where each cycle of the second the particular biological sample being analyzed. In some series comprises (i) incubating the reaction mixture at about 30 examples, a biological sample may be preheated for no more 92° C. to about 95°C. for no more than 30 seconds followed than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, by (ii) incubating the reaction mixture at about 40° C. to 25 minutes. 20 minutes, 15 minutes, 10 minutes, 9 minutes, about 60° C. for no more than about 1 minute. In this 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 particular example, the first and second series differ in their minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 elongation temperature condition. The example, however, is 35 seconds, 15 seconds, 10 seconds, or 5 seconds. In some not meant to be limiting as any combination of different examples, a biological sample may be preheated at a tem elongation and denaturing conditions could be used. perature from about 80° C. to about 110° C. In some In some embodiments, the ramping time (i.e., the time the examples, a biological sample may be preheated at a tem thermal cycler takes to transition from one temperature to perature from about 90° C. to about 100° C. In some another) and/or ramping rate can be important factors in 40 examples, a biological sample may be preheated at a tem amplification. For example, the temperature and time for perature from about 90° C. to about 97° C. In some which amplification yields a detectable amount of amplified examples, a biological sample may be preheated at a tem product indicative of the presence of a target nucleic acid perature from about 92° C. to about 95°C. In still other can vary depending upon the ramping rate and/or ramping examples, a biological sample may be preheated at a tem time. The ramping rate can impact the temperature(s) and 45 perature of about 80°, 81° C., 82°C., 83° C., 84° C., 85°C., time(s) used for amplification. 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 920 C., 930 In some cases, the ramping time and/or ramping rate can C., 949 C., 95°C., 96° C., 97° C., 98° C. 990 C., or 100° C. be different between cycles. In some situations, however, the In some embodiments, reagents necessary for conducting ramping time and/or ramping rate between cycles can be the nucleic acid amplification, including reagents necessary for same. The ramping time and/or ramping rate can be adjusted 50 conduction of parallel nucleic acid amplification may also based on the sample(s) that are being processed. include a reporter agent that yields a detectable signal whose In some situations, the ramping time between different presence or absence is indicative of the presence of an temperatures can be determined, for example, based on the amplified product. The intensity of the detectable signal may nature of the sample and the reaction conditions. The exact be proportional to the amount of amplified product. In some temperature and incubation time can also be determined 55 cases, where amplified product is generated of a different based on the nature of the sample and the reaction condi type of nucleic acid than the target nucleic acid initially tions. In some embodiments, a single sample can be pro amplified, the intensity of the detectable signal may be cessed (e.g., Subjected to amplification conditions) multiple proportional to the amount of target nucleic acid initially times using multiple thermal cycles, with each thermal cycle amplified. For example, in the case of amplifying a target differing for example by the ramping time, temperature, 60 RNA via parallel reverse transcription and amplification of and/or incubation time. The best or optimum thermal cycle the DNA obtained from reverse transcription, reagents nec can then be chosen for that particular sample. This provides essary for both reactions may also comprise a reporter agent a robust and efficient method of tailoring the thermal cycles may yield a detectable signal that is indicative of the to the specific sample or combination of samples being presence of the amplified DNA product and/or the target tested. 65 RNA amplified. The intensity of the detectable signal may In Some embodiments, a target nucleic acid may be be proportional to the amount of the amplified DNA product Subjected to a denaturing condition prior to initiation of a and/or the original target RNA amplified. The use of a US 9,546,389 B2 21 22 reporter agent also enables real-time amplification methods, blocking the optical activity of an associated dye. Non including real-time PCR for DNA amplification. limiting examples of probes that may be useful used as Reporter agents may be linked with nucleic acids, includ reporter agents include TaqMan probes, TaqMan Tamara ing amplified products, by covalent or non-covalent means. probes, TaqMan MGB probes, or Lion probes. Non-limiting examples of non-covalent means include ionic In some embodiments and where a reporter agent may be interactions, Vander Waals forces, hydrophobic interactions, an RNA oliognucleotide probe that includes an optically hydrogen bonding, and combinations thereof. In some active dye (e.g., fluorescent dye) and a quencher positioned embodiments, reporter agents may bind to initial reactants adjacently on the probe. The close proximity of the dye with and changes in reporter agent levels may be used to detect the quencher can block the optical activity of the dye. The amplified product. In some embodiments, reporter agents 10 probe may bind to a target sequence to be amplified. Upon may only be detectable (or non-detectable) as nucleic acid amplification progresses. In some embodiments, an opti the breakdown of the probe with the exonuclease activity of cally-active dye (e.g., a fluorescent dye) may be used as may a DNA polymerase during amplification, the quencher and be used as a reporter agent. Non-limiting examples of dyes dye are separated, and the free dye regains its optical activity include SYBR green, SYBR blue, DAPI, propidium iodine, 15 that can Subsequently be detected. Hoeste, SYBR gold, ethidium bromide, acridines, profla In some embodiments, a reporter agent may be a molecu vine, acridine orange, acriflavine, fluorcoumanin, ellipticine, lar beacon. A molecular beacon includes, for example, a daunomycin, chloroquine, distamycin D. chromomycin, quencher linked at one end of an oligonucleotide in a hairpin homidium, mithramycin, ruthenium polypyridyls, anth conformation. At the other end of the oligonucleotide is an ramycin, phenanthridines and acridines, ethidium bromide, optically active dye, Such as, for example, a fluorescent dye. propidium iodide, hexidium iodide, dihydroethidium, In the hairpin configuration, the optically-active dye and ethidium homodimer-1 and -2, ethidium monoazide, and quencher are brought in close enough proximity Such that ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, the quencher is capable of blocking the optical activity of the DAPI, acridine orange, 7-AAD, actinomycin D. LDS751, dye. Upon hybridizing with amplified product, however, the hydroxystilbamidine, SYTOX Blue, SYTOX Green, 25 oligonucleotide assumes a linear conformation and hybrid SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, izes with a target sequence on the amplified product. Lin TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, earization of the oligonucleotide results in separation of the PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO optically-active dye and quencher, Such that the optical 1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, activity is restored and can be detected. The sequence YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, 30 specificity of the molecular beacon for a target sequence on SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, the amplified product can improve specificity and sensitivity SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, of detection. -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, In some embodiments, a reporter agent may be a radio -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, active species. Non-limiting examples of radioactive species -60, -63 (red), fluorescein, fluorescein isothiocyanate 35 include “C, 2I, 12*I, 125I, II, Tc99m, SS, or H. (FITC), tetramethyl rhodamine isothiocyanate (TRITC), In some embodiments, a reporter agent may be an enzyme rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, that is capable of generating a detectable signal. Detectable Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, signal may be produced by activity of the enzyme with its allophycocyanin (APC), Sybr Green I, Sybr Green II, Sybr Substrate or a particular substrate in the case the enzyme has Gold, CellTracker Green, 7-AAD, ethidium homodimer I, 40 multiple Substrates. Non-limiting examples of enzymes that ethidium homodimer II, ethidium homodimer III, ethidium may be used as reporter agents include alkaline phosphatase, bromide, umbelliferone, eosin, green fluorescent protein, horseradish peroxidase, I’-galactosidase, alkaline phos erythrosin, coumarin, methyl coumarin, pyrene, malachite phatase, B-galactosidase, acetylcholinesterase, and green, Stilbene, lucifer yellow, cascade blue, dichlorotriazi luciferase. nylamine fluorescein, dansyl chloride, fluorescent lan 45 In various aspects, amplified product (e.g., amplified thanide complexes such as those including europium and DNA product, amplified RNA product) may be detected. terbium, carboxy tetrachlorofluorescein, 5 and/or 6-carboxy Detection of amplified product, including amplified DNA, fluorescein (FAM), 5- (or 6-) iodoacetamidofluorescein, may be accomplished with any suitable detection method 5-2 (and 3)-5-(Acetylmercapto)-succinyl known in the art. The particular type of detection method aminofluorescein (SAMSA-fluorescein), lissamine rhod 50 used may depend, for example, on the particular amplified amine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine product, the type of reaction vessel used for amplification, (ROX), 7-amino-methyl-coumarin, 7-Amino-4-methylcou other reagents in a reaction mixture, whether or not a marin-3-acetic acid (AMCA), BODIPY fluorophores, reporter agent was included in a reaction mixture, and if a 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3.6- reporter agent was used, the particular type of reporter agent Disulfonate-4-amino-naphthalimide, phycobiliproteins, 55 use. Non-limiting examples of detection methods include AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, optical detection, spectroscopic detection, electrostatic 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, detection, electrochemical detection, and the like. Optical DyLight 350, 405, 488, 550, 594,633, 650, 680, 755, and detection methods include, but are not limited to, fluorimetry 800 dyes, or other fluorophores. and UV-vis light absorbance. Spectroscopic detection meth In some embodiments, a reporter agent may be a 60 ods include, but are not limited to, mass spectrometry, sequence-specific oligonucleotide probe that is optically nuclear magnetic resonance (NMR) spectroscopy, and infra active when hybridized with an amplified product. Due to red spectroscopy. Electrostatic detection methods include, sequence-specific binding of the probe to the amplified but are not limited to, gel based techniques, such as, for product, use of oligonucleotide probes can increase speci example, gel electrophoresis. Electrochemical detection ficity and sensitivity of detection. A probe may be linked to 65 methods include, but are not limited to, electrochemical any of the optically-active reporter agents (e.g., dyes) detection of amplified product after high-performance liquid described herein and may also include a quencher capable of chromatography separation of the amplified products. US 9,546,389 B2 23 24 In any of the various aspects, the time required to com medical records), a public health worker, other medical plete the elements of a method may vary depending upon the personnel, and other medical facilities. particular steps of the method. For example, an amount of In an aspect, the disclosure provides a system that imple time for completing the elements of a method may be from ments a method according to any of the methods disclosed about 5 minutes to about 120 minutes. In other examples, an 5 herein. In another aspect, the disclosure provides a system amount of time for completing the elements of a method for amplifying a target ribonucleic acid (RNA) present in a may be from about 5 minutes to about 60 minutes. In other biological sample obtained directly from a subject. The examples, an amount of time for completing the elements of system comprises: (a) an input module that receives a user a method may be from about 5 minutes to about 30 minutes. request to amplify the target RNA in the biological sample: 10 (b) an amplification module that, in response to the user In other examples, an amount of time for completing the request: receives, in a reaction vessel, a reaction mixture elements of a method may be less than or equal to 120 comprising the biological sample and reagents necessary for minutes, less than or equal to 90 minutes, less than or equal conducting reverse transcription amplification in parallel to 75 minutes, less than or equal to 60 minutes, less than or with deoxyribonucleic acid (DNA) amplification, the equal to 45 minutes, less than or equal to 40 minutes, less 15 reagents comprising (i) a reverse transcriptase, (ii) a DNA than or equal to 35 minutes, less than or equal to 30 minutes, polymerase, and (iii) a primer set for the target RNA; and less than or equal to 25 minutes, less than or equal to 20 Subjects the reaction mixture in the reaction vessel to minutes, less than or equal to 15 minutes, less than or equal multiple cycles of a primer extension reaction to generate to 10 minutes, or less than or equal to 5 minutes. amplified DNA product that is indicative of the presence of In some embodiments, information regarding the pres the target RNA, each cycle comprising (i) incubating the ence of and/or an amount of amplified product (e.g., ampli reaction mixture at a denaturing temperature for a denatur fied DNA product) may be outputted to a recipient. Infor ing duration that is less than or equal to 60 seconds, followed mation regarding amplified product may be outputted via by (ii) incubating the reaction mixture at an elongation any Suitable means known in the art. In some embodiments, temperature for an elongation duration that is less than or such information may be provided verbally to a recipient. In 25 equal to 60 seconds, thereby amplifying the target RNA; and Some embodiments, such information may be provided in a (c) an output module operatively coupled to the amplifica report. A report may include any number of desired ele tion module, wherein the output module outputs information ments, with non-limiting examples that include information regarding the target RNA or the DNA product to a recipient. regarding the Subject (e.g., sex, age, race, health status, etc.) In another aspect, the disclosure provides a system for raw data, processed data (e.g. graphical displays (e.g., 30 amplifying a target ribonucleic acid (RNA) present in a figures, charts, data tables, data Summaries), determined biological sample obtained directly from a subject. The cycle threshold values, calculation of starting amount of system comprises: (a) an input module that receives a user target polynucleotide), conclusions about the presence of the request to amplify the target RNA in the biological sample: target nucleic acid, diagnosis information, prognosis infor (b) an amplification module that, in response to the user mation, disease information, and the like, and combinations 35 request: (i) receives, in a reaction vessel, a reaction mixture thereof. The report may be provided as a printed report (e.g., comprising the biological sample that has been obtained a hard copy) or may be provided as an electronic report. In from the Subject and reagents necessary for conducting Some embodiments, including cases where an electronic reverse transcription amplification and optionally deoxyri report is provided, such information may be outputted via an bonucleic acid (DNA) amplification, the reagents compris electronic display (e.g., an electronic display Screen). Such 40 ing (1) a reverse transcriptase and (2) a primer set for the as a monitor or television, a screen operatively linked with target RNA; and (ii) subjects the reaction mixture to multiple a unit used to obtain the amplified product, a tablet computer cycles of a primer extension reaction to yield a detectable screen, a mobile device screen, and the like. Both printed amount of amplified DNA product that is indicative of the and electronic reports may be stored in files or in databases, presence of the target RNA in the biological sample; (iii) respectively, Such that they are accessible for comparison 45 detects the amount of amplified DNA product of (iii); and with future reports. (iv) outputs information regarding the amount of amplified Moreover, a report may be transmitted to the recipient at DNA product to a recipient, wherein an amount of time for a local or remote location using any suitable communication completing (i)-(iv) is less than or equal to about 30 minutes; medium including, for example, a network connection, a and (c) an output module operatively coupled to the ampli wireless connection, or an internet connection. In some 50 fication module, wherein the output module transmits the embodiments, a report can be sent to a recipient’s device, information to a recipient. Such as a personal computer, phone, tablet, or other device. In another aspect, the disclosure provides a system for The report may be viewed online, saved on the recipients amplifying a target nucleic acid present in a biological device, or printed. A report can also be transmitted by any sample obtained from a Subject. The system comprises: (a) other Suitable means for transmitting information, with 55 an input module that receives a user request to amplify the non-limiting examples that include mailing a hard-copy target RNA in the biological sample; (b) an amplification report for reception and/or for review by a recipient. module that, in response to the user request: receives, in a Moreover, such information may be outputted to various reaction vessel, a reaction mixture comprising the biological types of recipients. Non-limiting examples of Such recipi sample and reagents necessary for conducting nucleic acid ents include the subject from which the biological sample 60 amplification, the reagents comprising (i) a DNA poly was obtained, a physician, a physician treating the Subject, merase and optionally a reverse transcriptase, and (ii) a a clinical monitor for a clinical trial, a nurse, a researcher, a primer set for the target nucleic acid; and Subjects the laboratory technician, a representative of a pharmaceutical reaction mixture in the reaction vessel to a plurality of series company, a health care company, a biotechnology company, of primer extension reactions to generate amplified product a hospital, a human aid organization, a health care manager, 65 that is indicative of the presence of the target nucleic acid in an electronic system (e.g., one or more computers and/or one the biological sample, each series comprising two or more or more computer servers storing, for example, a subjects cycles of (i) incubating the reaction mixture under a dena US 9,546,389 B2 25 26 turing condition characterized by a denaturing temperature specifically designed to amplify one or more sequences of and a denaturing duration, followed by (ii) incubating the the target nucleic acid molecule. In some embodiments, the reaction mixture under an elongation condition character amplification protocol may further include selecting a ized by an elongation temperature and an elongation dura reporter agent (e.g., an oligonucleotide probe comprising an tion, wherein an individual series differs from at least one optically-active species or other type of reporter agent other individual series of the plurality with respect to the described elsewhere herein) that is specific for one or more denaturing condition and/or the elongation condition; and sequences of the target nucleic acid molecule. Moreover, in (c) an output module operatively coupled to the amplifica Some embodiments, the reagents may comprise any Suitable tion module, wherein the output module outputs information reagents necessary for nucleic acid amplification as regarding the target RNA or the DNA product to a recipient. 10 described elsewhere herein, Such as, for example, a deoxy In another aspect, the disclosure provides a system for ribonucleic acid (DNA) polymerase, a primer set for the amplifying a target nucleic acid in a biological sample target nucleic acid, and (optionally) a reverse transcriptase. obtained from a Subject. The system can include an elec In some embodiments, the user interface can display a tronic display Screen that has a user interface that displays a plurality of graphical elements. Each of the graphical ele graphical element that is accessible by a user to execute an 15 ments can be associated with a given amplification protocol amplification protocol to amplify the target nucleic acid in among a plurality of amplification protocols. Each of the the biological sample. The system can also include a com plurality of amplification protocols may include a different puter processor (including any Suitable device having a combination of series of primer extension reaction. In some computer processor as described elsewhere herein) coupled cases, though, a user interface may display a plurality of to the electronic display screen and programmed to execute graphical elements associated with the same amplification the amplification protocol upon selection of the graphical protocol. An example of a user interface having a plurality element by the user. The amplification protocol can com of graphical elements each associated with a given ampli prise Subjecting a reaction mixture comprising the biological fication protocol is shown in FIG. 28A. As shown in FIG. sample and reagents necessary for conducting nucleic acid 28A, an example electronic display screen 2800 associated amplification to a plurality of series of primer extension 25 with a computer processor includes a user interface 2801. reactions to generate amplified product. The amplified prod The user interface 2801 includes a display of graphical uct can be indicative of the presence of the target nucleic elements 2802, 2803 and 2804. Each of the graphical acid in the biological sample. Moreover, each series of elements can be associated with a particular amplification primer extension reactions can comprise two or more cycles protocol (e.g., “Prot. 1 for graphical element 2802, “Prot. of incubating the reaction mixture under a denaturing con 30 2 for graphical element 2803 and “Prot. 4 for graphical dition that is characterized by a denaturing temperature and element 2804). Upon user selection (e.g., user touch when a denaturing duration, followed by incubating the reaction the electronic display screen 2800 includes a touch-screen mixture under an elongation condition that is characterized having the user interface) of particular graphical element, by an elongation temperature and an elongation duration. An the particular amplification protocol associated with the individual series can differ from at least one other individual 35 graphical element can be executed by an associated com series of the plurality with respect to the denaturing condi puter processor. For example, when a user selects graphical tion and/or the elongation condition. element 2803, amplification “Prot. 2 is executed by the In some embodiments, the target nucleic acid may be associated computer processor. Where only three graphical associated with a disease. The disease may be, for example, elements are shown in the example user interface 2801 of associated with an RNA virus or a DNA virus. Examples of 40 FIG. 28A, a user interface may have any suitable number of viruses are provided elsewhere herein. In some embodi graphical elements. Moreover, where each graphical ele ments, the disease may be associated with a pathogenic ment shown in the user interface 2801 of FIG. 28A is bacterium (e.g., Mycobacterium tuberculosis) or a patho associated with only one amplification protocol, each genic protozoan (e.g., Plasmodium as in Malaria), including graphical element of a user interface can be associated with examples of Such pathogens described elsewhere herein. In 45 one or more amplification protocols (e.g., a series of ampli Some embodiments, the amplification protocol can be fication protocols) such that an associated computer proces directed to assaying for the presence of said disease based on Sor executes a series of amplification protocols upon user a presence of the amplified product. interaction with the graphical element. In some cases, a user interface can be a graphical user In some embodiments, each of the graphical elements interface. Moreover, a user interface can include one or more 50 and/or may be associated with a disease, and a given graphical elements. Graphical elements can include image amplification protocol among the plurality of amplification and/or textual information, Such as pictures, icons and text. protocols may be directed to assaying a presence of the The graphical elements can have various sizes and orienta disease in the Subject. Thus, in Such cases, a user can select tions on the user interface. Furthermore, an electronic dis a graphical element in order to run an amplification protocol play screen may be any suitable electronic display including 55 (or series of amplification protocols) to assay for a particular examples described elsewhere herein. Non-limiting disease. In some embodiments, the disease may be associ examples of electronic display screens include a monitor, a ated with a virus such as, for example, any RNA virus or mobile device Screen, a laptop computer screen, a television, DNA virus including examples of such viruses described a portable video game system screen and a calculator screen. elsewhere herein. Non-limiting examples of viruses include In some embodiments, an electronic display screen may 60 human immunodeficiency virus I (HIV I), human immuno include a touch screen (e.g., a capacitive or resistive touch deficiency virus II (HIV II), an orthomyxovirus, Ebola virus, screen) Such that graphical elements displayed on a user Dengue virus, influenza viruses (e.g., H1N1 virus, H3N2 interface of the electronic display screen can be selected via virus, H7N9 virus or H5N1 virus), hepevirus, hepatitis A user touch with the electronic display screen. virus, hepatitis B virus, hepatitis C virus (e.g., armored In some embodiments, the amplification protocol may 65 RNA-hepatitis C virus (RNA-HCV)), hepatitis D virus, further include selecting a primer set for the target nucleic hepatitis E virus, hepatitis G virus, Epstein-Barr virus, acid. In Such cases, the primer set may be a primer set mononucleosis virus, cytomegalovirus, SARS virus, West US 9,546,389 B2 27 28 Nile Fever virus, polio virus, measles virus, herpes simplex line, the internet, a local intranet, a wireless connection, etc., virus, Smallpox virus, adenovirus (e.g., adenovirus type 55 or via a transportable medium, Such as a computer readable (ADV55), adenovirus type 7 (ADV7)) and Varicella virus. disk, flash drive, etc. The various steps may be implemented In some embodiments, the disease may be associated with a as various blocks, operations, tools, modules or techniques pathogenic bacterium (e.g., Mycobacterium tuberculosis) or 5 which, in turn, may be implemented in hardware, firmware, a pathogenic protozoan (e.g., Plasmodium as in Malaria), software, or any combination thereof. When implemented in including examples of Such pathogens described elsewhere hardware, Some or all of the blocks, operations, techniques, herein. etc. may be implemented in, for example, a custom inte An example of a user interface having a plurality of grated circuit (IC), an application specific integrated circuit graphical elements each associated with a given amplifica 10 (ASIC), a field programmable logic array (FPGA), a pro tion protocol is shown in FIG. 28B. As shown in FIG. 28B, an example electronic display screen 2810 associated with a grammable logic array (PLA), etc. computer processor includes a user interface 2811. The user In some embodiments, the input module is configured to interface 2811 includes a display of graphical elements receive a user request to perform amplification of the target 2812, 2813 and 2814. Each of the graphical elements can be 15 nucleic acid. The input module may receive the user request associated with a particular disease (e.g., "Ebola' for graphi directly (e.g. by way of an input device Such as a keyboard, cal element 2812, “H1N1 for graphical element 2813 and mouse, or touch screen operated by the user) or indirectly "Hep C (Hepatitis C) for graphical element 2814) that is, in (e.g. through a wired or wireless connection, including over turn, associated with one or more amplification protocols the internet). Via output electronics, the input module may directed toward the particular disease. Upon user selection provide the user's request to the amplification module. In (e.g., user touch when the electronic display screen 2810 Some embodiments, an input module may include a user includes a touch-screen having the user interface) with a interface (UI). Such as a graphical user interface (GUI), that particular graphical element, the particular amplification is configured to enable a user provide a request to amplify protocol(s) associated with the disease associated with the the target nucleic acid. A GUI can include textual, graphical graphical element can be executed by an associated com 25 and/or audio components. A GUI can be provided on an puter processor. For example, when a user interacts with electronic display, including the display of a device com graphical element 2812, one or more amplification protocols prising a computer processor. Such a display may include a associated with assaying for Ebola virus can be executed by resistive or capacitive touch screen. the associated computer processor. Where only three graphi Non-limiting examples of users include the Subject from cal elements are shown in the example user interface 2811 30 which the biological sample was obtained, medical person of FIG. 28B, a user interface may have any suitable number nel, clinicians (e.g., doctors, nurses, laboratory technicians), of graphical elements each corresponding to a various laboratory personnel (e.g., hospital laboratory technicians, disease. Moreover, where each graphical element shown in research scientists, pharmaceutical scientists), a clinical the user interface 2811 of FIG. 28B is associated with only monitor for a clinical trial, or others in the health care one disease, each graphical element of a user interface can 35 industry. be associated with one or more diseases such that an In various aspects, the system comprises an amplification associated computer processor executes a series of amplifi module for performing nucleic acid amplification reaction cation protocols (e.g., each individual amplification protocol on target nucleic acid or a portion thereof, in response to a directed to a particular disease) upon user selection of the user request received by the input module. The amplification graphical element. For example, a graphical element may 40 module may be capable of executing any of the methods correspond to Ebola virus and H1N1 virus such that selec described herein and may include any of a fluid handling tion of the graphical element results in an associated com device, one or more thermocyclers, means for receiving one puter processor executing amplification protocols for both or more reaction vessels (e.g., wells of a thermal block of a Ebola virus and H1N1 virus. thermocycler), a detector (e.g., optical detector, spectro In various aspects, the system comprises an input module 45 scopic detector, electrochemical detector) capable of detect that receives a user request to amplify a target nucleic acid ing amplified product, and means for outputting information (e.g., target RNA, target DNA) present in a biological (e.g., raw data, processed data, or any other type of infor sample obtained direct from a subject. Any suitable module mation described herein) regarding the presence and/or capable of accepting such a user request may be used. The amount of amplified product (e.g., amplified DNA product) input module may comprise, for example, a device that 50 to a recipient. In some cases, the amplification module may comprises one or more processors. Non-limiting examples comprise a device with a computer processor as described of devices that comprise processors (e.g., computer proces elsewhere herein and may also be capable of analyzing raw sors) include a desktop computer, a laptop computer, a tablet data from detection, with the aid of appropriate software. computer (e.g., Apple R iPad, Samsung R Galaxy Tab), a cell Moreover, in some embodiments, the amplification module phone, a Smart phone (e.g., Apple(R) iPhone, Android R. 55 may comprise input electronics necessary to receive instruc enabled phone), a personal digital assistant (PDA), a video tions from the input module and may comprise output game console, a television, a music playback device (e.g., electronics necessary to communicate with the output mod Apple(R) iPod), a video playback device, a pager, and a ule. calculator. Processors may be associated with one or more In some embodiments, one or more steps of providing controllers, calculation units, and/or other units of a com 60 materials to a reaction vessel, amplification of nucleic acids, puter system, or implanted in firmware as desired. If imple detection of amplified product, and outputting information mented in Software, the routines (or programs) may be may be automated by the amplification module. In some stored in any computer readable memory Such as in RAM, embodiments, automation may comprise the use of one or ROM, flash memory, a magnetic disk, a laser disk, or other more fluid handlers and associated software. Several com storage medium. Likewise, this software may be delivered to 65 mercially available fluid handling systems can be utilized to a device via any known delivery method including, for run the automation of Such processes. Non-limiting example, over a communication channel Such as a telephone examples of such fluid handlers include fluid handlers from US 9,546,389 B2 29 30 Perkin-Elmer, Caliper Life Sciences, Tecan, Eppendorf, a device comprising a processor may be included in both the Apricot Design, and Velocity 11. input module and the output module. A user may use the In some embodiments, an amplification module may device to request that a target nucleic acid be amplified and include a real-time detection instrument. Non-limiting may also be used as a means to transmit information examples of such instruments include a real-time PCR regarding amplified product to a recipient. In some cases, a thermocycler, ABI PRISM(R) 7000 Sequence Detection Sys device comprising a processor may be included in all three tem, ABI PRISM(R) 7700 Sequence Detection System, modules, such that the device comprising a processor may Applied Biosystems 7300 Real-Time PCR System, Applied also be used to control, provide instructions to, and receive Biosystems 7500 Real-Time PCR System, Applied Biosys information back from instrumentation (e.g., a thermocycler, tems 7900 HT Fast Real-Time PCR System (all from 10 a detector, a fluid handling device) included in the amplifi Applied Biosystems); LightCyclerTM System (Roche Diag cation module or any other module. nostics GmbH); MX3000PTM Real-Time PCR System, An example system for amplifying a target nucleic acid MX3005PTM Real-Time PCR System, and Mx4000R) Mul according to methods described herein is depicted in FIG. 1. tiplex Quantitative PCR System (Stratagene, La Jolla, The system comprises a computer 101 that may serve as part Calif.); and Smart Cycler System (Cepheid, distributed by 15 of both the input and output modules. A user enters a Fisher Scientific). In some embodiments, an amplification reaction vessel 102 comprising a reaction mixture ready for module may comprise another automated instrument Such nucleic acid amplification into the amplification module as, for example, a COBASR, AmpliPrep/COBASR) Taq 104. The amplification module comprises a thermocycler Man(R) system (Roche Molecular Systems), a TIGRIS DTS 105 and a detector 106. The input module 107 comprises system (Hologic Gen-Probe, San Diego, Calif.), a PAN computer 101 and associated input devices 103 (e.g., key THER system (Hologic Gen-Probe, San Diego, Calif.), a BD board, mouse, etc.) that can receive the user's request to MAX system (Becton Dickinson), a GeneXpert System amplify a target nucleic acid in the reaction mixture. The (Cepheid), a Filmarray(R) (BioFire Diagnostics) system, an input module 107 communicates the user's request to the iCubate system, an IDBOX System (Luminex), an Encom amplification module 104 and nucleic acid amplification passMDXTM (Rheonix) system, a Liat TM Aanlyzer (IQuum) 25 commences in the thermocycler 105. As amplification pro system, a Biocartis Molecular Diagnostic Platform system, ceeds, the detector 106 of the amplification module detects an Enigma(R) ML system (Enigma Diagnostics), a T2DX(R) amplified product. Information (e.g., raw data obtained by system (T2 Biosystems), a Verigene(R) system (NanoSphere), the detector) regarding the amplified product is transmitted a Great Basin’s Diagnostic System, a UnyveroTM System from the detector 106 back to the computer 101, which also (Curetis), a PanNAT system (Micronics), or a SpartanTM RX 30 serves as a component of the output module 108. The system (Spartan BioScience). computer 101 receives the information from the amplifica In various aspects, the system comprises an output mod tion module 104, performs any additional manipulations to ule operatively connected to the amplification module. In the information, and then generates a report containing the Some embodiments the output module may comprise a processed information. Once the report is generated, the device with a processor as described above for the input 35 computer 101 then transmits the report to its end recipient module. The output module may include input devices as 109 over a computer network (e.g., an intranet, the internet) described herein and/or may comprise input electronics for via computer network interface 110, in hard copy format via communication with the amplification module. In some printer 111, or via the electronic display 112 operatively embodiments, the output module may be an electronic linked to computer 101. In some cases, the electronic display display, in Some cases the electronic display comprising a 40 112 UI. In some embodiments, the output module is a commu In one aspect, the disclosure provides a computer-read nication interface operatively coupled to a computer net able medium comprising machine executable code that, work Such as, for example, the internet. In some embodi upon execution by one or more processors, implements a ments, the output module may transmit information to a method according to any of the methods disclosed herein. In recipient at a local or remote location using any suitable 45 another aspect, the disclosure provides a computer readable communication medium, including a computer network, a medium comprising machine executable code that, upon wireless network, a local intranet, or the internet. In some execution by one or more computer processors, implements embodiments, the output module is capable of analyzing a method of amplifying a target ribonucleic acid (RNA) data received from the amplification module. In some cases, present in a biological sample obtained directly from a the output module includes a report generator capable of 50 Subject, the method comprising: (a) providing a reaction generating a report and transmitting the report to a recipient, vessel comprising the biological sample and reagents nec wherein the report contains any information regarding the essary for conducting reverse transcription amplification in amount and/or presence of amplified product as described parallel with deoxyribonucleic acid (DNA) amplification, elsewhere herein. In some embodiments, the output module the reagents comprising (i) a reverse transcriptase, (ii) a may transmit information automatically in response to infor 55 DNA polymerase, and (iii) a primer set for the target RNA, mation received from the amplification module. Such as in to obtain a reaction mixture; and (b) Subjecting the reaction the form of raw data or data analysis performed by software mixture in the reaction vessel to multiple cycles of a primer included in the amplification module. Alternatively, the extension reaction to generate amplified DNA product that is output module may transmit information after receiving indicative of the presence of the target RNA, each cycle instructions from a user. Information transmitted by the 60 comprising (i) incubating the reaction mixture at a denatur output module may be viewed electronically or printed from ing temperature for a denaturing duration that is less than or a printer. equal to 60 seconds, followed by (ii) incubating the reaction One or more of the input module, amplification module, mixture at an elongation temperature for an elongation and output module may be contained within the same device duration that is less than or equal to 60 seconds, thereby or may comprise one or more of the same components. For 65 amplifying the target RNA. example, an amplification module may also comprise an In another aspect, the disclosure provides a computer input module, an output module, or both. In other examples, readable medium comprising machine executable code that, US 9,546,389 B2 31 32 upon execution by one or more computer processors, imple readable media may be involved in carrying one or more ments a method of amplifying a target ribonucleic acid sequences of one or more instructions to a processor for (RNA) present in a biological sample obtained directly from execution. a subject, the method comprising: (a) receiving the biologi cal sample that has been obtained from the subject; (b) EXAMPLES providing a reaction vessel comprising the biological sample and reagents necessary for conducting reverse transcription Example 1 amplification and optionally deoxyribonucleic acid (DNA) amplification, the reagents comprising (i) a reverse tran Amplification and Detection of Nucleic Acids in scriptase and (ii) a primer set for the target RNA, to obtain 10 Viral Stock Samples and Biological Samples a reaction mixture; (c) Subjecting the reaction mixture to multiple cycles of a primer extension reaction to yield a Amplification and detection experiments were performed detectable amount of amplified DNA product that is indica to compare results obtained from viral standard samples and biological samples. Biological samples comprising an RNA tive of the presence of the target RNA in the biological 15 sample; (d) detecting the amount of DNA product of (c); and viral pathogen and standard samples of the viral pathogen (e) outputting information regarding the amount of DNA were subject to amplification conditions, such that RNA of product to a recipient, wherein an amount of time for the pathogen was amplified. A set of experiments was completing (a)-(e) is less than or equal to about 30 minutes. conducted for each of the H3N2 and H1N1 (2007) influenza In one aspect, the disclosure provides a computer readable viruses. Each biological sample was obtained directly from medium comprising machine executable code that, upon a Subject via an oropharyngeal Swab. Each viral standard execution by one or more computer processors, implements sample was obtained as a serial dilution of a stock Solution a method of amplifying a target ribonucleic acid (RNA) comprising the virus. The concentrations of H3N2 and present in a biological sample obtained from a subject, the H1N1(2007) were 10 IU/mL. For H5N1 and H1N1 (2007), method comprising: (a) providing a reaction vessel com 25 dilutions of /2, /20, /200, /2000, and /20000 were subject to prising the biological sample and reagents necessary for amplification. In each experimental set, a negative control conducting nucleic acid amplification, the reagents compris (e.g., a sample comprising no viral RNA) was also subject ing (i) a DNA polymerase and optionally a reverse tran to amplification. Scriptase, and (ii) a primer set for the target nucleic acid, to Five microliters of each sample were combined in a 25 L 30 reaction tube with reagents necessary to conduct reverse obtain a reaction mixture; and (b) Subjecting the reaction transcription of the viral RNA and reagents necessary to mixture in the reaction vessel to a plurality of series of complete amplification of the complementary DNA obtained primer extension reactions to generate amplified product from reverse transcription (e.g., parallel nucleic acid ampli from the target nucleic acid, each series comprising two or fication). The reagents necessary to conduct reverse tran more cycles of (i) incubating the reaction mixture under a 35 Scription and DNA amplification were Supplied as a com denaturing condition characterized by a denaturing tempera mercially available pre-mixture (e.g., Qiagen One-Step ture and a denaturing duration, followed by (ii) incubating RT-PCR or One-Step RT-qPCR kit) comprising reverse the reaction mixture under an elongation condition charac transcriptases (e.g., Sensiscript and Omniscript tran terized by an elongation temperature and an elongation scriptases), a DNA Polymerase (e.g., HotStarTaq DNA duration, wherein an individual series differs from at least 40 Polymerase), and dNTPs. Moreover, the reaction tubes also one other individual series of the plurality with respect to the included a TaqMan probe comprising a FAM dye for detec denaturing condition and/or the elongation condition. tion of amplified DNA product. To generate amplified DNA Computer readable medium may take many forms, product, each reaction mixture was incubated according to a including but not limited to, a tangible (or non-transitory) protocol of denaturing and elongation conditions comprising storage medium, a carrier wave medium, or physical trans 45 5 min at 95°C., followed by 20 min at 45° C., followed by mission medium. Non-volatile storage media include, for 2 min at 95°C., and followed by 40 cycles of 5 seconds at example, optical or magnetic disks, such as any of the 95° C. and 30 seconds at 55° C. in a real-time PCR storage devices in any computer(s) or the like. Such as may thermocycler. Detection of amplified product occurred dur be used to implement the calculation steps, processing steps, ing incubations. etc. Volatile storage media include dynamic memory, such as 50 Amplification results for H3N2 are graphically depicted main memory of a computer. Tangible transmission media in FIG. 2 (FIG. 2A corresponds to the various viral standard include coaxial cables; copper wire and fiber optics, includ samples, FIG. 2B corresponds to the biological samples) and ing the wires that comprise a bus within a computer system. amplification results for H1N1 (2007) are graphically Carrier-wave transmission media can take the form of elec depicted in FIG. 3 (FIG. 3A corresponds to the various viral tric or electromagnetic signals, or acoustic or light waves 55 standard samples, FIG. 3B corresponds to the biological Such as those generated during radio frequency (RF) and samples). Recorded fluorescence of the FAM dye is plotted infrared (IR) data communications. Common forms of com against the number of cycles. puter-readable media therefore include for example: a floppy As shown in FIG. 2A, each of the H3N2 viral standard disk, a flexible disk, hard disk, magnetic tape, any other samples showed detectable signal over the negative control, magnetic medium, a CD-ROM, DVD or DVD-ROM, any 60 with Ct values ranging from 18 to 32. As shown in FIG. 2B, other optical medium, punch cards paper tape, any other each of the viral H3N2 biological samples showed detect physical storage medium with patterns of holes, a RAM, a able signal over the negative control, with Ct values ranging PROM and EPROM, a FLASH-EPROM, any other memory from 29-35. chip or cartridge, a carrier wave transporting data or instruc As shown in FIG. 3A and with the exception of the /20000 tions, cables or links transporting Such a carrier wave, or any 65 dilution, each of the H1N1 (2007) viral standard samples other medium from which a computer can read program showed detectable signal over the negative control, with Ct ming code and/or data. Many of these forms of computer values ranging from 24-35. As shown in FIG. 3B, each of the US 9,546,389 B2 33 34 H1N1 (2007) biological samples showed detectable signal Example 3 over the negative control, with Ct values ranging from 28-35. Amplification and Detection of Hepatitis B Virus In general, the data shown in FIG. 2 and FIG. 3 indicate (HBV) in Plasma Samples that the tested viruses could be detected, via amplified DNA 5 product, with good sensitivity, at concentrations as low as 50 Amplification experiments were performed to determine IU/mL and over a 4-log concentration range with cycle the robustness of an amplification method to detect target threshold values of no more than about 40. Moreover, data nucleic acid in a biological sample. Diluted human blood also indicate that detection of viral RNA obtained from a plasma samples comprising hepatitis B virus (HBV) at biological sample obtained from a subject could also be 10 various concentrations (e.g., 50 infective units per milliliter detected in a similar fashion. (IU/mL), 200 IU/mL, 2000 IU/mL, 20000 IU/mL) were each subject to amplification reactions. HBV is a DNA virus Example 2 that replicates via an RNA intermediate. HBV is detectable via direct PCR of the DNA virus. Multiple samples (n=2-4) Amplification and Detection of Viral Nucleic Acid 15 were tested for each concentration in additional to multiple in Different Buffer Systems samples of a negative control (e.g., plasma not comprising HBV). Amplification and detection experiments were performed 2.5 LL of each sample with reagents necessary to conduct reverse transcription of the RNA and reagents necessary to to compare results obtained from using different buffer o complete amplification of the complementary DNA obtained systems for amplification. A set of experiments was con from reverse transcription (e.g., parallel nucleic acid ampli ducted for two different buffer systems, S1 and S2. The S1 fication) in a 50 uL reaction tube to obtain a reaction buffer included a Zwitterionic buffering agent and BSA, and mixture. The reagents necessary to conduct reverse tran the S2 buffer included Zwitterionic buffering agent and Scription and DNA amplification were Supplied as a com Sodium hydroxide. Experiments for each buffer were com- 25 mercially available pre-mixture (e.g., Qiagen One-Step RT pleted using a set of H5N1 influenza virus standard samples PCR or One-Step RT-qPCR kit) comprising reverse obtained as serial dilutions of a stock Solution comprising transcriptases (e.g., Sensiscript and Omniscript tran the virus. The concentration H5N1 was 10 IU/mL. Dilu scriptases), a DNA Polymerase (e.g., HotStarTaq DNA tions of /2, /20, /200, /2000, /20000, /200000, and a negative Polymerase), and dNTPs. Moreover, the mixture also control were Subject to amplification. 30 included a TaqMan probe comprising a FAM dye for detec Five microliters of each sample were combined in a 25uIL tion of amplified DNA product. Also, the reaction mixture reaction tube with reagents necessary to conduct reverse included a Zwitterionic buffering agent and a uracil-DNA transcription of the viral RNA and reagents necessary to glycosylase (UNG) enzyme to prevent inhibitory effects of complete amplification of the complementary DNA obtained amplification inhibitors found in plasma. Each reaction from reverse transcription (e.g., parallel nucleic acid ampli 35 mixture was incubated according to a protocol of denaturing fication). The reagents necessary to conduct reverse tran and elongation conditions comprising 1 min at 94° C. scription and DNA amplification included reverse tran followed by 10 min at 50° C., followed by 2 min at 94° C., scriptases, a DNA polymerase, dNTPs, and the appropriate and followed by 50 cycles of 5 seconds at 94° C. and 35 S1 or S2 buffer. Moreover, the reaction tubes also included a seconds at 58° C. in a real-time PCR thermocyler. Detection a TaqMan probe comprising a FAM dye for detection of of amplified product occurred during incubations. amplified DNA product. To generate amplified DNA prod Amplification results are graphically depicted in FIG. 5 and determined Ct values tabulated in Table 1. Recorded uct, each reaction mixture incubated according to a protocol relative fluorescence units (RFU) of the FAM dye is plotted of denaturing and elongation conditions comprising 5 min at against the number of cycles in FIG. 5. As shown in FIG. 5 95°C., followed by 20 min at 45° C., followed by 2 min at 45 and Table 1, HBV could be detected at each concentration 95°C., and followed by 40 cycles of 5 seconds at 95°C. and tested with cycle threshold values ranging from 28.99 to 30 seconds at 55° C. in a real-time PCR thermocycler. 39.39. In general, higher concentration samples corre Detection of amplified product occurred during incubations. sponded to lower cycle threshold values. Amplification results for buffer system S1 are graphically In general, the data shown in FIG. 5 and Table 1 indicate depicted in FIG. 4A and amplification results for buffer 50 HBV could be detected, via amplified DNA product, with system S2 were graphically depicted in FIG. 4B. Recorded good sensitivity, at concentrations as low as 50 IU/mL (the fluorescence of the FAM dye is plotted against the number lowest tested) with cycle threshold values of no more than of cycles. about 40. While the highest concentration tested (20000 As shown in FIG. 4A each of the viral standard samples IU/mL) was 400 times more concentrated than the lowest amplified in buffer system S1 showed detectable signal over 55 concentration tested (50 IU/mL), cycle threshold values the negative control, with Ct values ranging from 25 to 36. were only about 25% higher for lower concentrations, As shown in FIG. 4B, each of the viral standard samples indicating that the amplification scheme was generally amplified in buffer S2 showed detectable signal over the robust. negative control, with Ct values ranging from 25-35. 60 In general, the data shown in FIG. 4 indicate that the TABLE 1. tested virus could be detected, via amplified DNA product, Ct Results from Experiments in Example 3 with good sensitivity, at concentrations as low as 50 IU/mL and over a S-log concentration range with cycle threshold Sample # IU/mL, Ct values of no more than about 40. Moreover, data also 65 1 2OOO 33.09 indicate that similar amplification results can be obtained 2 50 39.39 with different buffer systems. US 9,546,389 B2 35 36 TABLE 1-continued incubated according to a protocol of denaturing and elon gation conditions comprising 1 min at 95°C., followed by Ct Results from Experiments in Example 3 10 minutes at 55° C., followed by 2 minutes at 95° C., Sample # IU/mL, Ct followed by 40 cycles of 5 seconds at 95°C. and 30 seconds at 55° C. in a real-time PCR thermocycler. Detection of 3 2.OOE-04 29 4 2OOO 32.97 amplified product occurred during incubations. These reac 5 2OO 35.51 tion mixtures are referred to as PTC-1 mixtures. 6 2OOO 33.07 For the fourth reaction mixture for each pathogenic spe 7 2.OOE-04 30.03 cies, the pathogenic species was not pre-heated prior to 8 2OO 35.78 10 9 50 37.91 being added to the reaction mixture. These reaction mixtures 10 2.OOE-04 29.37 were subjected to a protocol comprising a plurality of series 11 2OO 35.73 of amplification reactions, with each series comprising mul 12 2.OOE-04 28.99 tiple cycles of denaturing and elongation conditions. Reac 15 tion mixtures were incubated according to Such a protocol comprising 1 minute at 95°C., followed by 10 cycles of Example 4 Series 1 (95° C. for 5 seconds, 20 seconds of 60-50° C., stepping down 1° C./cycle, and 60° C. for 10 seconds), Pre-Heating a Biological Sample Prior to followed by 2 minutes 95°C. for 2 minutes, followed by 40 Amplification of Nucleic Acid in the Biological cycles of Series 2 (95° C. for 5 seconds, 55° C. for 30 Sample and Series of Amplification Reactions seconds) in a real-time PCR thermocycler. Series 1 and Series 2 differ in their elongation temperature and elongation Amplification experiments were conducted to determine duration. Detection of amplified product occurred during the effect of pre-heating a biological sample on detection incubations. These reaction mixtures are referred to as sensitivity and also to determine the effect of using multiple 25 PTC-2 mixtures. series of amplification reactions on detection sensitivity. Twenty 25ull reaction mixtures were prepared, with each TABLE 2 reaction mixture comprising 1 of a pathogenic species, reagents necessary to complete appropriate nucleic acid Experimental Conditions of Example 4 amplification reactions (e.g., reverse transcription and DNA 30 Reaction amplification for RNA species, and DNA amplification for Mixture DNA species), and a TaqMan probe comprising a FAM dye. Type Protocol Four of the reaction mixtures contained H1N1 (2007) (i.e., PH-1 95° C. 10 minute preheating on pathogenic species before an RNA virus four of the reaction mixtures contained H3N2 adding to the reaction mixture, then 95° C. for 2 minutes, 35 (95° C. for 5 seconds, 55° C. for 30 (i.e., an RNA virus), four of the reaction mixtures contained seconds) x 40 cycles H1N1 (2009), four of the reaction mixtures contained tuber PH-2 50° C. 30 minute preheating on pathogenic before adding culosis (TB) (i.e., a bacterial sample), and four of the to the reaction mixture, then 95°C. for 2 minutes, (95° C. for reaction mixtures contained Aleutian disease virus (ADV) 5 seconds, 55° C. for 30 seconds) x 40 cycles (i.e., a DNA virus). H1N1(2007), H1N1 (2009), H3N2, and 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 ADV pathogenic species were from oropharyngeal Swabs 40 seconds) x 40 cycles obtained from subjects. TB was obtained from a bacterium PTC-2 95° C. for 1 minute, (95° C. for 5 seconds, 60-50° C., stock. stepping down 1 C.fcycle, for 20 seconds, 60° C. for 10 Various combinations of pre-heating and amplification seconds) x 10 cycles, then 95° C. for 2 minutes, (95° C. protocols were utilized and are summarized in Table 2. For for 5 seconds, 55° C. for 30 seconds) x 40 cycles the first reaction mixture for each pathogenic species, the 45 pathogenic species was pre-heated 10 min at 95°C. prior to Results from each pathogenic species are graphically being added to the reaction mixture. After addition of the depicted in FIG. 6 (H1N1 (2007), FIG. 7 (H3N2), FIG. 8 pathogenic species to the reaction mixture, the reaction (H1N1 (2009)), FIG. 9 (TB), and FIG. 10 (ADV). Item A in mixture was incubated according to a protocol of denaturing each of FIGS. 6-10 represents results obtained for reaction and elongation conditions comprising 2 minutes at 95° C. 50 mixtures PH-1 and PH-2, whereas Item B in each of FIGS. followed by 40 cycles of 5 seconds at 95°C. and 30 seconds 6-10 represents results obtained for reaction mixture PTC-1 at 55° C. in a real-time PCR thermocycler. Detection of and PTC-2. Ct values determined for each experiments are amplified product occurred during incubations. These reac summarized in Table 3. Ct values could not be determined tion mixtures are referred to as PH-1 mixtures. for PH-1 and PH-2 ADV reaction mixtures, commensurate For the second reaction mixture for each pathogenic 55 with the data shown in FIG. 10A. species, the pathogenic species was pre-heated 30 min at 50° According to data shown in Table 3, Ct values between C. prior to being added to the reaction mixture. After were fairly similar between PH-1 and PH-2 reaction mix addition of the pathogenic species to the reaction mixture, tures, indicating that a pathogenic species (or biological the reaction mixture was incubated according to a protocol sample comprising a pathogenic species) could be pre of denaturing and elongation conditions comprising 2 min 60 heated at a range of conditions to obtain similar detection utes at 95° C. followed by 40 cycles of 5 seconds at 95°C. sensitivity. Moreover, PTC-1 reaction mixtures had Ct val and 30 seconds at 55° C. in a real-time PCR thermocycler. ues similar to those determined for PH-1 and PH-2 reaction Detection of amplified product occurred during incubations. mixtures. PTC-1 and PH-1/PH-2 protocols were similar, These reaction mixtures are referred to as PH-2 mixtures. except that PTC-1 did not include a pre-heating step. Thus, For the third reaction mixture for each pathogenic species, 65 a comparison of PTC-1 data with PH-1/PH-2 data indicates the pathogenic species was not pre-heated prior to being that pre-heating of a pathogenic species prior to providing it added to the reaction mixture. These reaction mixtures to a reaction mixture may not be necessary for obtaining US 9,546,389 B2 37 38 results with good sensitivity. However, in Some cases with TABLE 4 TB and ADV samples, pre-heating can be even worse than Results from H3N2 and ADV Multiplexing without pre-heating. Experiment in Example 5 However, for all pathogenic species tested, PTC-2 Ct values were lower than any of PH-1, PH-2, or PTC-1. A Type Sample Ct comparison of PTC-1 and PTC-2 data indicate that subject RNA virus H3N2 26.03 ing reaction mixtures to a multiple series of amplification DNA virus ADV 3O.S RNA & DNA H3N2 & ADV 26(H3N2) & 30(ADV) reactions, with each series comprising multiple cycles of virus denaturing and elongation conditions, may improve detec 10 tion sensitivity. In another experiment to assess the multiplexing capa TABLE 3 bilities of an amplification protocol, three reaction mixtures, each comprising one of H3N2, TB, or a mixture of H3N2 Ct Results from Experiments in Example 4 and TB were incubated according to an amplification pro 15 tocol comprising 2 min at 95°C., followed by 40 cycles of PH-1 PH-2 PTC-1 PTC-2 5 seconds at 95° C. and 30 seconds at 55° C. in a real time Type Sample (Ct) (Ct) (Ct) (Ct) PCR thermocycler. Detection of amplified product occurred RNA virus H1N1 (2007) 27 30 28 22 during incubations. RNA virus H3N2 34 33 32 23 RNA virus H1N1 (2009) 32 32 32 24 Results of the experiments are graphically depicted in DNA bacteria TB 34 32 26 2O FIG. 12 and shown below in Table 5. As shown in FIG. 12, DNA virus ADV 36 30 both H3N2 and TB could be detected similarly when in combination or in the absence of the other. 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 Example 5 25 recorded for the TB reaction mixture. When both of H3N2 and TB were combined into a single reaction mixture, Ct values of 29 (H3N2) and 30 (TB) were obtained. Ct values Multiplexing Samples were similar for the combined reaction mixture when com pared to the single component reaction mixtures. Results Amplification and detection experiments were performed 30 indicate that multiplexing is achievable with good sensitiv to benchmark various amplification protocols and to deter ity and that both RNA and DNA species can be detected in mine whether multiplexing could be achieved. Biological a multiplexing scheme. samples comprising RNA (e.g., H1N1 (2007), H1N1 (2009), TABLE 5 H3N2) or DNA (e.g., ADV, human bocavirus (HBoV) viral 35 pathogens or DNA bacterial pathogens (e.g., TB) were Results from H3N2 and TB Multiplexing Experiment in Example 5 Subject to various amplification conditions. Each biological sample was obtained directly from a subject via an oropha Type Sample Ct ryngeal Swab, except for TB samples which were from a RNA virus H3N2 32 bacterium stock. One microliter of each sample was com 40 DNA virus TB 32 bined in a 25 Jull reaction tube with reagents necessary to RNA & DNA H3N2 & TB 29(H3N2) & 30(TB) conduct nucleic acid amplification and to detect amplified virus product as described herein to obtain a reaction mixture. To assess the multiplexing capabilities of an amplification 45 Example 6 protocol, three reaction mixtures, each comprising one of H3N2, ADV, or a mixture of H3N2 and ADV were incubated Benchmarking Multiple Series of Amplification according to an amplification protocol comprising 2 min at Reactions 94° C., 20 min at 45° C., 1 min at 94° C., followed by 50 cycles of 5 seconds at 94° C. and 35 seconds at 55° C. in a 50 Amplification and detection experiments were performed real time PCR thermocycler. Detection of amplified product to benchmark various amplification protocols comprising occurred during incubations. multiple series of amplification reactions. Biological Results of the experiments are graphically depicted in samples comprising RNA (e.g., H1N1 (2007), H1N1 (2009), FIG. 11 and shown below in Table 4. As shown in FIG. 11, H3N2) or DNA (e.g., ADV, human bocavirus (HBoV) viral 55 pathogens or DNA bacterial pathogens (e.g., TB) were both H3N2 and TB could be detected similarly when in Subject to various amplification conditions. Each biological combination or in the absence of the other. In the absence of sample was obtained directly from a subject via an oropha ADV, a Ct value of 26.03 was recorded for the H3N2 ryngeal swab, except for TB samples which were from a reaction mixture and in the absence of H3N2, a Ct value of 30.5 was recorded for the ADV reaction mixture. When both bacterium stock. One microliter of each sample was com 60 bined in a 25 Jull reaction tube with reagents necessary to of H3N2 and ADV were combined into a single reaction conduct nucleic acid amplification and to detect amplified mixture, Ct values of 26 (H3N2) and 30 (ADV) were product as described herein to obtain a reaction mixture. obtained. Ct values were nearly identical for the combined In one set of experiments, amplification mixtures were reaction mixture when compared to the single component Subjected to an amplification protocol comprising two series reaction mixtures. Results indicate that multiplexing is 65 of amplification reactions, each series comprising different achievable with good sensitivity and that both RNA and denaturation and elongation conditions. Six reaction mix DNA species can be detected. tures (two comprising H3N2, two comprising ADV, two US 9,546,389 B2 39 40 comprising HBoV) were incubated according to an ampli multiple series of amplification reactions. Biological fication protocol comprising 1 second at 94°C., followed by samples comprising H3N2 were subject to various amplifi 11 cycles of Series 1 (1 second at 94° C. and 10 seconds at cation conditions. Each biological sample was obtained 45° C.), followed by 1 minute at 95° C., followed by 40 directly from a subject via an oropharyngeal Swab. One cycles of Series 2 (5 seconds at 95°C. and 30 seconds at 55° 5 microliter of each sample was combined in a 25 LIL reaction C.) in a real time PCR thermocycler. Detection of amplified tube with reagents necessary to conduct nucleic acid ampli product occurred during incubations. fication and to detect amplified product as described herein Results of the experiments are shown below in Table 6. As to obtain a reaction mixture. shown in Table 6, determined Ct values ranged from 8.35 to Amplification mixtures were Subjected to amplification 23. Results indicate that protocols comprising multiple protocols, Some comprising one of three different first series series of amplification reactions can be useful in achieving 10 of amplification reactions and the same second series, the good sensitivity. Moreover, results also indicate that both three first series comprising different denaturation and elon RNA and DNA species can be detected with protocols gation conditions than the second series. Each of first series comprising multiple series of amplification reactions. and the second series comprised multiple cycles. Another experiment was conducted without a first series, comprising TABLE 6 15 only the second series. In a real time PCR thermocycler, each of four reaction mixtures comprising H3N2 were Results from H3N2. ADV, and HBOV Experiment in Example 6 incubated according to one of the amplification protocols Type Sample Ct shown below in Table 8: RNA virus H3N2-1 17 TABLE 8 RNA virus H3N2-2 2O DNA virus ADV-1 18.8 Experimental Protocols in Example 7 DNA virus ADV-2 23 DNA virus HBOV-1 8.35 Reaction DNA virus HBOV-2 1837 Mixture Protocol 25 1 94° C. for 1 minute, (Series 1A -- 94° C. for 1 second, In another set of experiments, amplification mixtures 45° C. for 2 minutes) x 5 cycles, 95° C. for 1 minute, were Subjected to an amplification protocol comprising three (Series 2 -- 95° C. for 5 seconds, 55° C. for series of amplification reactions, the series differing from the 30 seconds) x 50 cycles others with respect to their denaturation and/or elongation 2 80° C. for 2 minutes, (Series 1B -- 80° C. for 1 second, 30 45° C. for 2 minutes) x 5 cycles, 95° C. for 1 minute, condition. Five reaction mixtures (one comprising sh1N1 (Series 2 -- 95° C. for 5 seconds, 55° C. for (2007), one comprising H3N2, one comprising pH1N1 30 seconds) x 50 cycles (2009), one comprising ADV, and one comprising TB) were 3 80° C. for 2 minutes, 45° C. for 30 minutes, 95° C. for incubated according to an amplification protocol comprising 1 minute, (Series 2 -- 95° C. for 5 seconds, 55° C. for 1 minute at 94° C., followed by 5 cycles of Series 1 (5 30 seconds) x 50 cycles 35 4 94° C. for 1 second, (Series 1C -- 94° C. for 1 second, seconds at 94° C. and 30 seconds at 60-50° C. stepped down 45° C. for 30 seconds) x 50 cycles, (Series 2 -- 95° C. 1°C./cycle), followed 5 cycles of Series 2 (5 seconds at 94° for 5 seconds, 55° C. for 30 seconds) x 50 cycles C. and 30 seconds at 50° C.), followed by 2 minutes at 95° C., followed by 40 cycles of Series 3 (5 seconds at 95° C. and 30 seconds at 55° C.) in a real time PCR thermocycler. Results of the experiments are graphically depicted in Detection of amplified product occurred during incubations. 40 FIG. 13 and tabulated below in Table 9. As shown in FIG. Results of the experiments are shown below in Table 7. As 13, reaction mixture 3 had the highest Ct value (28.59). The shown in Table 7, determined Ct values ranged from 20 to others comprising multiple series had lower values ranging 30. Results indicate that protocols comprising multiple from 8.5 to 26.5. Results indicate that protocols comprising series of amplification reactions can be useful in achieving multiple series of amplification reactions can be useful in good sensitivity. Moreover, results also indicate that both 45 achieving good sensitivity. Moreover, results also indicate RNA and DNA species can be detected with protocols that protocols comprising multiple series of amplification comprising multiple series of amplification reactions. reactions may achieve better sensitivity when compared to protocols with only a single series. TABLE 7 50 TABLE 9 Results from sFH1N1 (2007), H3N2, pH1N1 (2009), ADV, and TB Experiment in Example 6 Experimental Results of Example 7 Type Sample Ct Reaction Mixture Ct RNA virus sH1N1 (2007) 22 RNA virus H3N2 23 55 1 22.97 RNA virus pH1N1 (2009) 24 2 26.5 DNA virus ADV 30 3 28.59 DNA bacteria TB 2O 4 8.5

60 Example 7 Example 8 Benchmarking Multiple Series of Amplification Benchmarking Multiple Series of Amplification Reactions Reactions 65 Amplification and detection experiments were performed Amplification and detection experiments were performed to benchmark various amplification protocols comprising to benchmark various amplification protocols comprising US 9,546,389 B2 41 42 multiple series of amplification reactions. Biological reactions can be useful in achieving good sensitivity. More samples comprising H3N2 were subject to various amplifi over, results also indicate that protocols comprising multiple cation conditions. Each biological sample was obtained series of amplification reactions may achieve better sensi directly from a subject via an oropharyngeal Swab. One tivity when compared to protocols with only a single series. microliter of each sample was combined in a 25 LIL reaction In some cases, multiple series of amplification reactions may tube with reagents necessary to conduct nucleic acid ampli be necessary for producing detectable quantities of amplified fication and to detect amplified product as described herein product. to obtain a reaction mixture. Amplification mixtures were Subjected to amplification TABLE 11 protocols, some comprising one of six first series of ampli 10 fication reactions and the same second series, the six first Experimental Results of Example 8 series comprising different denaturation and elongation con Reaction ditions than the second series. Another six experiments were Mixture Ct conducted without a first series. In a real time PCR thermo 15 1 26.03 cycler, each of twelve reaction mixtures comprising H3N2 2 were incubated according to one of the amplification pro 3 tocols shown below in Table 10: 4 5 6 27.28 TABLE 10 7 21.64 8 1956 Experimental Protocols in Example 8 9 17.2 10 14.53 Reaction 11 19.2 Mixture Protoco 12 1 95° C. for 3 minutes, 45° C. for 5 minutes, 95° C. 25 or 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. Example 9 or 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. Comparing Results with Purified and Unpurified or 1 minute, (Series 2 -- 95° C. for 5 seconds, 30 Sample 55° C. for 30 seconds) x 40 cycles 4. 95° C. for 10 minutes, 45° C. for 20 minutes, 95°C. Amplification and detection experiments were performed or 1 minute, (Series 2 -- 95° C. for 5 seconds, 55° C. for 30 seconds) x 40 cycles to compare results obtained with purified and unpurified 5 95° C. for 10 minutes, 45° C. for 3 minutes, 95° C. samples. Purified and un-purified biological samples com or 1 minute, (Series 2 -- 95° C. for 5 seconds, 35 prising H1N1 were subject an amplification protocol. Each 55° C. for 30 seconds) x 40 cycles 6 45° C. for 20 minutes, 95°C. for 1 minute, biological sample was obtained directly from a subject via (Series 2 - 95° C. for 5 seconds, 55° C. for an oropharyngeal Swab. One microliter of each sample was 30 seconds) x 40 cycles combined in a 25 Jull reaction tube with reagents necessary 7 94° C. for 2 minutes, (Series 1A -- 94° C. for 1 second, 45° C. for 10 seconds) x 10 cycles, 40 to conduct nucleic acid amplification and to detect amplified 95° C. for 1 minute, (Series 2 -- 95° C. for 5 seconds, product as described herein to obtain a reaction mixture. 55° C. for 30 seconds) x 50 cycles Three reaction mixtures were generated, with two of the 8 94° C. for 10 seconds, (Series 1B -- 94° C. for 1 second, 45° C. for 10 seconds) x 10 cycles, reaction mixtures comprising sample purified by one of 95° C. for 1 minute, (Series 2 -- 95° C. for 5 seconds, column purification or magnetic purification. The third reac 55° C. for 30 seconds) x 50 cycles 45 tion mixture comprised unpurified sample. 9 94° C. for 2 minutes, (Series 1C -- 94° C. for The reaction mixtures were incubated according to an 10 seconds, 45° C. for 20 seconds) x 10 cycles, 95°C. for 1 minute, (Series 2 - 95°C. for amplification protocol comprising 2 minutes at 94° C. 20 5 seconds, 55° C. for 30 seconds) x 50 cycles minutes at 45°C., 1 minute at 94°C., followed by 50 cycles 10 94° C. for 10 seconds, (Series 1D -- 94° C. for of 5 seconds at 94° C. and 35 seconds at 55° C. in a real time 10 seconds, 45° C. for 20 seconds) x 10 PCR thermocycler. Detection of amplified product occurred cycles, 95°C. for 1 minute, (Series 2 - 95°C. for 50 5 seconds, 55° C. for 30 seconds) x 50 cycles during incubations. 11 94° C. for 2 minutes, (Series 1E -- 94° C. for Results of the experiments are graphically depicted in 30 seconds, 45° C. for 60 seconds) x 10 FIG. 14 and shown below in Table 12. As shown in Table 12, cycles, 95°C. for 1 minute, (Series 2 - 95°C. for determined Ct values ranged from 27 to 31 and were similar 5 seconds, 55° C. for 30 seconds) x 50 cycles 12 94° C. for 10 seconds, (Series 1F -- 94° C. for 55 between unpurified sample and sample purified by various 30 seconds, 45° C. for 60 seconds) x 10 means. Results indicate that purification of Sample may be cycles, 95°C. for 1 minute, (Series 2 - 95°C. for not necessary to achieve similar detection sensitivity. 5 seconds, 55° C. for 30 seconds) x 50 cycles TABLE 12 Results of the experiments are tabulated below in Table 60 11. Ct values ranged from 14.53 to 27.28, with reaction Experimental Results of Example 9 mixtures 2-5 having no detected product. Generally speak Sample Type Ct ing, reaction mixtures not subjected to multiple series of Column Purification 31 amplification reactions had either no detectable product or Magnetic Beads Purification 27 had higher Ct values than reaction mixtures subjected to 65 Unpurified 28 multiple series of amplification reaction. Results indicate that protocols comprising multiple series of amplification US 9,546,389 B2 43 44 Example 10 is added to the second run reaction mixture. Amplification results for H1N1 are graphically depicted in FIG. 17. The Analysis of Whole Blood and Saliva Samples figure shows recorded relative fluorescence units (RFU) as a function of cycle number. Plots for each of the eight Amplification and detection experiments were performed samples (1-8) have been indicated in the figure. Samples on H3N2 virus-containing blood and saliva samples. Four with detectable signals have Ct values indicated in Table 13. different samples were tested. Two samples comprising either of the whole blood or saliva samples and two samples TABLE 13 comprising a 10-fold dilution (in PBS) of either of the whole blood or saliva samples. Each of the four samples was 10 Experimental Results of Example 11 combined with reagents necessary to conduct reverse tran Scription of the viral RNA and reagents necessary to com i 1 2 3 4 5 6 7 8 plete amplification of the complementary DNA obtained Sample /10 /100 /1000 O /10 /100 /1000 O from reverse transcription. The reagents necessary to con dilution Ct 18 21 27 — 11 17 24 duct reverse transcription and DNA amplification were 15 Cycles 10 cycles 15 Cycles Supplied as a commercially available pre-mixture (e.g., of the Takara One-Step RT-PCR or One-Step RT-qPCR kit) com first run prising reverse transcriptases (e.g., Sensiscript and Omnis cript transcriptases), a DNA Polymerase (e.g., HotStarTaq DNA Polymerase), and dNTPs. Moreover, the reaction tubes also included a TaqMan probe comprising a FAM dye for Example 12 detection of amplified DNA product. To generate amplified DNA product, each reaction mixture was incubated accord ing to a protocol of denaturing and elongation conditions Amplification and Detection of Ebola Recombinant comprising 20 minutes at 45° C., followed by 2 minutes at 25 Plasmid 94°C., followed by 42 cycles of 5 seconds at 94° C. and 35 seconds at 55° C. in a real-time PCR thermocycler. Detec Amplification and detection experiments were performed tion of amplified product occurred during incubations. on human whole blood samples comprising various copy Amplification results for H3N2 are graphically depicted numbers of recombinant plasmid corresponding to the Zaire in FIG. 15 (FIG. 15A corresponds to non-diluted blood, FIG. 30 Ebola Virus (Zaire-EBOV). Eight samples were tested. Six 15B corresponds to diluted blood) and FIG. 16 (FIG. 16A of the samples included the recombinant plasmid at a corresponds to non-diluted saliva, FIG. 16B corresponds to particular copy number (250000, 25000, 2500, 250, 25 and diluted saliva). Recorded fluorescence of the FAM dye is 2.5 copies) and two of the samples (one having blood only, plotted against the number of cycles. one having water only) served as control samples. Whole As shown in FIG. 15 and FIG. 16, both non-diluted and 35 diluted blood and saliva reaction mixtures showed detect blood samples were analyzed without sample purification. able signal, with Ct values ranging from 24-33. Thus, the Each sample was combined with reagents necessary for data shown in FIGS. 15 and 16 indicate that non-dilute nucleic acid amplification (e.g., DNA polymerase, dNTPs. biological samples could be analyzed with good sensitivity, primers, co-factors, Suitable buffer, primers, etc.) and a with Ct values of no more than about 40. Moreover, data also 40 reporter agent (e.g., an oligonucleotide probe comprising indicate that, in cases where dilution of Sample is necessary FAM dye) into a reaction mixture. A summary of the various for analysis, amplified product may also be detected in a reaction mixtures by sample number, including copy number similar fashion. In some cases, if the inhibitors from Samples of recombinant plasmid, is shown in Table 14. To generate are too much, dilution may be another way to eliminate the amplified product, each reaction mixture was subjected to inhibition from the sample, for example, whole blood. 45 two series of denaturing and elongation conditions. The two series were as follows: (i) in a first series, 15 cycles of 1 Example 11 second at 95°C. and 1 second at 45° C., followed by 1 min Nested PCR at 95°C.; and (ii) in a second series, 45 cycles of 5 seconds 50 at 95°C. and 10 seconds at 55° C. During the second series, Amplification and detection experiments are performed signal from the reporter agent was recorded to generate on H1N1 virus-containing samples. Eight samples are amplification curves and obtain Ct values. Amplification tested. The samples each include H1N1 (2007) virus stock. curves for the experiments are graphically depicted in FIG. The samples are each diluted in PBS, at dilutions indicated 18, each labeled by Sample number corresponding to sample in Table 13 below. The concentration of virus stock is 1x10' 55 numbers shown in Table 14. Results depicted in FIG. 18 IU/mL. To generate amplified DNA product, a reaction show recorded relative fluorescence units (RFU) as a func mixture comprising a given sample is incubated according to tion of cycle number. Ct values obtained from the curves a protocol of denaturing and elongation conditions. The shown in FIG. 18 are summarized in Table 15. protocol comprises: (i) in a first run, heating the mixture in As shown in FIG. 18, recombinant plasmid was detected a thermocycler at 94° C. for 1 minute followed by 10 or 15 60 via amplified products for all of the samples that included cycles (as indicated in Table 13 below) of 5 seconds at 94° recombinant plasmid except for Sample 6. Moreover, recom C. and 10 seconds at 57°C.; and (ii) in a second run, heating binant plasmid was not detected in either of the control the mixture in the thermocycler at 94° C. for 1 minute samples (samples 7 and 8). Accordingly, results shown in followed by 35 cycles of 5 seconds at 94° C. and 30 seconds FIG. 18 indicate that, in some cases, a detection sensitivity at 57°C. A 1 uL series dilution sample is added to a Takara 65 of 25 copies of plasmid/rXn can be obtained using multiple One-step qPCR pre-mixture in a 25 uL reaction volume. series of denaturing and elongation conditions and without After the first run for certain cycles, 1 uL from the reaction sample purification. US 9,546,389 B2 45 46 TABLE 1.4 As shown in FIG. 19A and FIG. 19B. pseudovirus was detected, in both duplicate sets, via amplified products for all Experimental reaction mixtures of Example 12 of the samples that included pseudovirus (samples 1-6). plasmid Moreover, pseudovirus was not detected in any of the Sample (copies/rxn) control samples (samples 7 and 8). Accordingly, results 2SOOOO shown in FIG. 19A and FIG. 19B indicate that, in some 2SOOO cases, a detection sensitivity of 25 copies of virus/rXn can be 2SOO obtained using multiple series of denaturing and elongation 250 conditions without sample purification. 25 10 2.5 O (blood only) TABLE 16 0 (water only) Experimental reaction mixtures of Example 13

15 Pseudovirus TABLE 1.5 Sample (copies/rxn) 2SOOOOO Determined Ct values from experiments in Example 12 2SOOOO 2SOOO Sample Copies/rxn Ct 2SOO 1 2SOOOO 26.12 250 2 2SOOO 33.61 25 3 2SOO 37.61 O (blood only) 4 250 40.61 0 (water only) 5 25 42.97 6 2.5 7 O 25 8 O TABLE 17 Determined Ct values from experiments in Example 13 Example 13 Sample Copies/rxn Ct. 1 Ct. 2 30 1 2SOOOOO 8.57 8.44 Amplification and Detection of Ebola Virus 2 2SOOOO 12.09 11.27 3 2SOOO 1S.O3 14.99 Amplification and detection experiments were performed 4 2SOO 1890 18.87 on human whole blood samples comprising various copy 5 250 21.71 21.71 numbers of Zaire Ebola Virus (Zaire-EBOV) pseudovirus. 35 6 25 27.86 39.42 Eight samples were tested in duplicate (duplicate set #1 and 7 O (blood only) duplicate set #2) for a total of sixteen samples. Six of the 8 0 (water only) samples included the pseudovirus at a particular copy num ber (2500000, 250000, 25000, 2500, 250 and 25 copies) and two of the samples (one having blood only, one having water 40 Example 14 only) served as control samples. Whole blood samples were analyzed without sample purification. Amplification and Detection of Ebola Virus Each sample was combined with reagents necessary for reverse transcription and nucleic acid amplification (e.g., Amplification and detection experiments were performed reverse transcriptase, DNA polymerase, dNTPs, co-factors, 45 on human whole blood samples comprising various copy primers, Suitable buffer, etc.) and a reporter agent (e.g., an numbers of Zaire Ebola Virus (Zaire-EBOV) pseudovirus. oligonucleotide probe comprising FAM dye) into a 30 ul Eight samples were tested. Six of the samples included the reaction mixture. A Summary of the reaction mixtures by pseudovirus at a particular copy number (2500000, 250000, sample number, including copy number of pseudovirus, is 25000, 2500, 250 and 25) and two of the samples (one shown in Table 16. To generate amplified product from the 50 having 20000 copies of a pseudovirus positive control, one pseudovirus, each reaction mixture was subjected to two having water only) served as control samples. Whole blood series of denaturing and elongation conditions. The two samples were analyzed without sample purification. series were as follows: (i) in a first series, 15 cycles of 1 Each sample was combined with reagents necessary for second at 95°C. and 1 second at 45° C., followed by 1 min reverse transcription and nucleic acid amplification (e.g., at 95°C.; and (ii) in a second series, 45 cycles of 5 seconds 55 reverse transcriptase, DNA polymerase, primers, dNTPs. at 95°C. and 10 seconds at 55° C. During the second series, co-factors, Suitable buffer, etc.) and a reporter agent (e.g., an signal from the reporter agent was recorded to generate oligonucleotide probe comprising FAM dye) into a 30 uL amplification curves and obtain Ct values. Amplification reaction mixture. A Summary of the various reaction mix curves for the experiments are graphically depicted in FIG. tures by sample number, including pseudovirus copy num 19A (duplicate set #1) and FIG. 19B (duplicate set #2), each 60 ber, is shown in Table 18. To generate amplified product labeled by sample number corresponding to those shown in from the pseudovirus, each reaction mixture was Subjected Table 16. Results depicted in FIG. 19A and FIG. 19B show to two series of denaturing and elongation conditions. The recorded relative fluorescence units (RFU) as a function of two series were as follows: (i) in a first series, 15 cycles of cycle number. Ct values obtained from the curves shown in 1 second at 95°C. and 1 second at 45° C., followed by 1 min FIG. 19A and FIG. 19B are summarized in Table 17, with 65 at 95°C.; and (ii) in a second series, 35 cycles of 5 seconds “Ct 1” corresponding to duplicate set #1 and "Ct 2 corre at 95°C. and 10 seconds at 55° C. During the second series, sponding to duplicate set #2. signal from the reporter agent was recorded to generate US 9,546,389 B2 47 48 amplification curves and obtain Ct values. Amplification number of pseudovirus and reagent system, is shown below curves for the experiments are graphically depicted in FIG. in Table 20. To generate amplified product from the 20, each labeled by sample number corresponding to those pseudovirus, each reaction mixture was subjected to two shown in Table 18. Results depicted in FIG. 20 show series of denaturing and elongation conditions. The two recorded relative fluorescence units (RFU) as a function of 5 series were as follows: (i) in a first series, 15 cycles of 1 cycle number. Ct values obtained from the curves shown in second at 95°C. and 1 second at 45° C., followed by 1 min FIG. 20 are Summarized in Table 19. at 95°C.; and (ii) in a second series, 40 cycles of 5 seconds As shown in FIG. 20, pseudovirus was detected via at 95°C. and 10 seconds at 55° C. During the second series, amplified products for all of the samples that included signal from the reporter agent was recorded to generate pseudovirus (samples 1-6), including the sample including 10 amplification curves and obtain Ct values. Amplification positive control pseudovirus (sample 7). Moreover, curves for the experiments are graphically depicted in FIG. pseudovirus was not detected in the water only control 21, each labeled by sample number corresponding to those sample (sample 8). Accordingly, results shown in FIG. 20 shown in Table 20. Results depicted in FIG. 21 show indicate that, in some cases, a detection sensitivity of 25 15 recorded relative fluorescence units (RFU) as a function of copies of virus/rxin can be obtained using multiple series of cycle number. Ct values obtained from the curves shown in denaturing and elongation conditions without sample puri FIG. 21 are summarized in Table 21. fication. As shown in FIG. 21, pseudovirus was detected via amplified products for all of the samples, including samples TABLE 1.8 having 25 copies/rxn. Accordingly, results shown in FIG. 21 Experimental reaction mixtures of Example 14 indicate that, in some cases, a detection sensitivity of 25 copies of virus/rxin can be obtained using multiple series of Pseudovirus denaturing and elongation conditions, with different reagent Sample (copies/rxn) systems and without sample purification. 2SOOOOO 25 2SOOOO 2SOOO TABLE 20 2SOO 250 Experimental reaction mixtures of Example 15 25 20000 (positive control pseudovirus) 30 Pseudovirus Reagent 0 (water only) Sample (copies/rxn) System

1 250 B-1 2 25 B-1 TABLE 19 3 250 B-2 35 4 25 B-2 Determined Ct values from experiments in Example 14 5 250 B-3 6 25 B-3 Sample Copies/rxn Ct 7 250 B-4 1 2SOOOOO 10.44 8 25 B-4 2 2SOOOO 13.30 40 3 2SOOO 16.14 4 2SOO 1962 5 250 22.92 TABLE 21 6 25 30.00 7 20000 (positive control) 15.94 Determined Ct values from experiments in Example 15 8 0 (water only) 45 Sample Copies/rxn Ct 1 250 20.38 Example 15 2 25 24.82 3 250 20.62 4 25 24.05 Amplification and Detection of Ebola Virus 50 5 250 20.26 6 25 25.09 Amplification and detection experiments were performed 7 250 1986 on human whole blood samples comprising one of two copy 8 25 24.00 numbers (250 copies/rxn or 25 copies/rxn) of Zaire Ebola Virus (Zaire-EBOV) pseudovirus. Each whole blood sample 55 was tested using one of four reagent systems, for a total of eight samples. Each of the reagent systems (B-1, B-2, B-3 Example 16 and B-4) included reagents necessary for reverse transcrip tion and nucleic acid amplification (e.g., reverse tran Real-Time PCR Detection for Zaire Ebola Virus scriptase, DNA polymerase, primers, dNTPs, co-factors, 60 Suitable buffer, etc.) and a reporter agent (e.g., an oligo nucleotide probe comprising FAM dye). Each of the differ A one-step qPCR method of the present disclosure was ent reagent systems contained different concentrations of the used to analyze patient blood serum samples for the Zaire various components in the reagent systems. Each whole Ebola virus. The samples were not purified. The samples blood sample was combined with its appropriate reagent 65 included nine Zaire Ebola virus positive samples and seven system into a 30 uL reaction mixture. A Summary of the Zaire Ebola virus negative samples. A Roche LC96 real-time various reaction mixtures by sample number, including copy PCR system was used. US 9,546,389 B2 49 50 The program employed in this example to analyze the mixtures by Sample number, including dilution, for the samples is shown in Table 22. second set of experiments is shown in Table 25. To generate amplified product from Malaria pathogens, each reaction TABLE 22 mixture was subjected to two series of denaturing and 5 elongation conditions. The two series were as follows: (i) in Thermal cycling program a first series, 13 cycles of 1 second at 95°C. and 1 second Step Term Time Cycle NO. at 45° C., followed by 1 min at 95°C.; and (ii) in a second series, 45 cycles of 5 seconds at 95°C. and 10 seconds at 55° 1 42° C. 1 min 1 cycle C. During the second series, signal from the reporter agent 2 95° C. 5 second 10 cycles 10 45° C. 10 second was recorded to generate amplification curves. Amplifica 3 95° C. 1 min 1 cycle 4 95° C. 5 second 40 cycles tion curves for the first set of experiments are graphically 55o C. 10 second depicted in FIG.22A and amplification curves for the second (Reading) set of experiments are graphically depicted in FIG. 22B. 15 Each curve is labeled by its corresponding sample number in The results of the one-step qPCR method are shown in Tables 24 and 25, respectively. Results depicted in FIG.22A Table 23. The one-step qPCR method testing for the Zaire and FIG. 22B show recorded relative fluorescence units Ebola virus showed 100% consistency as compared to a (RFU) as a function of cycle number. verified reagent and method. As shown in FIG. 22A, Malaria pathogens were detected via amplified products for the two reaction mixtures com TABLE 23 prising whole blood sample (samples 1 and 2) and for the positive control comprising recombinant plasmid (sample Results 3). Moreover, Malaria pathogens were not detected in the Sample Coyote One-Step Verified reagent water only control sample (sample 4). Accordingly, results i QPCR Method (Cd) and method (Cd) consistency 25 shown in FIG. 22A indicate that Malaria pathogens can, in 1 NA NA Yes Some cases, be detected using multiple series of elongation 2 26.53 29.73 Yes and denaturation conditions without sample purification. 3 17.68 1953 Yes 4 NA NA Yes As shown in FIG. 22B, Malaria pathogens were detected 5 NA NA Yes 30 via amplified products for all reaction mixtures containing 6 NA NA Yes whole blood sample (samples 1-6). Moreover, Malaria 7 NA NA Yes 8 21.52 20.98 Yes pathogens were not detected in the water only and blood 9 18.97 1888 Yes only control samples (sample 7 and 8). Accordingly, results 10 24.97 24.44 Yes shown in FIG. 22B indicate that a pathogen(s), including 11 18.92 1891 Yes 35 12 26.32 25.22 Yes Malaria pathogens, can, in Some cases, be detected at 13 20.48 20.85 Yes dilutions of up to 1:400000 using multiple series of dena 14 18.5 20.45 Yes turing and elongation conditions and without sample puri 15 NA NA Yes fication.

40 TABLE 24 Example 17 Experimental reaction mixtures for first Amplification and Detection of Malaria set of experiments in Example 17 Amplification and detection experiments were performed 45 Sample Dilution on a human whole blood sample comprising an unknown 1 1:4 concentration of Malaria pathogens. Two sets of experi 2 1:4 ments were completed. In the first set of experiments, 3 1:2 (plasmid in whole duplicate experiments were completed for a 1:4 dilution (in 50 blood control) 1xPBS) of the human whole blood sample; an experiment 4 None (water only) was completed for a sample comprising whole blood and a plasmid corresponding to Malaria pathogens; and an experi ment was completed for a water only control. In the second TABLE 25 set of experiments, experiments were completed for Samples 55 comprising various dilutions (1:4, 1:40, 1:400, 1:4000, Experimental reaction mixtures for second 1:40000 and 1:400000) of the human whole blood sample in set of eXperiments in Example 17 1xPBS along with blood only and water only control Sample Dilution samples. Whole blood samples were analyzed without 1:4 sample purification. 60 1:40 Each sample was combined with reagents necessary for 1:400 nucleic acid amplification (e.g., DNA polymerase, primers, 1:4000 1:40OOO dNTPs, co-factors, suitable buffer, etc.) and a reporter agent 1:4OOOOO (e.g., an oligonucleotide probe comprising FAM dye) into a O (blood only) 30 uL reaction mixture. A Summary of the reaction mixtures 65 0 (water only) by sample number, including dilution, for the first set of experiments is shown in Table 24. A Summary of the reaction US 9,546,389 B2 51 52 Example 18 As shown in FIG. 23C, virus was detected via amplified products for reaction mixtures containing Dengue virus and Amplification and Detection of Dengue Virus either not diluted (sample 1) or diluted up to 1:1000 (samples 2, 3 and 4). A Ct value, however, was not deter Amplification and detection experiments were performed mined for the 1:1000 reaction mixture. Virus was not on samples obtained from a culture comprising an unknown detected in higher dilutions (sample 5) or in the water only concentration of Dengue virus. Three sets of experiments control sample (sample 6). Accordingly, results shown in were completed. In the first set of experiments, duplicate FIG. 23C indicate that virus can, in some cases, be detected experiments were completed for undiluted culture; an at dilutions of up to 1:1000, where Ct values can be experiment was completed for 1:10 dilution of the culture: 10 generated at dilutions up to 1:100 using multiple series of and an experiment was completed for a water only control. denaturing and elongation conditions and without sample In the second set of experiments, experiments were com purification. pleted for various dilutions (no dilution, 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000) of the culture along with TABLE 26 a water only control sample. In the third set of experiments, 15 experiments were completed for various dilutions (no dilu Experimental reaction mixtures and determined Ct tion, 1:10, 1:100, 1:1000 and 1:10000) of the culture along values for first set of experiments in Example 18 with a water only control sample. Culture samples were analyzed without sample purification. Sample Dilution Ct value 2 LL of each sample was combined with reagents neces 1 Ole 19.32 sary for reverse transcription and nucleic acid amplification 2 Ole 20.40 (e.g., reverse transcriptase, DNA polymerase, primers, 3 1:10 23.23 dNTPs, co-factors, suitable buffer, etc.) and a reporter agent 4 no virus (e.g., an oligonucleotide probe comprising FAM dye) into a (water only) 30LL reaction mixture. A Summary of the reaction mixtures, 25 including dilution, for the first set of experiments is shown in Table 26, for the second set of experiments in Table 27 TABLE 27 and for the third set of experiments in Table 28. To generate Experimental reaction mixtures and determined Ct values amplified product from the virus, each reaction mixture was for second set of experiments in Example 18 Subjected to two series of denaturing and elongation condi 30 tions. The two series were as follows: (i) in a first series, 1 Sample Dilution Ct value min at 42° C., 10 cycles of 5 seconds at 95° C. and 10 1 Ole 20.85 seconds at 45° C., followed by 1 min at 95°C.; and (ii) in 2 1:10 25.14 a second series, 45 cycles of 5 seconds at 95° C. and 10 3 1:100 31.57 35 4 1:1000 seconds at 55° C. During the second series, signal from the 5 1:1OOOO reporter agent was recorded to generate amplification 6 1:1OOOOO curves. Amplification curves for the first set of experiments 7 1:1OOOOOO are graphically depicted in FIG. 23A, amplification curves 8 no virus for the second set of experiments are graphically depicted in (water only) FIG. 23B and amplification curves for the third set of 40 experiments are graphically depicted in FIG. 23C. Each curve is labeled by its corresponding sample number in TABLE 28 Tables 26, 27 and 28 respectively. Results depicted in FIG. 23A, FIG. 23B and FIG. 23C show recorded relative fluo Experimental reaction mixtures and determined Ct rescence units (RFU) as a function of cycle number. Ct 45 values for third set of experiments in Example 18 values obtained from the curves shown in FIG. 23A, FIG. Sample Dilution Ct value 23B and FIG. 23C are shown in Tables 26, 27 and 28 respectively. 1 None 1922 2 1:10 22:43 As shown in FIG. 23A, virus was detected via amplified 2 1:100 26.55 products for the three reaction mixtures comprising virus 50 4 1:1000 (samples 1-3). Moreover, virus was not detected in the water 5 1:1OOOO only control sample (sample 4). Accordingly, results shown 6 no virus in FIG. 23A indicate that Dengue virus can, in Some cases, (water only) be detected using multiple series of elongation and denatur ation conditions. 55 As shown in FIG. 23B, virus was detected via amplified Example 19 products for reaction mixtures containing Dengue virus and either not diluted (sample 1) or diluted up to 1:1000 Detection of Single Nucleotide Polymorphisms (samples 2, 3 and 4). A Ct value, however, was not deter (SNPs) mined for the 1:1000 reaction mixture (sample 4). Virus was 60 not detected in higher dilutions (samples 5, 6 and 7) or in the Amplification and detection experiments were performed water only control sample (sample 8). Accordingly, results on human oropharyngeal Swab or blood samples comprising shown in FIG. 23B indicate that virus can, in some cases, be a particular genotype of cytochrome P450 2C19, detected at dilutions of up to 1:1000, where Ct values can be CYP2C19*2 (having a “GA” genotype) or CYP2C19*3 generated at dilutions up to 1:100 using multiple series of 65 (having a “GG’ genotype). Two sets of experiments were denaturing and elongation conditions and without sample conducted—one set for samples obtained from human oro purification. pharyngeal Swabs and one set for samples obtained from US 9,546,389 B2 53 54 blood. In the first set of experiments, seven different samples using nucleic acid sequencing. Thus, results shown in FIG. obtained from human oropharyngeal Swabs were analyzed 25A and FIG. 25B suggest that, in some cases, SNPs can be without sample purification. In the second set of experi detected via real-time amplification in samples obtained ments, five different blood samples were analyzed without from blood without sample purification. sample purification. Each sample was combined with reagents necessary for TABLE 29 nucleic acid amplification (e.g., DNA polymerase, primers, dNTPs, co-factors, suitable buffer, etc.) and two reporter Determined Ct values and genotypes for oropharyngeal agents (e.g., an oligonucleotide probe comprising FAM dye Swab experiments in Example 19 to detect amplification of nucleic acids, an oligonucleotide 10 Reaction Ct- FAM Ct-Texas Red probe comprising Texas Red dye to detect the “GA geno Mixture Reporter Reporter Genotype type) into a reaction mixture. To generate amplified product, 1 38.70 40.25 GA each reaction mixture was Subjected to a thermocycling 2 38.28 GG protocol that included 5 min at 95°C. followed by 50 cycles 3 3416 GG of 5 seconds at 95° C. and 10 seconds at 49° C. During 15 4 33.18 33.75 GA thermocycling, signals from the reporter agents were 5 35.20 GG recorded to generate amplification curves. Amplification 6 33.08 33.59 GA curves for the first set of experiments (human oropharyngeal 7 36.45 37.01 GA Swabs) are graphically depicted in FIG. 24A (signal corre sponding to the FAM oligonucleotide probe) and FIG. 24B (signal corresponding to the Texas Red oligonucleotide TABLE 30 probe). Amplification curves for the second set of experi Determined Ct values and genotypes ments (blood samples) are graphically depicted in FIG. 25A for blood experiments in Example 19 (signal corresponding to the FAM oligonucleotide probe) and FIG. 25B (signal corresponding to the Texas Red 25 Reaction Ct- FAM Ct-Texas Red oligonucleotide probe). Results depicted in FIG. 24A, FIG. Mixture Reporter Reporter Genotype 24B, FIG. 25A and FIG. 25B show recorded relative fluo 1 38.36 36.24 GA rescence units (RFU) as a function of cycle number. Each 2 39.97 39.67 GA 3 41.25 GG curve is labeled by its corresponding reaction mixture num 4 33.96 GG ber in Table 29 (oropharyngeal Swab experiments) or Table 30 30 (blood experiments). Ct values determined for amplifi 5 35.68 34:12 GA cation curves are also shown in Table 29 or Table 30 along with determined genotype. In amplification curves where signal from Texas Red was observed in FIG. 24B or FIG. Example 20 25B, it was determined that the corresponding reaction 35 mixture had the “GA' genotype. Moreover, in amplification Amplification and Detection of Adenovirus Type 55 curves where signal from Texas Red was not observed in (ADV55) and Adenovirus Type 7 (ADV7) FIG. 24B or FIG. 25B, it was determined that the corre sponding reaction mixture had the “GG’ genotype. Amplification and detection experiments were performed As shown in FIG. 24A, amplified product was observed 40 on Samples obtained from oropharyngeal Swabs comprising for each of the reaction mixtures having sample obtained various copy numbers of adenovirus type 55 (ADV55) or from oropharyngeal Swabs, Suggesting that amplification of unknown concentrations of adenovirus type 7 (ADV7). Two nucleic acids occurs. However, as shown in FIG. 24B, sets of experiments were completed—one set for Samples amplified product was observed for only some of the reac having ADV55 and one set for experiments having ADV7. tion mixtures (reaction mixtures 1, 4, 6 and 7) having sample 45 In the first set of experiments, six different experiments obtained from oropharyngeal Swabs, these reaction mixtures having samples comprising differing copy numbers (1, 10. corresponding to the 'GA' genotype. In the other reaction 100, 1000, 10000, and 100000 copies) of ADV55 were mixtures (reaction mixtures 2, 3 and 5), amplified products completed without sample purification along with an experi were not observed, these reaction mixtures corresponding to ments for a negative control. In the second set of experi the “GG’ genotype. Results shown in FIG. 24A and FIG. 50 ments, eight different experiments having samples compris 24B were validated via amplification and detection experi ing unknown copy number of ADV7 were completed ments using DNA extracted from oral Swab samples (data without sample purification. not shown). Thus, results shown in FIG. 24A and FIG. 24B Each sample was combined with reagents necessary for Suggest that, in some cases, SNPs can be detected via nucleic acid amplification (e.g., DNA polymerase, primers, real-time amplification in Samples obtained from oropha 55 dNTPs, co-factors, suitable buffer, etc.) and a reporter agent ryngeal Swabs without sample purification. (e.g., an oligonucleotide probe comprising FAM dye) into a As shown in FIG. 25A, amplified product was observed reaction mixture. A Summary of the reaction mixtures, for each of the reaction mixtures having sample obtained including ADV55 copy number, for the first set of experi from blood, Suggesting that amplification of nucleic acids ments is shown in Table 31. To generate amplified product occurs. However, as shown in FIG. 25B, amplified product 60 from viruses, each reaction mixture was Subjected to two was observed for only some of the reaction mixtures (reac series of denaturing and elongation conditions. The two tion mixtures 1, 2 and 5) having sample obtained from series were as follows: (i) in a first series, 20 cycles of 1 blood, these reaction mixtures corresponding to the “GA' second at 95°C. and 1 second at 45° C., followed by 1 min genotype. In the other reaction mixtures (reaction mixtures at 95°C.; and (ii) in a second series, 35 cycles of 5 seconds 3 and 4), amplified products were not observed, these 65 at 95°C. and 34 seconds at 60° C. During the second series, reaction mixtures corresponding to the “GG’ genotype. signal from the reporter agent was recorded to generate Results shown in FIG. 25A and FIG. 25B were validated amplification curves and obtain Ct values. Amplification US 9,546,389 B2 55 56 curves for the first set of experiments are graphically Each sample was combined with reagents necessary for depicted in FIG. 26A, each labeled by reaction mixture reverse transcription and nucleic acid amplification (e.g., number corresponding to those shown in Table 31. Ampli reverse transcriptase, DNA polymerase, primers, dNTPs. fication curves for the second set of experiments are graphi co-factors, Suitable buffer, etc.) and a reporter agent (e.g., an cally depicted in FIG. 26B and corresponding Ct values oligonucleotide probe comprising FAM dye) into a reaction shown in Table 32. Amplification curves in FIG. 26B are mixture. A Summary of the reaction mixtures, including labelled as they correspond to reaction mixture number RNA-HCV copy number is shown in Table 33. To generate shown in Table 32. Results depicted in FIG. 26A and FIG. amplified DNA product from viruses, each reaction mixture 26B show recorded relative fluorescence units (RFU) as a was Subjected to two series of denaturing and elongation 10 conditions. The two series were as follows: (i) in a first function of cycle number. series, 20 cycles of 1 second at 95° C. and 1 second at 45° As shown in FIG. 26A, ADV55 was detected via ampli C., followed by 1 min at 95°C.; and (ii) in a second series, fied products for all of the reaction mixtures comprising 55 cycles of 5 seconds at 95° C. and 34 seconds at 60° C. sample containing virus (reaction mixtures 1-6). Moreover, During the second series, signal from the reporter agent was virus was not detected in the negative control reaction 15 recorded to generate amplification curves. Amplification mixture (reaction mixture 7). Accordingly, results shown in curves for the first set of experiments are graphically FIG. 26A indicate that ADV55 virus can, in some cases, be depicted in FIG. 27, each labeled by number corresponding detected using multiple series of elongation and denaturation to reaction mixture numbers shown in Table 33. Results conditions without sample purification and at various levels depicted in FIG. 27 show recorded relative fluorescence of dilution. units (RFU) as a function of cycle number. As shown in FIG. 26B, ADV7 was detected via amplified As shown in FIG. 27, RNA-HCV was detected via products for all of the reaction mixtures. Accordingly, results amplified products for all of the reaction mixtures compris shown in FIG. 26B indicate that ADV7 virus can, in some ing sample containing virus (reaction mixture 1-3). More cases, be detected using multiple series of elongation and over, RNA-HCV was not detected in the negative control denaturation conditions and without sample purification. 25 reaction mixture (reaction mixture 4). Accordingly, results shown in FIG. 27 indicate that RNA-HCV can, in some TABLE 31 cases, be detected using multiple series of elongation and denaturation conditions without sample purification. A Experimental reaction mixtures for ADVSS experiments in Example 20 detection sensitivity of 10 copies/rxin can also be achieved. 30 Reaction ADV55 Copy TABLE 33 Mixture Number RXn Experimental reaction mixtures for 1 1 RNA-HCV experiments in Example 21 2 10 3 100 35 Reaction RNA-HCV Copy 4 1OOO Mixture Number, Rxn 5 1OOOO 6 1OOOOO 1 10 7 O (negative control) 2 100 3 500 40 4 O (negative control) TABLE 32 While preferred embodiments of the present invention Determined Ct values for ADV7 experiments in Example 20 have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided Reaction 45 Mixture Ct Value by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art 1 S.12 without departing from the invention. It should be under 2 7.16 stood that various alternatives to the embodiments of the 3 10.97 4 14.15 invention described herein may be employed in practicing 5 17.58 50 the invention. It is intended that the following claims define 6 20.29 the scope of the invention and that methods and structures 7 22.13 within the scope of these claims and their equivalents be 8 17.66 covered thereby.

55 What is claimed is: Example 21 1. A method of amplifying a target ribonucleic acid (RNA) present in a biological sample obtained from a Amplification and Detection of Armored RNA Subject, comprising: Hepatitis C Virus (RNA-HCV) (a) providing a reaction vessel comprising said biological 60 sample and reagents necessary for conducting reverse Amplification and detection experiments were performed transcription amplification in parallel with deoxyribo on blood plasma samples comprising various copy numbers nucleic acid (DNA) amplification, which biological of armored RNA Hepatitis C Virus (RNA-HCV). Three sample is obtained directly from said Subject and different experiments having samples comprising differing provided in said reaction vessel without further pro copy numbers (10, 100 and 500 copies) of RNA-HCV were 65 cessing, wherein said reagents comprise (i) a reverse completed without sample purification along with an experi transcriptase, (ii) a DNA polymerase, and (iii) a primer ment completed for a negative control. set for said target RNA, to obtain a reaction mixture, US 9,546,389 B2 57 58 and wherein said reverse transcriptase and said DNA cycles of (i) incubating said reaction mixture under a polymerase are separate enzymes; and denaturing condition characterized by a denaturing (b) without first Subjecting said reaction mixture to a temperature and a denaturing duration, followed by (ii) condition Suitable for said reverse transcription ampli incubating said reaction mixture under an elongation fication separate from said DNA amplification, subject condition characterized by an elongation temperature ing said reaction mixture in said reaction vessel to and an elongation duration, wherein an individual multiple cycles of a primer extension reaction to series differs from at least one other individual series of reverse transcribe said target RNA and generate ampli said plurality with respect to said denaturing condition fied DNA product in parallel, wherein presence of said and/or said elongation condition. amplified DNA product is indicative of a presence of 10 14. The method of claim 13, wherein said reagents are for said target RNA, each cycle comprising (i) incubating conducting reverse transcription amplification in parallel said reaction mixture at a denaturing temperature for a with deoxyribonucleic acid amplification. denaturing duration that is less than or equal to 60 15. The method of claim 13, wherein, in (a), said bio seconds, followed by (ii) incubating said reaction mix logical sample has not undergone nucleic acid extraction ture at an elongation temperature for an elongation 15 and/or is not purified. duration that is less than or equal to 60 seconds, thereby 16. The method of claim 13, wherein said reaction mix amplifying said target RNA. ture does not comprise a detergent. 2. The method of claim 1, wherein said reagents further 17. The method of claim 13, further comprising subject comprise a reporter agent that yields a detectable signal ing said target nucleic acid to one or more denaturing indicative of a presence of said amplified DNA product, and conditions prior to (b), wherein said one or more denaturing wherein the intensity of the detectable signal is proportional conditions are selected from the group consisting of a to the amount of said amplified DNA product or target RNA. denaturing temperature profile and a denaturing agent. 3. The method of claim 1, wherein said primer set 18. The method of claim 13, further comprising subject comprises a first primer to generate a strand that is comple ing said target nucleic acid to one or more denaturing mentary to said target RNA. 25 conditions between a first series and a second series of said 4. The method of claim 1, wherein said target RNA is plurality of series of primer extension reactions. pathogenic RNA. 19. The method of claim 13, wherein said individual 5. The method of claim 1, wherein said reaction vessel series differ with respect to at least any one of ramping rate comprises two or more thermal Zones. between denaturing temperature and elongation tempera 6. The method of claim 1, wherein said denaturing tem 30 ture, denaturing temperature, denaturing duration, elonga perature is from about 90° C. to 100° C., and/or wherein said tion temperature and elongation duration. elongation temperature is from about 35° C. to 72° C. 20. The method of claim 13, wherein said plurality of 7. The method of claim 1, wherein, in (a), said biological series of primer extension reactions comprises a first series sample has not undergone RNA extraction and/or is not and a second series, wherein each cycle of said first series purified. 35 comprises (i) incubating said reaction mixture at about 92 8. The method of claim 1, wherein, prior to (b), said C.-95° C. for no more than 30 seconds, followed by (ii) reaction mixture does not comprise a detergent. incubating said reaction mixture at about 35° C.-65 C. for 9. The method of claim 1, wherein said amplifying yields no more than 1 minute, and wherein each cycle of said a detectable amount of DNA product indicative of a pres second series comprises (i) incubating said reaction mixture ence of said target RNA in said biological sample at a cycle 40 at about 92° C.-95° C. for no more than 30 seconds, threshold value (Ct) of less than 40. followed by (ii) incubating said reaction mixture at about 10. The method of claim 1, wherein said amplifying yields 40° C.-60° C. for no more than 1 minute. a detectable amount of DNA product indicative of a pres 21. The method of claim 13, further comprising, prior to ence of said target RNA in said biological sample at a time (b), pre-heating said biological sample at a pre-heating period of 10 minutes or less. 45 temperature from 90° C. to 100° C. for a pre-heating 11. The method of claim 1, wherein said target RNA is duration of no more than 10 minutes. released from said biological sample during one or more 22. The method of claim 13, wherein said target nucleic cycles of said primer extension reaction. acid is a nucleic acid derived from a pathogen. 12. The method of claim 1, wherein the denaturing 23. A system for amplifying a target ribonucleic acid duration is less than or equal to 30 seconds, and/or wherein 50 (RNA) present in a biological sample obtained from a the elongation duration is less than or equal to 30 seconds. Subject, comprising: 13. A method of amplifying a target nucleic acid present (a) an input module that receives a user request to amplify in a biological sample obtained from a Subject, comprising: said target RNA in said biological sample; (a) providing a reaction vessel comprising said biological (b) a reaction vessel comprising said biological sample sample and reagents necessary for conducting nucleic 55 and reagents necessary for conducting reverse tran acid amplification, which biological sample is obtained scription amplification in parallel with deoxyribo directly from said Subject and provided in said reaction nucleic acid (DNA) amplification, which biological vessel without further processing, wherein said sample is obtained directly from said Subject and reagents comprise (i) a deoxyribonucleic acid (DNA) received in said reaction vessel without further pro polymerase and optionally a reverse transcriptase, and 60 cessing, wherein said reagents comprise (i) a reverse (ii) a primer set for said target nucleic acid, to obtain a transcriptase, (ii) a DNA polymerase, and (iii) a primer reaction mixture; and set for said target RNA, and wherein said reverse (b) Subjecting said reaction mixture in said reaction vessel transcriptase and said DNA polymerase are separate to a plurality of series of primer extension reactions to enzymes; generate amplified product that is indicative of a pres 65 (c) an amplification module that, in response to said user ence of said target nucleic acid in said biological request, implements an amplification protocol compris sample, each series comprising two or more than ten ing, without first Subjecting said reaction mixture to a US 9,546,389 B2 59 60 condition suitable for said reverse transcription ampli plurality of series of primer extension reactions to fication separate from said DNA amplification, subject generate amplified product that is indicative of a pres ing said reaction mixture in said reaction vessel to ence of said target nucleic acid in said biological multiple cycles of a primer extension reaction to Sample, which biological sample is obtained directly reverse transcribe said target RNA and generate ampli from said subject and provided in said reaction vessel fied DNA product in parallel, wherein presence of said without further processing, wherein said reagents com amplified DNA product is indicative of a presence of prise (i) a deoxyribonucleic acid (DNA) polymerase said target RNA, each cycle comprising (i) incubating and optionally a reverse transcriptase, and (ii) a primer said reaction mixture at a denaturing temperature for a set for said target nucleic acid, to obtain a reaction denaturing duration that is less than or equal to 60 10 Seconds, followed by (ii) incubating said reaction mix mixture, ture at an elongation temperature for an elongation wherein each series comprises more than ten cycles of (i) duration that is less than or equal to 60 seconds, thereby incubating said reaction mixture under a denaturing amplifying said target RNA; and condition characterized by a denaturing temperature (d) an output module operatively coupled to said ampli 15 and a denaturing duration, followed by (ii) incubating fication module, wherein said output module outputs said reaction mixture under an elongation condition information regarding said target RNA or said DNA characterized by an elongation temperature and an product to a recipient. elongation duration, wherein an individual series dif 24. The system of claim 23, wherein said input module fers from at least one other individual series of said includes a user interface that displays a plurality of graphical plurality with respect to said denaturing condition elements associated with amplification protocols, wherein a and/or said elongation condition. given graphical element is associated with said amplification 26. The system of claim 25, wherein said amplification protocol implemented by said amplification module. protocol further comprises selecting a primer set for said 25. A system for amplifying a target nucleic acid in a target nucleic acid. biological sample obtained from a subject, comprising: 25 27. The system of claim 25, wherein said user interface an electronic display screen comprising a user interface displays a plurality of graphical elements associated with that displays a graphical element that is accessible by a amplification protocols, wherein a given graphical element user to execute an amplification protocol to amplify is associated with said amplification protocol. said target nucleic acid in said biological sample; and 28. The system of claim 27, wherein each of said graphi cal elements is associated with a disease, and wherein said a computer processor coupled to said electronic display 30 amplification protocol is directed to assaying a presence of Screen and programmed to execute said amplification said disease in said subject. protocol upon selection of said graphical element by 29. The system of claim 25, wherein said biological said user, which amplification protocol comprises: sample has not undergone RNA extraction and/or is not Subjecting a reaction mixture in a reaction vessel and purified. comprising said biological sample and reagents neces 35 sary for conducting nucleic acid amplification to a K CK CK ck ck