Compositions, methods and kits for nucleic acids synthesis and amplification

Abstract

The present disclosure is directed to compositions, methods and kits useful for the synthesis of nucleic acid molecules. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in an one-step qPCR or RT-qPCR procedure comprising a reverse transcriptase, a DNA polymerase, at least one dNTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

Description

COMPOSITIONS, METHODS AND KITS FOR NUCLEIC ACIDS SYNTHESISAND AMPLIFICATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 524,605, filed June 30, 2023, which is herein incorporated by reference in its entirety.FIELD

[0002] The disclosure relates to compositions, methods and kits for the synthesis of nucleic acids. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in a real-time / quantitative polymerase chain reaction (qPCR) procedure, such as, for example, a one-step qPCR or a one-step reverse transcription qPCR (RT-qPCR) procedure, including one or more agents used to increase stability.BACKGROUND

[0003] The transcription, amplification, detection and analysis of nucleic acids are some of the most important procedures in modem molecular biology. The application of such procedures for nucleic acid analysis is especially important in the investigation of gene expression, diagnosis of infectious agents or genetic diseases, the generation of complementary deoxyribonucleic acid (cDNA), and analysis of retroviruses, to name but a few applications. The reverse transcription of ribonucleic acid (RNA), followed by polymerase chain reaction (PCR) cDNA amplification, commonly referred to as RT-PCR, has become widely used for the detection and quantification of nucleic acid targets and is particularly important for gene expression analysis.

[0004] Quantitative or “real-time” reverse transcription polymerase chain reaction (RT-qPCR) is a version of PCR that detects and quantifies RNA. Total RNA or messenger RNA (mRNA) is first transcribed into cDNA. The cDNA is then used as the template for the quantitative PCR or real-time PCR reaction (qPCR). In qPCR, the amount of amplification product is measured in each PCR cycle using fluorescence from fluorescent labels. RT-qPCR has a variety of applications, including gene expression analysis, RNA interference (RNAi) validation, microarray validation, pathogen detection, genetic testing, and disease research.

[0005] RT-PCR and RT-qPCR typically involve two separate molecular syntheses: (i) the synthesis of cDNA from an RNA template; and (ii) the replication of newly synthesized cDNA through PCR amplification. RT-PCR and RT-qPCR can be performed by one-step (or coupled) RT-PCR and RT-qPCR methods using two or more enzymes, in which at least two separate enzymes (e.g., a reverse transcriptase and a polymerase) are employed for initial cDNA synthesis and subsequent amplification, respectively.

[0006] In one-step RT-qPCR, reverse transcription and PCR amplification are combined into a single reaction mixture which provides numerous advantages over two-step RT-qPCR (where the synthesis and amplification steps are performed using two different or separate reactions). One- step RT-qPCR requires less handling of the reaction mixture reagents and nucleic acid products than two-step RT-qPCR (e.g., opening of the reaction tube for component, or enzyme addition in between the two reaction steps), and is therefore less labor intensive and time consuming. One-step RT-qPCR also allows for less sample to be used if necessary, and fewer pipetting steps reduces the risk of contamination (Sellner and Turbett, Biotechniques 25:234-238 (1998)). Furthermore, there is less experimental variation since both reactions take place in the same tube.

[0007] Moreover, samples from which nucleic acids are extracted often contain additional compounds that are inhibitory to PCR. Humic acid in soil and feces, hematin in blood, immunoglobin G in serum, and various blood anticoagulants, like heparin and citrate, are all examples of such inhibitors. Such inhibitors may not be completely removed during the nucleic acid extraction and purification process, thus negatively impacting downstream PCR amplification, as reflected by an increase in Ct (i.e., threshold cycle) and decrease in dRn (z.e., difference in normalized reporter signal) when assayed by real time PCR.

[0008] A high Ct coupled with low dRn usually indicates low target nucleic acid concentration in reactions for quantitative PCR (qPCR) and reverse transcriptase-qPCR (RT-qPCR) applications. A reaction that exhibits reduced or no amplification indicates that the target nucleic acid is absent, or present in such small amounts that it is not detectable. A reaction that contains detectable amounts of target, but is inhibited by the presence of qPCR inhibitors may show an artificially high threshold cycle (Ct) and low fluorescence signal (dRn), which can lead the user to believe that the amount of target nucleic acid is less than the actual amount present. If the level of inhibition is severe enough, the reaction may fail to amplify completely, thus leading to a false-negative result.

[0009] Current one-step RT-qPCR methods have limited multiplex ability, limited at least in part by the benchtop stability and the reaction efficiency of the process.

[0010] Because of the importance of nucleic acid synthesis applications to the fields of molecular biology and cellular biology, a one-step RT-qPCR system, in the form of a generalized ready -to-use composition, which exhibits high sensitivity, is not restricted by the amount of sample, reduces the amount of practitioner manipulation, minimizes the risks of contamination, minimizes the expense of reagents, maximizes multiplex validation, and / or maximizes the amount of nucleic acid end product, is needed in the art. In addition, a method to increase or maximize the stability of assembled reactions and the reaction efficiency, especially when multiple targets are present in the sample, is necessary to ensure accurate results.SUMMARY

[0011] The present disclosure is generally directed to compositions, methods and kits useful for the synthesis of nucleic acids. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in a one-step qPCR or RT-qPCR procedure using one or more reverse transcriptases, such as Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, in combination with one or more DNA polymerases, such as DNA polymerase from Thermophilus aquations (Taq). Preferably, the compositions further comprise one or more agents used to increase stability of assembled reactions of a polymerase chain reaction. Such stabilizing agents can include, for example, an RNA aptamer and / or a salt. The present disclosure thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of target sequences which can be used for a variety of industrial, medical, forensic and diagnostic purposes. The embodiments disclosed herein are especially useful for the rapid amplification and detection of viral genes, including both RNA and DNA targets.

[0012] In particular, the present disclosure is directed to compositions comprising at least one active DNA polymerase and at least one active reverse transcriptase (RT). In some embodiments, such compositions further comprise at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction.

[0013] In some preferred embodiments, the reverse transcriptases are thermostable. For instance, the thermostable reverse transcriptases can be M-MLV reverse transcriptases, mutants, variants, or derivatives thereof. In some embodiments, the M-MLV RTs can comprise one or moremutations. Such mutations can include, for example: Y64, R116, DI 24, Hl 26, Y133, KI 52, Q190, T197, H204, V223, M289, T306, or F309. In some embodiments, the concentration of the reverse transcriptase(s) is between about 0.5 U / pL to about 5 U / pL.

[0014] In some preferred embodiments the DNA polymerases are thermostable DNA polymerases. For example, the thermostable DNA polymerases can be Taq DNA polymerases, mutants, variants, or derivatives thereof, such as, for example, AmpliTaq® Fast DNA polymerase or a mutant, variant, or derivative thereof. In some embodiments, the concentration of the DNA polymerase(s) is between about 0.005 U / pL to about 3.0 U / pL.

[0015] In some embodiments, the stabilizing agents of the present compositions include RNA aptamers. In some preferred embodiments, the RNA aptamers interact with the reverse transcriptase.

[0016] In some embodiments, the present composition includes qPCR inhibitor blocking agents of the present compositions include a neutral compound with cationic group lacking hydrogen. In some embodiments, the qPCR inhibitor blocking agents of the present compositions include a nonionic polyoxyethylene surfactant.

[0017] In some embodiments, the present compositions can further comprise one or more nucleotides. Such nucleotides can be, for example, deoxynucleoside triphosphates (dNTPs), for example, dTTP, dATP, dCTP, dGTP or dUTP. In some embodiments the concentration of each of the nucleotides in the composition is about 0.5 mM to about 5 mM.

[0018] In some embodiments, the present compositions can further comprise glycerol.

[0019] In some embodiments of the present compositions, the compositions can further comprise RNase inhibitor protein (RIP). In some embodiments the concentration of RIP is between about 0.1 U / pL to about 1.0 U / pL.

[0020] In some embodiments, the present compositions can further comprise one or more passive reference control. In some embodiments, the one or more passive reference control can be, for example, a ROX dye.

[0021] In some embodiments, the present compositions can be formulated as concentrated stock solutions. In some embodiments, such concentrated stock solutions can be from about a 2X to about a 6X stock solution. In some embodiments, such stock solutions can be diluted for subsequent use in, for example, nucleic acid synthesis methods. In some preferred embodiments the compositions can be formulated as at least a 4X stock solution.

[0022] The present disclosure are also directed to methods for performing qPCR or RT-qPCR of a nucleic acid sample. In some embodiments, the present methods can comprise the steps of: (i) mixing a composition comprising at least one reverse transcriptase, at least one DNA polymerase, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or RT-qPCR, with: (a) a nucleic acid sample; (b) one or more labeled probes; and (c) one or more primers; and (ii) performing said qPCR or RT-qPCR on said nucleic acid sample.

[0023] In some embodiments of the present methods, qPCR or RT-qPCR can be performed in a single vessel (e.g., tube, compartment, well) or in a single reaction mixture.

[0024] In some embodiments of the present methods, compositions comprising at least one reverse transcriptase, at least one DNA polymerase, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction, are mixed with (a) a nucleic acid sample; (b) one or more labeled probes; or (c) one or more primers. In some embodiments of the present methods, the one or more labeled probes can be a TaqMan® probe.

[0025] In some embodiments of the present methods, the use of compositions comprising at least one qPCR inhibitor blocking agent can increase tolerance to various qPCR inhibitors (herein referred to as “inhibitor tolerance”), if present. In some embodiments, the increase in inhibitor tolerance can be indicated by a decrease in Ct. In some preferred embodiments of the present methods, Ct is decreased by at least about 10% when compared to methods using compositions that do not comprise any qPCR inhibitor blocking agents. In some embodiments, Ct can be decreased by at least 1. . In embodiments, the composition is free or substantially free of fish gelatin.

[0026] In another aspect, the present disclosure is directed to methods for amplifying a nucleic acid molecule. In some embodiments, the methods for amplification can comprise the steps of: (i) mixing a nucleic acid template with a composition comprising one or more reverse transcriptases, one or more DNA polymerases, and one or more stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction, to form a reaction mixture; and (ii) incubating the reaction mixture under conditions sufficient to amplify a nucleic acid molecule complementary to all or a portion of said nucleic acid template. In some embodiments, the nucleic acid template can be RNA or DNA.

[0027] In some embodiments of the present methods, nucleic acid amplification can be performed by PCR. In some embodiments, the PCR can be qPCR. In some embodiments, the PCR can be performed by RT-qPCR. In other embodiments, the PCR can be endpoint PCR. In some other embodiments the PCR can be multiplex PCR. In yet other embodiments, the PCR can comprise thermal cycling. In some embodiments, the thermal cycling can be optimized for fast thermal cycling.

[0028] In another aspect, the present disclosure is directed to methods for nucleic acid synthesis. In some embodiments the methods for nucleic acid synthesis can comprise: (i) mixing one or more first nucleic acid molecules with one or more reverse transcriptases, one or more polymerases, and one or more stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction; and (ii) incubating the mixture under conditions sufficient to produce or synthesize one or more second nucleic acid molecules complementary to all or a portion of one or more first nucleic acid molecules. In some embodiments, the first nucleic acid molecules are RNA molecules. In some embodiments, the second nucleic acid molecules are DNA molecules. In some embodiments, the methods for nucleic acid synthesis can further comprise incubating one or more first nucleic acid molecules under conditions sufficient to produce or synthesize one or more second nucleic acid molecules complementary to all or a portion of one or more first nucleic acid molecules.

[0029] The present disclosure is also directed to reaction mixtures comprising: (a) at least one reverse transcriptase; (b) at least one polymerase; (c) at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction; and (d) at least one primer. In some embodiments the reaction mixture can further comprise a nucleic acid template (e.g., RNA or DNA). In yet other embodiments, the reaction mixtures can further comprise a labeled probe (e g., TaqMan® probe).

[0030] The present disclosure is also directed to kits comprising, for example, in a single container, a composition having at least one reverse transcriptase, at least one DNA polymerase, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of a polymerase chain reaction. In some embodiments, the composition can be housed in a single tube or vessel.

[0031] In some embodiments of the present kits, the compositions can be formulated as 4X stock solutions. In some preferred embodiments of the kits, the compositions can be used forqPCR, RT-qPCR methods, nucleic acid synthesis methods, or nucleic acid amplification (e.g., PCR) methods. In some embodiments, the qPCR or RT-qPCR methods can comprise multiplexing.

[0032] Disclosed herein is a composition comprising a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

[0033] In an embodiment, said reverse transcriptase comprises a thermostable reverse transcriptase.

[0034] In an embodiment, said thermostable reverse transcriptase comprises an Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or a mutant, variant, or derivative thereof.

[0035] In an embodiment, said M-MLV reverse transcriptase comprises one or more mutations selected from the group consisting of: Y64, R116, D124, H126, Y133, KI 52, Q190, T197, H204, V223, M289, T306, and F309.

[0036] In an embodiment, said DNA polymerase comprises a thermostable DNA polymerase.

[0037] In an embodiment, said thermostable DNA polymerase comprises Taq DNA polymerase or a mutant, variant, or derivative thereof.

[0038] In an embodiment, said thermostable DNA polymerase comprises a recombinant Taq DNA polymerase or a mutant, variant, or derivative thereof.

[0039] In an embodiment, said at least one stabilizing agent comprises an RNA aptamer.

[0040] In an embodiment, said RNA aptamer interacts with the reverse transcriptase.

[0041] In an embodiment, said at least one stabilizing agent further comprises a sulfate additive.

[0042] In an embodiment, said sulfate additive is at least one selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0043] In an embodiment, said sulfate additive is at least one selected from the group consisting of magnesium sulfate and potassium sulfate.

[0044] In an embodiment, the composition further comprises at least one qPCR inhibitor blocking agent.

[0045] In an embodiment, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0046] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0047] In an embodiment, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0048] In an embodiment, the composition further comprises a silicone-based emulsifier.

[0049] In an embodiment, the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0050] In an embodiment, the composition further comprises a hot-start component that reversibly inactivates the at least one DNA polymerase.

[0051] In an embodiment, the composition excludes gelatin or fish gelatin.

[0052] In an embodiment, the composition excludes uracil-DNA glycosylase (UDG) or uracil-N-glycosylase (UNG).

[0053] In an embodiment, the composition further comprises a passive reference control.

[0054] In an embodiment, said passive reference control comprises a ROX dye.

[0055] In an embodiment, said composition is for use in nucleic acid synthesis, nucleic acid amplification, a quantitative polymerase chain reaction (qPCR), or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR).

[0056] Disclosed herein is a method of performing a quantitative polymerase chain reaction (qPCR) or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) on a nucleic acid sample, the method comprising mixing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or said RT-qPCR, with the nucleic acid sample; at least one primer pair having specificity for a nucleic acid in the nucleic acid sample; at least one labeled probe having specificity for amplicons complementary to all or a portion of the nucleic acid; and performing said qPCR or said RT-qPCR on said nucleic acid sample.

[0057] In an embodiment, said at least one stabilizing agent comprises an RNA aptamer that interacts with the reverse transcriptase to increase the stability of the assembled reactions.

[0058] In an embodiment, said at least one stabilizing agent further comprises a sulfate additive.

[0059] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0060] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0061] In an embodiment, said composition further comprises at least one qPCR inhibitor blocking agent.

[0062] In an embodiment, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0063] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0064] In an embodiment, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0065] In an embodiment, the composition further comprises an non-ionic silicone emulsifier.

[0066] In an embodiment, said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0067] In an embodiment, said labeled probe is a dual -labeled probe with a reporter dye and a quencher dye.

[0068] In an embodiment, said qPCR or said RT-qPCR is performed in a single tube or a single reaction.

[0069] In an embodiment, said performing said qPCR or said RT-qPCR comprises thermal cycling or fast thermal cycling.

[0070] In an embodiment, said quantitative polymerase chain reaction (qPCR) or said quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is a multiplex reaction capable of detecting up to six target nucleic acids.

[0071] Disclosed herein is a method for amplifying a nucleic acid comprises mixing a nucleic acid template with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incubating said reaction mixture underconditions sufficient to amplify the nucleic acid, the nucleic acid being complementary to all or a portion of said nucleic acid template.

[0072] In an embodiment, said nucleic acid template is a template having RNA.

[0073] In an embodiment, said nucleic acid is DNA.

[0074] In an embodiment, said at least one stabilizing agent comprises an RNA aptamer.

[0075] In an embodiment, said RNA aptamer interacts with the reverse transcriptase.

[0076] In an embodiment, said at least one stabilizing agent further comprises a sulfate additive.

[0077] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0078] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0079] In an embodiment, said composition further comprises at least one qPCR inhibitor blocking agent.

[0080] In an embodiment, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0081] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0082] In an embodiment, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0083] In an embodiment, the composition further comprises an non-ionic silicone emulsifier.

[0084] In an embodiment, the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0085] Disclosed herein is a method for nucleic acid synthesis comprising mixing a sample having first nucleic acid with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; and incubating said mixture under conditions sufficient to produce a second nucleic acid that is complementary to all or a portion of said first nucleic acid.

[0086] In an embodiment, said first nucleic acid is RNA.

[0087] In an embodiment, said second nucleic acid is DNA.

[0088] In an embodiment, said at least one stabilizing agent comprises an RNA aptamer.

[0089] In an embodiment, said RNA aptamer interacts with the reverse transcriptase.

[0090] In an embodiment, said at least one stabilizing agent further comprises a sulfate additive.

[0091] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0092] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0093] In an embodiment, said composition further comprises at least one qPCR inhibitor blocking agent.

[0094] In an embodiment, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0095] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0096] In an embodiment, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0097] In an embodiment, the composition further comprises an non-ionic silicone emulsifier.

[0098] In an embodiment, said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0099] Disclosed herein is a reaction mixture comprising a master mix composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; at least one primer having a sequence specific to a target nucleic acid in a sample mixed with said reaction mixture in the assembled polymerase chain reaction; and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

[0100] In an embodiment, said at least one labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.

[0101] In an embodiment, said target nucleic acid is RNA, and the amplicons are DNA.

[0102] In an embodiment, said at least one stabilizing agent comprises an RNA aptamer.

[0103] In an embodiment, said RNA aptamer interacts with said at least one reverse transcriptase.

[0104] In an embodiment, said at least one stabilizing agent further comprises a sulfate additive.

[0105] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0106] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0107] In an embodiment, said composition further comprises at least one qPCR inhibitor blocking agent.

[0108] In an embodiment, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0109] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0110] In an embodiment, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.[0U1] In an embodiment, wherein the master mix composition further comprises an non-ionic silicone emulsifier.

[0112] In an embodiment, said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0113] Disclosed herein is a kit comprising a single container containing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

[0114] In an embodiment, said kit is used for qPCR or RT-qPCR.

[0115] In an embodiment, said kit is used for nucleic acid synthesis.

[0116] In an embodiment, said kit is used for nucleic amplification.

[0117] In an embodiment, the kit further comprises another container containing a second composition including at least one primer having a sequence specific to a target nucleic acid in asample to be subjected to the assembled polymerase chain reaction, and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

[0118] In an embodiment, the kit further comprises another container containing at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction; and a further container containing at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

[0119] In an embodiment, said at least one probe is a dual-labeled probe with a reporter dye and a quencher dye.

[0120] In an embodiment, said reverse transcriptase is a Moloney Murine Leukemia virus (M- MLV) reverse transcriptase or any mutant, variant or derivative thereof having reverse transcriptase activity.

[0121] In an embodiment, said polymerase is Taq DNA polymerase or any mutant, variant or derivative thereof having DNA polymerase activity.

[0122] In an embodiment, said at least one stabilizing agent comprises an RNA aptamer.

[0123] In an embodiment, said RNA aptamer interacts with said reverse transcriptase.

[0124] In an embodiment, said at least one stabilizing agent further comprises a sulfate additive.

[0125] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0126] In an embodiment, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0127] In an embodiment, said composition further comprises at least one qPCR inhibitor blocking agent.

[0128] In an embodiment, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanedioL

[0129] In an embodiment, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0130] In an embodiment, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0131] In an embodiment, the composition further comprises an non-ionic silicone emulsifier.

[0132] In an embodiment, the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0133] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0134] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several exemplary embodiments of the disclosure and together with the description, serve to explain certain teachings. The skilled artisan will understand that the described drawings are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0135] FIG. 1 depicts an one-step RT-PCR according to exemplary embodiments.

[0136] FIGS. 2A and 2B are amplification plots at t=Q and / =24 hrs of a PCR reaction performed using a first exemplary composition.

[0137] FIGS. 3A and 3B are amplification plots at t=Q and Z=24 hrs of a PCR reaction performed using a second exemplary composition.

[0138] FIG. 4 shows amplification plots of different targets using an exemplary composition.

[0139] FIG. 5 is a graph showing tolerance of exemplary compositions including an inhibitor blocking agent to the inhibitor heparin.

[0140] FIG. 6 is a graph showing tolerance of exemplary compositions including a polyoxyethylene surfactant to the inhibitor heparin.DETAILED DESCRIPTIONOverview

[0141] The present disclosure is directed to compositions, methods, and kits for use in the production or analysis of nucleic acids. In particular, the present disclosure offers several advantages compared to known compositions or methods for the generation or amplification of nucleic acids by, for example, qPCR or RT-qPCR. These advantages include, but are not limited to:a) providing increase multiplex capacity to allow for simultaneous amplification of more nucleic acid targets (e.g., up to six nucleic acid targets) in a single reaction; b) providing increased DNA polymerase concentrations for improved reaction efficiency when more than one target is present; c) providing surfactant optimized to improve tolerance to common PCR inhibitors; d) providing a non-ionic silicone emulsifier to counter surfactant compositions, improving fluorescence baseline consistency, and reducing reaction replicate variability; e) providing an osmoprotectant that improve amplification of GC-rich targets and increase tolerance to common PCR inhibiters; f) providing product validation up to 6-plex in a 1-step qPCR or RT-qPCR; g) providing increased stability of assembled reactions by reducing DNA polymerase activity prior to starting a PCR process; h) providing an RNA aptamer to interact with the reverse transcriptase, reducing residual activity prior to reverse transcription; j) providing higher surfactant concentration to enhance stability of the master mix reagent; k) providing a true “one tube / one step” procedure for qPCR or RT-qPCR (versus having two separate reactions for the synthesis of DNA from an RNA template and the subsequent amplification of the DNA), which eliminates premixing or additional aliquoting steps since reverse transcriptase and DNA polymerase are contained in the master mix together and users do not have to add the reverse transcriptase to the master mix prior to performing qPCR or RT-qPCR; l) providing more concentrated compositions for use in nucleic acid synthesis (e.g. qPCR or RT-qPCR), which allow for higher volume sample input if or when sample template concentrations are low (which is often the case for viral targets or forensic analysis); m) providing compositions for use in nucleic acid synthesis (e.g. qPCR or RT-qPCR) that have increased tolerance to various PCR inhibitors; n) providing compositions for use in nucleic acid synthesis (e.g. qPCR or RT-qPCR) that provide for maximal specificity and sensitivity; o) providing nucleic acid synthesis (e.g., qPCR or RT-qPCR) compositions and methods that can be used with fast thermal cycling protocols for quicker read-outs; p) providing nucleic acid synthesis (e.g., qPCR or RT-qPCR) compositions and methods which allow for the increased capability to multiplex (e.g., amplify a multiplicity of targets using asingle sample (e.g., two targets on one sample) or multiple samples (e.g., two targets on two different samples) in a single reaction at substantially the same time); and / or q) providing increased multiplexed nucleic acid synthesis (e.g., qPCR or RT-qPCR) compositions and methods which allow for the type of identification and quantification of nucleic acids (e.g., oncoviral nucleic acid) in a single reaction thereby reducing the amount of sample used as well as reducing costs and the amount of time it takes to obtain results.

[0142] FIG. 1 depicts an one-step RT-PCR according to exemplary embodiments.

[0143] Referring to FIG. 1, the present disclosure provides for a true “one tube / one step” procedure for qPCR, RT-PCR, or RT-qPCR. In the one-step RT-qPCR, the reverse transcriptase and the DNA polymerase are premixed into a single tube, allowing both the RT and the subsequent PCR step to be performed in a single reaction. The advantages of one-step RT-PCR over two-step RT-PCR include fast and simple analysis, less pipetting steps, lower risk of errors and contamination, and suitability for high-throughput applications.

[0144] One-step RT-PCR combines first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube performed in a single reaction. This reaction setup can help simplify workflow, reduces variation, and minimizes possible contamination. One-step RT-PCR allows easy processing of large numbers of samples, making it amenable to high-throughput applications. One-step RT-PCR uses gene-specific primers for amplification, limiting the analysis to a few genes per RNA sample. Use of a gene-specific primer in RT-PCR can help maximize the yield of the target cDNA and minimize background amplification.

[0145] In exemplary embodiments, “an assembled PCR reaction” is a PCR reaction including all of the necessary components for performing first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube. In exemplary embodiments, “an assembled PCR reaction” is a PCR reaction including all of the necessary components for performing first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction.Compositions

[0146] The present disclosure provides compositions comprising a variety of components in various combinations. Such components can include one or more enzymes having reverse transcriptase activity, one or more DNA polymerases, one or more osmoprotectants, such as a nonionic polyoxyethylene surfactant and / or inhibitor blocking agent, or one or more stabilizingagents. Additional components can also include, for example, one or more primers, one or more deoxyribonucleoside triphosphates (dATPs), one or more antibodies, one or more ribonuclease inhibitors, one or more preservative, and one or more passive reference dyes, as well as suitable PCR buffer components, including, but not limited to, glycerol and tris-buffered saline, which may include bovine serum albumin (BSA). The composition may further include nuclease-free water.

[0147] Such compositions may be formulated for high-order multiplex reactions, use of RNA or DNA as a starting material, lab material or liquid handlers that require benchtop stability, and inhibitor intolerance.

[0148] Such compositions can be formulated as concentrated stock solutions (e.g., 2X, 3X, 4X, 6X, 10X, etc.) or as working solutions (e.g., IX). In some embodiments, the concentrated stock solution is at 4X. In some embodiments, having the composition as a concentrated (e.g., 4X) stock solution allows a greater amount of nucleic acid sample to be added (such as, for example, when the compositions are used for nucleic acid synthesis). These compositions can be used in the present methods to produce, analyze, quantitate, and otherwise manipulate nucleic acid molecules using a one-step (or coupled) qPCR or RT-qPCR procedure.

[0149] In some embodiments, the compositions of the present disclosure can be formulated as master mixes. Master mixes improve the efficiency and reduce the errors associated with the assembly of large number of reactions required for high-throughput analysis. In some embodiments, master mixes can contain combination of reagents common to all reactions. For example, the master mix can contain a buffer, a salt, such as magnesium sulfate (MgSO4) or potassium sulfate (K2SO4), nucleotides, a labeled probe or dye, a reverse transcriptase, a thermostable DNA polymerase, a stabilizing agent, and / or an osmoprotectant. Each reaction would then contain an aliquot of the common master mix and a specific target nucleic acid and primer pair. Master mixes can be manufactured and distributed as a concentrated solution. The master mix can then be diluted when final reactions are assembled.

[0150] In some embodiments, the present compositions can be packaged in a suitable container or vessel capable of holding the composition and which does not significantly interact with components of the composition. The container or vessel can be designed to permit easy dispensing of the dosage form by individuals or by a liquid handling instrument. The containers or vessels of such composition can be further packaged into multi-pack units.Reverse Transcriptases

[0151] The compositions of the present disclosure comprise polypeptides having reverse transcriptase activity (i.e., reverse transcriptases). In some preferred embodiments, the polypeptides having reverse transcriptase activity are thermostable. As used herein, the term "thermostable” refers to an enzyme that is heat stable or heat resistant. For the purposes of this disclosure, a thermostable polypeptide having reverse transcriptase activity can also be defined as a polypeptide having reverse transcriptase activity which retains a greater percentage of its activity after a heat treatment than is retained by a polypeptide having reverse transcriptase activity that has wild-type thermostability after an identical treatment.

[0152] According to some embodiments, enzymes having reverse transcriptase activity can be, for example, retroviral reverse transcriptases such as M-MLV reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV) reverse transcriptase, Avian Sarcoma Leukosis Virus (ASLV) reverse transcriptases, as well as Lenti virus reverse transcriptases, or corresponding mutants, variants or derivatives thereof having reverse transcriptase activity. As used herein, “mutants, variants, or derivatives” refer to all permutations of a chemical species, which can exist or be produced, that still retains the definitive chemical activity of that chemical species. Some preferred embodiments include enzymes that are RNase H+ enzymes such as, for example, RNase H+ M-MLV or RNase H+ AMV reverse transcriptases. Alternatively, the reverse transcriptases used in the present compositions can have reduced, substantially reduced, or eliminated RNase H activity (see, e.g., U.S. Pat. No. 7,078,208, the disclosure of which is fully incorporated by reference in its entirety). RNase H is a processive 5’ and 3’ ribonuclease that is specific for the RNA strand of RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons, pp.23-24 (1984)). RNase H activity can be determined by a variety of assays, such as those described, for example, in U.S. Pat. No. 5,244,797, in Kotewicz, et al., Nucl. Acids Res. 16:265-277 (1988) and in Gerard, et al., FOCUS (Life Technologies) 14:91-93 (1992), the disclosures of which are fully incorporated herein by reference in there entireties.

[0153] Additional enzymes having reverse transcriptase activity can be used in accordance with the present disclosure, such as Thermus thermophilus (Tth) reverse transcriptase, which has reversetranscriptase activity in the presence of Mn2+and DNA polymerase activity in the presence of Mg2+(Myers and Gelfand, Biochemistry 30:7661-7666 (1991), the disclosure of which is fully incorporated herein by reference in its entirety). Methods for the isolation or purification of reverse transcriptases have been described, for example, in U.S. Pat. Nos. 4,943,531 and 5,017,492, the disclosures of which are fully incorporated herein by reference in their entireties. It is to be understood that a variety of reverse transcriptases can be used in the present disclosure, including reverse transcriptases not specifically disclosed above, without departing from the scope or preferred embodiments thereof.

[0154] In accordance with the present disclosure, any number of mutations can be made to the reverse transcriptases and, in a preferred embodiment, multiple mutations can be made which result in an increased reverse transcriptase stability or functionality. Enzymes for use herein can also include those in which terminal deoxynucleotidyl transferase (TdT) activity has been reduced, substantially reduced, or eliminated. Reverse transcriptases which exhibit such increased or decreased functionalities are described in, for example, U.S. Pat. Nos. 7,056,716 and 7,078,208 (the disclosures of which are fully incorporated by reference in their entireties). In some embodiments, such mutated reverse transcriptases can include reverse transcriptases with one or more alterations at amino acid positions equivalent or corresponding to Y64, R116, D124, H126, Y133, K152, Q190, T197, H204, V223, M289, T306, or F309 of M-MLV reverse transcriptase. Such mutations can include point mutations, frame shift mutations, deletions and insertions, with one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty, thirty, etc.) point mutations preferred.

[0155] Mutations can be introduced into the reverse transcriptases of the present disclosure using any methodology known to those of skill in the art. In one embodiment, mutant or modified reverse transcriptases can be made by recombinant techniques. A number of cloned reverse transcriptase genes are available or can be obtained using standard recombinant techniques (see, e.g., U.S. Pat. No. 5,668,005 and PCT Publication No. WO 98 / 47912). For example, oligonucleotide site-directed mutagenesis can be used to create a mutant polymerase which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule. Alternatively, mutations can also be introduced randomly by, for example, conducting a PCR reaction in the presence of manganese as a divalent metal ion cofactor.

[0156] Polypeptides having reverse transcriptase activity can be added to the present compositions to give a final concentration in a working solution of about 0.001 U / pL to about 500U / pL, about 0.005 U / pL to about 100 U / pL, about 0.01 U / pL to about 50 U / pL, about 0.05 U / pL to about 20 U / pL, about 0.1 U / pL to about 10 U / pL, about 0.2 U / pL to about 5 U / pL, or preferably at a concentration of about 0.2 U / pL, about 0.4 U / pL, about 0.8 U / pL, about 1.0 U / pL, about 1.2 U / pL, about 1.4 U / pL, about 1.8 U / pL, about 2 U / pL, about 3 U / pL, about 4 U / pL, or about 5 U / pL.Polymerases

[0157] The compositions of the present disclosure can also comprise one or more polymerases. Such polymerases can be any enzyme capable of replicating a DNA molecule. Preferably, the DNA polymerases are thermostable DNA polymerases. Thermostable DNA polymerases, as used herein, are not irreversibly inactivated when subjected to elevated temperatures for the time necessary to effect destabilization of single-stranded nucleic acids or denaturation of doublestranded nucleic acids during PCR amplification. Irreversible denaturation of the enzyme refers to substantial loss of enzyme activity. Preferably a thermostable DNA polymerase will not irreversibly denature at about 90 °C- 100 °C under conditions such as is typically required for PCR amplification.

[0158] DNA polymerases in accordance with the present disclosure can be isolated from natural or recombinant sources, by techniques that are well-known in the art (see, e.g., PCT Publication Nos. WO 92 / 06200; WO 96 / 10640; U.S. Patent Nos. 5,455,170; 5,912,155; and 5,466,591, the disclosures of which are fully incorporated herein by reference in their entireties), from a variety of thermophilic bacteria that are available commercially (for example, from American Type Culture Collection, Rockville, Md.) or can be obtained by recombinant DNA techniques (see, e.g., PCT Publication No. WO 96 / 10640 and U.S. Patent No. 5,912,155). Suitable for use as sources of thermostable polymerases or the genes thereof for expression in recombinant systems are, for example, the thermophilic bacteria Thermus thermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcus woosii and other species of the Pyrococcus genus, Bacillus sterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum, Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima and other species of the Thermotoga genus, and Methanobacterium thermoautotrophicum, and mutants, variants or derivatives thereof. It is to be understood, however, that DNA polymerases from other organisms can also be used herein without departing from the scope or preferred embodimentsthereof. As an alternative to isolation, DNA polymerases are available commercially from, for example, Life Technologies (Carlsbad, CA), New England BioLabs (Beverly, MA), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, CA), Boehringer Mannheim Biochemicals (Indianapolis, IN) and Perkin Elmer Cetus (Norwalk CT).

[0159] Particularly preferred thermostable DNA polymerases for use in the present compositions and methods include, but are not limited to, Taq, Tne, Tma, Tfi / VENT, DEEPVENT, Pfu, Pwo, Tfi or Tth DNA polymerases, or mutants, variants or derivatives thereof having DNA polymerase activity. Taq DNA polymerase and mutant forms thereof are commercially available, for example, from Life Technologies (Carlsbad, CA), or can be isolated from their natural source, the thermophilic bacterium Thermus aquaticus, as described previously (see, e.g., U.S. Patent Nos. 4,889,818 and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties). One particularly preferred commercially-available thermostable DNA polymerase is AmpliTaq™ Fast DNA Polymerase (Roche Molecular Systems, Inc., Pleasanton, CA) or AmpliTaq™ Gold Fast PCR (Applied Biosystems). Tne DNA polymerase can be isolated from its natural source, the thermophilic bacterium Thermotoga neapolitana (see, e.g., PCT Publication No. WO 96 / 10640 and U.S. Patent No. 5,912,155), and Tma DNA polymerase can be isolated from its natural source, the thermophilic bacterium Thermotoga maritima (see, e.g., U. S. Patent No. 5,374,553, the disclosure of which is incorporated herein by reference in its entirety). It is to be understood that a variety of DNA polymerases can be used in the present compositions, methods and kits, including polymerases not specifically disclosed herein, without departing from the scope or preferred embodiments thereof.

[0160] Methods for producing mutants and derivatives of thermophilic DNA polymerases, particularly of Tne and Tma polymerases are disclosed, for example, in U.S. Application Nos. 08 / 689,807 and 08 / 689,818, both filed Sept. 6, 1996, both of which are incorporated by reference herein in their entireties. Tfi, Tli / VENT, and DEEPVENT are available commercially (e.g., from New England BioLabs; Beverly, MA), or can be produced as described (Bej and Mahbubani, in: PCR Technology: Current Innovations, Griffin, H. G., and Griffin, A. M., eds., CRC Press, pp. 219-237 (1994) for Tli / VENT; Flaman, et al., Nucl. Acids Res. 22:3259-3260 (1994) for DEEPVENT). Thermostable DNA polymerases of the present disclosure can be added to the present compositions to give a final concentration in a working solution of about 0.0001 U / pL to about 50 U / pL, about 0.0005 U / pL to about 10 U / pL, about 0.001 U / pL to about 5 U / pL, about0.005 U / pL to about 2 U / pL, about 0.01 U / pL to about 1 U / pL, about 0.02 U / pL to about 0.5 U / pL, or preferably at a concentration of about 0.02 U / pL, about 0.04 U / pL, about 0.08 U / pL, about 0.1 U / pL, about 0.12 U / pL, about 0.14 U / pL, about 0.18 U / pL, about 0.2 U / pL, about 0.3 U / pL, about 0.4 U / pL, or about 0.5 U / pL.

[0161] In some embodiments, the concentration of DNA polymerases can be determined as a ratio of the concentration of the enzymes having reverse transcriptase activity to the concentration of the enzymes having DNA polymerase activity. Thus, in some compositions the unit ratio of the reverse transcriptase enzymes to the DNA polymerase enzymes can range from about 500 U / pL to about 0.001 U / pL, about 250 U / pL to about 0.005 U / pL, about 100 U / pL to about 0.01 U / pL, about 50 U / pL to about 0.05 U / pL, about 25 U / pL to about 0.1 U / pL, or preferably about 5 U / pL to about 0.5 U / pL. Of course, other suitable ratios of unit activities of reverse transcriptase enzymes to DNA polymerases suitable for use in the present compositions, methods and kits will be apparent to one of ordinary skill in the art.Stabilizing Agents

[0162] In accordance with the present disclosure, one or more stabilizing agents can be included in the present compositions. In some embodiments, the stabilizing agent increases the stability of assembled reactions during a PCR process. In some embodiments, the stabilizing agent includes an RNA aptamer. In some embodiments, the stabilizing agent increases the reaction efficiency during a PCR process, especially when more than one target is present in the sample. In some embodiments, the stabilizing agent permits an increased level of the DNA polymerase to allow balancing of benchtop stability with multiplex capacity.PCR Inhibitor Blocking Agents, Surfactants, and Qsmoprotectants

[0163] In accordance with the present disclosure, one or more qPCR inhibitor blocking agents and / or osmoprotectants can be added to the present compositions to assist in overcoming the inhibition of PCR reactions by a variety of compounds often found in samples used for nucleic acid preparation, isolation or purification. Such inhibitors include, for example, heparin (blood); hematin (blood); EDTA (blood); citrate (blood); immunoglobin G (blood, serum); humic acid (soil, feces); lactoferrin (milk, saliva, other secretory fluids); urea (urine); plant polysaccharides (plants); melanin (skin, hair); myoglobin (tissue); and indigo dye (textiles). The addition of qPCR inhibitorblocking agents, both individually and in combination, can increase tolerance to such qPCR inhibitor contaminants. Thus, the present compositions can further comprise agents that work alone or in combination to increase tolerance to various qPCR inhibitors including, for example, humic acid, hematin, and heparin.

[0164] Such qPCR inhibitor blocking agents for use in the present disclosure can include nonionic surfactants such as, but not limited to, nonionic polyoxyethylene surfactants, such as polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate, or neutral compounds with a cationic group lacking a hydrogen, such as, but not limited to, ethylene glycol, betaine and 1,2-propanediol. In some embodiments, a silicone-based emulsifier (such as antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15) may be included to counter any foaming tendency of the nonionic surfactant.

[0165] Such qPCR inhibitor blocking agents for use in the present disclosure can also include proteins such as, but not limited to, albumin (e.g., BSA, recombinant BSA and albumins derived from other species), and DNA-binding proteins (e.g., phage T4 gene 32 (T4gP32)), or peptide or polypeptide variants, fragments or derivatives thereof. Other non-protein-based qPCR inhibitor blocking agents for use in the present disclosure include, for example, deferoxamine mesylate. In other embodiments, the composition is free of a gelatin or fish gelatin.

[0166] According to various embodiments, a Ct value can be determined using a derivative of a PCR curve. For example, a first, second, or nth order derivative method can be performed on a PCR curve in order to determine a Ct value. In various embodiments, a characteristic of a derivative can be used in the determination of a Ct value. Such characteristics can include, but are not limited to, a positive inflection of a second derivative, a negative inflection of a second derivative, a zero crossing of the second derivative, or a positive inflection of a first derivative. In various embodiments, a Ct value can be determined using a thresholding and baselining method. For example, an upper boundary to an exponential phase of a PCR curve can be established using a derivative method, while a baseline for a PCR curve can be determined to establish a lower boundary to an exponential phase of a PCR curve. From the upper and lower boundaries of a PCR curve, a threshold value can be established from which a Ct value is determined. Other methods for the determination of a Ct value include, but are not limited to, various embodiments of a fit point method, and various embodiments of a sigmoidal method (See, e g., U.S. Patent Nos. 6,303,305;6,503,720; 6,783,934; 7,228,237 and U.S. Application Publication No. 2004 / 0096819; the disclosures of which are herein incorporated by reference in their entireties).

[0167] In some embodiments, the higher the concentration of BSA used, the more tolerant the reaction is to hematin and humic acid inhibition. However, in some embodiments with increasing amounts of BSA, dRn decreases and baseline value (or background signal) increases. As used herein, the term “dRn” or “delta Rn” refers to the difference in the normalized reporter signal (Rn) subtracted from the background signal (baseline) which is then normalized by a passive reference signal. Delta Rn can be determined by the formula [Rn+- Rn"], where Rn+is the Rn value for a reaction involving all components, including the template, and Rn is the value for an unreacted sample.

[0168] qPCR inhibitor blocking compounds or agents can be added to the present compositions to give a final concentration in a working solution of about 1 ng / pL to about 10,000 ng / pL, about 50 ng / pL to about 8000 ng / pL, about 100 ng / pL to about 6000 ng / pL, about 200 ng / pL to about 3000 ng / pL or preferably about 500 ng / pL to about 1000 ng / pL. qPCR inhibitor blocking agents can also be added as a percentage of the final concentration in a working solution, for example, from about 0.001% to about 15%, about 0.05% to about 10%, about 0.01% to about 5%, or preferably about 0.1% to about 1%.

[0169] In some aspects, qPCR inhibitor blocking agents can reduce the amount of qPCR inhibition by such qPCR inhibitors by at least 1 to 100% compared to the level of inhibition observed in the absence of such qPCR inhibitor blocking agents. For example, inhibition can be reduced by at least about 1%, about 2%, about 5%, about 10%, about 20%, about 50%, about 75%, about 100% or any percentage in between.Nucleotides

[0170] The compositions of the present disclosure can further comprise one or more nucleotides (e.g., deoxynucleoside triphosphates (dNTPs)). The nucleotide components of the present compositions serve as the “building blocks” for newly synthesized nucleic acids, being incorporated therein by the action of the reverse transcriptases or DNA polymerases. Examples of nucleotides suitable for use in the present compositions include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, diTP, 7-deaza-dGTP, a-thio-dATP, a-thio-dTTP, a-thio-dGTP, a-thio- dCTP or derivatives thereof, all of which are available commercially from sources including LifeTechnologies (Carlsbad, CA), New England BioLabs (Beverly, MA) and Sigma Chemical Company (Saint Louis, MO). Such dNTPs may be unlabeled, or they may be detectably labeled by coupling them by methods known in the art with radioisotopes (e.g.,3H,14C,32P or35S), vitamins (e.g., biotin), fluorescent moieties (e.g., fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescent labels, dioxigenin (DIG) and the like. Labeled dNTPs may also be obtained commercially, for example from Life Technologies (Carlsbad, CA) or Sigma Chemical Company (Saint Louis, MO).

[0171] Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TA MRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dRl 10]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Life Technologies, Foster City, CA. FluoroLink™ DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham Arlington Heights, IL; Fluorescein- 15 -d ATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR.sub.770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15-2'-dATP available from Boehringer Mannheim Indianapolis, IN; and ChromaTide™ Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, OR. DIG labels include digoxigenin-11-UTP available from Boehringer Mannheim, Indianapolis, IN, and biotin labels include biotin-21-UTP and amino-7-dUTP available from Clontech, Palo Alto, CA. The term nucleotide includes modified nucleotides. Many examples of modified nucleotides are disclosed in U.S. Patent No. 6,200,757 (the disclosure of which is herein incorporated by reference in its entirety).

[0172] In some embodiments of the present compositions, dNTPs can be added to give a final concentration in a working solution of each dNTP of about .001 mM to about 100 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 1 mM, or preferably about 0.2 mM to about 0.6 mM.Primers

[0173] In addition to nucleotides, the present compositions can comprise one or more primers which facilitate the synthesis of a first DNA molecule complementary to all or a portion of an RNA template (e.g., a single-stranded cDNA molecule). Such primers can also be used to synthesize a DNA molecule complementary to all or a portion of the first DNA molecule, thereby forming a double-stranded cDNA molecule. Additionally, these primers can be used in amplifying nucleic acid molecules in accordance with the present disclosure. Oligonucleotide primers can be any oligonucleotide of two or more (e.g., 2, 3, 4, 5, 10, 20, etc.) nucleotides in length. Such primers include, but are not limited to, target-specific primers (which are preferably gene-specific primers), oligo (dT) primers, random primers or arbitrary primers. Additional primers that can be used for amplification of the DNA molecules according to the methods disclosed herein will be apparent to one of ordinary skill in the art. It is to be understood that a vast array of primers can be useful in the present compositions, methods and kits, including those not specifically disclosed herein, without departing from the scope or preferred embodiments thereof.

[0174] In some embodiments of the disclosed compositions, the final concentration of primers in a working solution can range from about 25 nM to about 2000 nM, such as about 50 nM to about 1700 nM, about 75 nM to about 1500 nM, about 100 nM to about 1200 nM, about 200 nM to about 1000 nM, or any range in between. In some exemplary embodiments, the concentration of the primers is between about 400 nM to about 900 nM.Probes

[0175] In accordance with the present disclosure, the compositions can further comprise probes for the detection of target nucleic acids. Various probes are known in the art, for example, TaqMan® probes (see, e.g., U.S. Patent No. 5,538,848), various stem-loop molecular beacons (see, e.g., U.S. Patent Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature Biotechnology 14:303-308 (1996)), stemless or linear beacons (see, e.g., PCT Publication No. WO 99 / 21881), PNA Molecular Beacons™ (see, e.g., U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., Proceedings in SPIE 4264:53-58 (2001)), non-FRET probes (see, e.g., U.S. Patent No. 6,150,097), Sunrise® / Amplifluor® probes (U.S. Patent No. 6,548,250), stemloop and duplex Scorpion™ probes (see, e.g., Solinas et al., Nucleic Acids Research 29:E96 (2001) and U.S. Patent No. 6,589,743), bulge loop probes (see, e.g., U.S. Patent No. 6,590,091), pseudo knot probes (see, e.g., U.S. Patent No. 6,589,250), cyclicons (see, e.g., U.S. Patent No. 6,383,752),MGB Eclipse™ probes (Epoch Biosciences, Bothell, WA), hairpin probes (see, e.g., U.S. Patent No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes and ferrocene-modified probes described, for example, in U.S. Patent No. 6,485,901; Mhlanga et al., Methods 25:463-471 (2001); Whitcombe et al., Nature Biotechnology 17:804-807 (1999); Isacsson et al., Molecular Cell Probes 14:321-328 (2000); Svanvik et al., Anal. Biochem. 281 :26- 35 (2000); Wolffs et al., Biotechniques 766:769-771 (2001); Tsourkas et al., Nucleic Acids Research 30:4208-4215 (2002); Riccelli et al., Nucleic Acids Research 30:4088-4093 (2002); Zhang et al., Shanghai 34:329-332 (2002); Maxwell et al., J. Am. Chem. Soc. 124:9606-9612 (2002); Broude et al., Trends Biotechnol. 20:249-56 (2002); Huang et al., Chem Res. Toxicol. 15: 118-126 (2002); and Yu et al., J. Am. Chem. Soc. 14: 11155-11161 (2001). Probes can comprise reporter dyes such as, for example, 6-carboxyfluorescein (6-FAM) or tetrachlorofluorescin (TET) and the like. Detector probes can also comprise quencher moieties such as tetramethylrhodamine (TAMRA), Black Hole Quenchers (Biosearch Technologies, Novato, CA), Iowa Black (IDT, Coralville, IA), QSY quencher (Molecular Probes, Eugene, OR), and DABSYL and DABCEL sulfonate / carboxylate Quenchers (Epoch Biosciences, Bothell, WA) and the like. Probes can also comprise two probes, wherein for example a fluorophore is on one probe, and a quencher on the other, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on a target alters the signal signature via a change in fluorescence.

[0176] Exemplary detectable labels include, for instance, a fluorescent dye or fluorophore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like.Suitable detectable labels may include, for example, fluoresceins (e.g., 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 6-HAT; 6- JOE; 6-carboxyfluorescein (6-FAM); FITC); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY® fluorophores (e.g., 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, Fl-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g., calcium crimson, calcium green, calcium orange, cal cofluor white), Cascade Blue, CascadeYellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e.g., GFP. EGFP), blue fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e.g., fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / dabcyl, fluorescein / fluorescein, BODIPY® FL / BODIPY® FL, Fluorescein / QSY7 and QSY9), LysoTracker and LysoSensor (e.g., LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153, LysoSensor Yellow / Blue DND-160, LysoSensor Yellow / Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra, 5- carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, 5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in, e.g., US Publication No. 2009 / 0197254), among others as would be known to those of skill in the art.

[0177] In some embodiments, the probes are designed according to the methods and principles described in, for example, U.S. Patent No. 6,727,356 (the disclosure of which is incorporated herein by reference in its entirety). Some probes can be sequence-based, for example 5' nuclease probes and some, such as SYBR® Green can be non-sequence specific DNA-binding dyes. In some preferred embodiments, the detector probe is a TaqMan® probe (Applied Biosystems, Foster City, CA). It is to be understood that a wide variety of probes are known in the art that can be used in the present compositions, methods and kits, including those not specifically disclosed herein.

[0178] In some embodiments of the disclosed compositions, the final probe concentration in a working solution can range from about 5 nM to about 750 nM, such as about 10 nM to about 600 nM, about 25 nM to about 500 nM, about 50 nM to about 400 nM, about 75 nM to about 300 nM, or any number in between. In some exemplary embodiments, the probe concentration is between about 100 nM to about 250 nM.Additional Components / Additives

[0179] Other additives capable of facilitating or enhancing reverse transcription, amplification, or a combination of both reactions (e.g., agents for facilitating or enhancing RT-qPCR), other than those disclosed herein, are known in the art. In accordance with the present compositions and methods, one or more of these additives can be incorporated in the present compositions to optimize the generation and replication of nucleic acids from a ribonucleic acid or deoxyribonucleic acid templates. Additives can be organic or inorganic compounds. Some additives useful in the present compositions, methods and kits include polypeptides as well as nonpolypeptide additives. Such additives can include, for example, RNase inhibitor protein (RIP), lectins, E. coli single- stranded binding (SSB) protein, tRNA, rRNA, 7-deaza-2'-deoxyguanosine (dC7GTP), sulfur-containing compounds, acetate-containing compounds, dimethylsulfoxide (DMSO), glycerol, formamide, betaine, tetramethylammonium chloride (TMAC), polyethylene glycol (PEG), various surfactants or generally any zwitterionic, cationic, anionic or non-ionic (e g., TWEEN 20, NP-40, Triton X-100, and CHAPS) detergents, ectoine, sodium azide, kathon, and polyols, to name just a few. Those of ordinary skill in the art will be able to identify additional additives for use in accordance with the present compositions, methods and kits.

[0180] The compositions and methods in accordance with the present disclosure can also include additional “hot start” PCR components or steps, as a means to further prevent, reduce or eliminate nonspecific nucleic acid synthesis. The term “hot start” , as used herein, refers to any modified form of PCR which prevents non-specific amplification of DNA by inactivating the polymerase activity at lower annealing temperatures and reactivating or activating the polymerase activity at higher temperatures during the extension phase. Many hot start mechanisms are well known to those of ordinary skill in the art and will be readily selectable based on their ability to work in accordance with the present disclosure. In some embodiments, the hot start components that can be optionally added to the present compositions can include, for example, an antibody or antibodies, specially designed primers, competitive oligonucleotides or aptamers, polymerase binding proteins or sequestration beads. Sequestration wax beads for hot start PCR are commercially available, e g., AmpliWax® PCR Gem 100 and AmpliWax® PCR Gem 50 (Applied Biosystems, Foster City, CA). Selection of a suitable aptamer can be performed by a method known in the art or a commercially available aptamer can be used. Similarly, selection of a suitable primer can be performed by a method known in the art or a commercially available primer can be used. In some cases, a suitable primer can be a primer specially designed to have secondarystructures that prevent the primers from annealing until cycling temperatures denature them. Antibodies for hot start PCR can be generated or selected by a method known in the art.Alternatively, a commercially available antibody can be used, for example, the TaqStart® Antibody (Clontech, Mountain View, CA) which is effective with any Taq-derived DNA polymerase, including native, recombinant, and N-terminal deletion mutants. An appropriate concentration of the reagent for hot start PCR in the assembled PCR can be determined by a method known in the art or, for a commercial product, as suggested by the manufacturer. Other examples of hot start components or mechanisms used for this purpose are known in the art (see, e.g., U.S. Patent No. 6,403,341 and U.S. Patent Application Publication No. 2009 / 0269766, the disclosures of which are fully incorporated herein by reference in their entireties.)

[0181] The compositions and methods in accordance with the present disclosure can also include a passive reference control. In some embodiments the passive reference control is used to minimize sample-to-sample or well-to-well variations in quantitative real-time nucleic aciddetection assays and can be included at a concentration allowing its use as detectable control. In an embodiment, a reference chromophore, specifically a fluorophore, is included as the passive reference control. In an embodiment, the reference chromophore is the dye ROX (Invitrogen, Carlsbad, CA). In one embodiment, ROX can be included in the composition at a concentration in a working solution of about 40 nM to about 80 nM, specifically about 60 nM.

[0182] It is to be understood that a wide variety of additional components known in the art can be useful in the present compositions, methods and kits, including those not specifically disclosed herein. Those of skill in the art will also understand the methods required to determine the particular conditions or concentrations to use of each component in accordance with the present disclosure.Buffers and Salts

[0183] To form the compositions of the present disclosure, one or more reverse transcriptases and one or more DNA polymerases are preferably mixed in a buffered salt solution. In accordance with the present disclosure, buffer agents or salt solutions used in the present compositions and reaction mixtures provide appropriate pH and ionic conditions to maintain stability of the enzymes having reverse transcriptase activity or DNA polymerase activity. The terms “stable” and “stability” , as used herein, generally mean the retention by a composition, such as an enzymecomposition, of at least 70%, preferably at least 80%, and most preferably at least 90%, of the original enzymatic activity (in units) after the enzyme or composition containing the enzyme has been stored for about 3 days at a temperature of about room temperature (e.g., about 20 °C to about 25 °C), about one week at a temperature of about 4 °C, about two to six months at a temperature of about -20 °C, and about six months or longer at a temperature of about -80 °C. Examples of such buffering agents can include, for example, TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. In some embodiments, the buffering agent includes tris with BSA to provide a buffered pH of about 8. Examples of such salt solutions can include, for example, potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, and lithium acetate. It is to be understood that a wide variety of buffers and salt solutions are known in the art that can be used in the present compositions, methods and kits, including those not specifically disclosed herein.

[0184] In some embodiments, the compositions can be provided as a concentrated stock. As used herein, the term “concentrated stock” means at a concentration that requires further dilution in order to achieve optimal concentration for use in a solution to perform a particular function (such as reverse transcription of nucleic acids). As used herein, “working solution” can be used to refer to the solution having an optimal concentration to perform a particular function. For example, compositions of the present disclosure can be stock solutions of about 2X, about 3X, about 4X, about 5X, about 6X, about 10X, and so on. In some preferred embodiments, the stock solution is about 4X. In some preferred embodiments, the compositions can require greater than 2X, greater than 3X, greater than 4X, greater than 5X, greater than 6X, greater than 10X, and so on, dilution to be at working, or optimal, concentration for use in nucleic acid synthesis methods.Nucleic Acid Samples

[0185] Nucleic acid samples suitable for use in accordance with the present disclosure can include any quantity of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules can be extracellular nucleic acid molecules. In other embodiments, such nucleic acid molecules can be derived from cells. In general, such cells can include, for example, any prokaryotic, eukaryotic or plant cell. Such cells can be normal cells, diseased cells, transformedcells, established cells, progenitor cells, precursor cells, fetal cells, embryonic cells, bacterial cells, yeast cells, animal cells (including human cells), avian cells, plant cells and the like, or tissue isolated from a plant or an animal (e.g., human, cow, pig, mouse, sheep, horse, monkey, canine, feline, rat, rabbit, bird, fish, insect, etc.). In some preferable embodiments, nucleic acid molecules can also be isolated from viruses. In accordance with the present methods, such nucleic acid samples can be extracted from a variety of sources. These include, but are not limited to, for example clothing, soil, skin, hair, blood, serum, feces, milk, saliva, urine, or other secretory fluids. These sources can also contain compounds that inhibit PCR amplification.

[0186] In accordance with the present disclosure, the nucleic acid samples or templates can be any ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) of interest, known or unknown, to the practitioner. Nucleic acid samples can be artificially synthesized or isolated from natural sources. In some preferred embodiments the nucleic acid sample is single stranded. Alternatively, the nucleic acid sample can be double stranded. In some embodiments the nucleic acid sample can be messenger RNA (mRNA), RNA, genomic DNA (gDNA) or cDNA. Many nucleic acid sample preparation or isolation methods are known in the art. A variety of nucleic acid isolation or preparation kits are also available commercially, for example, MagMAX™ (Applied Biosystems, Foster City, CA), iPrep™ (Invitrogen, Carlsbad, CA) and QIAmp MinElute (Qiagen, San Diego, CA).Methods of Nucleic Acid Synthesis

[0187] In accordance with the present disclosure, the above compositions can be used in methods for nucleic acid synthesis of one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty, etc.) nucleic acid molecules comprising mixing one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty, etc.) nucleic acid templates. In some embodiments, RNA or mRNA can serve as the template for nucleic acid synthesis by one or more reverse transcriptase. Alternatively, cDNA or gDNA can serve as the template for nucleic acid synthesis by one or more polymerases. In some embodiments, such methods can comprise incubating the mixture comprising one or more reverse transcriptases or one or more polymerases under conditions sufficient to make a nucleic acid molecule or molecules complementary to all or a portion of the one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty, thirty, etc.) templates. To make the nucleic acid molecule or moleculescomplementary to the one or more templates, a primer (e.g., an oligo(dT) primer) and one or more nucleotides are preferably used for nucleic acid synthesis in the 5' to 3' direction.

[0188] Additional embodiments provide methods for amplifying a nucleic acid molecule comprising contacting the nucleic acid molecule with a polymerase. In some embodiments, simplex (i.e., single) amplification reactions are performed at one time in a single reaction or vessel. In other embodiments, multiplex (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000 and so on) amplification reactions are performed at one time in a single reaction or vessel. As used herein, “multiplex” or “multiplexing” refers to the essentially simultaneous amplification or analysis of multiple targets in a single reaction. In some embodiments, multiplexing can involve the amplification of a single or multiple targets from one or multiple sample input(s) and an exogenous control target from one exogenous control template within the same reaction vessel (e.g., tube, compartment, well). In some preferred embodiments, such methods can comprise one or more polymerase chain reactions (PCRs). For example, such multiplex PCR reactions can comprise the essentially simultaneous amplification of greater than 1, greater than 2, greater than 3, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 20, greater than 50, greater than 100, greater than 1000, etc. nucleic acid targets within the same reaction.

[0189] In some embodiments, such PCR methods can be quantitative PCR (qPCR) or end point PCR amplification methods. In some preferred embodiments, such PCR methods are real time PCR amplification methods. In some embodiments, such PCR methods can comprise thermal cycling, which can comprise alternating heating and cooling of the mixture sufficient to amplify the DNA molecule and which most preferably comprises alternating from a first temperature range of from about 90 °C to about 100 °C, to a second temperature range of from about 45 °C to about 75 °C, from about 50 °C to about 70 °C, from about 55 °C to about 65 °C, or preferably at about 58 °C, at about 59 °C, at about 60 °C, at about 61 °C or at about 62 °C. In some embodiments, the thermal cycling can be performed any number of times, such as any number greater than about 10 times, greater than about 20 times, greater than about 30 times, or from about 5 to about 80 times, about 10 to about 70 times, about 20 to about 60 times, or preferably from about 30 to about 50 times. In some other embodiments, the thermal cycling can be optimized for fast thermal cycling. Such protocols and apparatuses for fast thermal cycling can be found in, for example, U.S. Patent Nos. 6,210,882, or Kopp, et al., Science 280: 1046-1048 (1998); Chiou, et al., Anal. Chem.73:2018-2021 (2001) (for modified electric heating elements); Kalinina, et al., Nucleic Acids Res. 25: 1999-2004 (1997) (for hot air cyclers); and Giordano, et al., Anal. Biochem. 291 : 124-132 (2001) (for infrared controlled reactions), the disclosures of which are fully incorporated herein by reference in their entireties.

[0190] Such PCR thermal cycling can be performed on a variety of instruments known to those of skill in the art. Some instruments can be commercially available, for example, from Applied Biosystems (e.g., AB SDS Instruments 7300 Real-Time PCR System, 7500 Real-Time PCR System, 7500 Fast Real-Time PCR System, 7900HT Real-Time PCR System, StepOne Real-Time PCR System and StepOne Plus Real-Time PCR System, or ViiA 7 Real-Time PCR System). It is to be understood that a wide variety of instruments are known in the art that may be useful in the present methods, including those not specifically disclosed herein.

[0191] In other embodiments, the present compositions can be used in methods for one-step (or coupled) RT-qPCR. In some embodiments, RT-qPCR reaction mixtures are incubated at a temperature sufficient to synthesize a DNA molecule complementary to all or portion of an RNA template (e.g., cDNA) and then incubated at a second temperature sufficient to amplify newly synthesized cDNA molecules. In accordance with the present methods, such temperatures used for cDNA synthesis can range from about 30 °C to about 75 °C, about 35 °C to about 70 °C, about 40 °C to about 60 °C, or preferably from about 45 °C to about 55 °C. In accordance with the present methods, such temperatures used for cDNA amplification can range from about 40 °C to about 80 °C, about 45 °C to about 75 °C, about 50 °C to about 70 °C or preferably from about 55 °C to about 65 °C.

[0192] In some embodiments of the present methods (including, for example, those methods for nucleic acid synthesis, nucleic acid amplification, qPCR, or RT-qPCR), the use of at least one stabilizing agent can increases the reaction efficiency during a PCR process, especially when more than one target is present in the sample. In some embodiments, the use of at least one stabilizing agent permits an increased level of the DNA polymerase to allow balancing of benchtop stability with multiplex capacity.

[0193] In some embodiments of the present methods (including, for example, those methods for nucleic acid synthesis, nucleic acid amplification, qPCR, or RT-qPCR), the use of at least one qPCR inhibitor blocking agent can increase tolerance to one or more qPCR inhibitors. In some embodiments, increased tolerance can be indicated by, for example, a decrease in Ct or increase indRn (e.g., when analyzed by real time PCR) or by an increase in the amount of amplified product (e.g., when analyzed by agarose gel electrophoresis).

[0194] In some embodiments of the present methods, qPCR inhibitor tolerance (as determined by Ct) can be increased by at least about 10% (e.g., about 10%, about 20%, about 30%, about 40%, about 60%, about 80%, etc.) when using at least one qPCR inhibitor blocking agent compared to methods that do not. In other embodiments of the present methods, Ct value is decreased at least one (e.g., at least 1, at least 2, at least 3 at least 5, at least 10, etc.) compared to the Ct value achieved for methods that employ compositions without qPCR inhibitor blocking agents. In other embodiments, methods that utilize compositions comprising at least 500 ng / pL BSA can decrease Ct by at least 8 for reactions comprising at least 40 pM hematin, or by at least 7 for reactions comprising at least 10 ng / pL humic acid. In yet other embodiments, methods utilize compositions that are free or substantially free of gelatins, including fish gelatin. In other embodiments, a neutral compound with a cationic group lacking a hydrogen, such as, for example, ethylene glycol, betaine and 1,2-propanediol, serves as a qPCR inhibitor blocking agent. In yet other embodiments, a nonionic polyoxyethylene surfactant, such as, for example, Brij 35, also serves as a qPCR inhibitor blocking agent.

[0195] In some embodiments of the present methods, an RNA sample is stored at -80 °C, a DNA sample is stored at -20 °C, and a master mix composition of the present disclosure is stored at -25 °C to -15 °C.

[0196] In some embodiments of the present methods, an RT-qPCR mix is prepared by first thawing a sample on ice, thawing assays on ice or on the benchtop, and thawing a master mix composition of the present disclosure on ice or on the benchtop. The assays are vortexed briefly to mix then centrifuged to collect. Thawed samples are mixed by gentle inversion or by flicking three to five times and then centrifuged to collect the contents at the bottom of the tube. These mixing and centrifuging steps may be repeated if the compositions are not sufficiently thawed. The total volume for each reaction component is then calculated according to Table 1. Working on ice, the components are added directly to each well of an optical reaction plate. The components may be mixed together without the sample then added to the plate. The sample may be added directly to the wells of the plate. The reaction plate is covered with an optical adhesive cover and inverted three to five times, making sure that the contents of the wells are moving back and forth between the seal and the bottom of the well to ensure proper mixing. The reaction plate is then centrifuged at 150 *g (1000 rpm) for one minute to collect the contents at the bottom of the wells and eliminate air bubbles.Table 1 Component Amountsincludes 10% overage.[2]Potential assays include the TaqMan™ Assay Mix, FAM™ dye; TaqMan™ Assay Mix, VIC™ dye; TaqMan™ Assay Mix, ABY™ dye; and TaqMan™ Assay Mix, JUN™ dye, Cy®5 dye, Cy®5.5 dye, and Alexa™647 dye. Assays can be at a concentration other than 20X. Scale the volume appropriately.

[0197] In some embodiments of the present methods, setting up and running the qPCR instrument includes setting up the following parameters in the qPCR system software: 20 pL recommended sample volume (may be varied), default auto increment settings value, default data collection value, and default ramp rate settings value. The thermal protocol is set up according to Table 2. The passive reference dye is selected, preferably between TaqPath™DuraPlex™ 1-Step RT-qPCR Master Mix and TaqPath™DuraPlex™ 1-Step RT-qPCR Master Mix (No ROX™). The reaction plate is then loaded into the qPCR system and the run is started.Table 2 Thermal Protocolhl RT enzyme functions best in the range of 48 to 55°C.[2]Required time for RT inactivation and initial denaturation, and to activate the DNA polymerase.[3]Annealing temperature should be consistent with melting temperature (Tm) of primer designs.

[0198] In some embodiments of the present methods, the results of the run are analyzed as appropriate for the assay and instrument. The standard curve method or the relative quantification method is preferably used to analyze the results. The qPCR system software can be used to set the baseline and threshold values for the amplification plot. They can be set automatically or manually. The baseline is the initial cycle in which there is a change in the fluorescence signal. The intersection of the threshold and the amplification plot defines the Ct value. In qPCR assays, the threshold is set above the background signal and within the exponential growth phase of the amplification curve. General guidelines for analysis include viewing the amplification plot and then, if needed, adjusting the baseline and threshold values and reviewing the replicates and outliers. In the well table or results table, the Ct (Cq) values are viewed for each well and for each replicate group. For standard curve experiments, the slope, amplification efficiency, R2value, y- intercept, Ct values, and outliers are viewed.Kits

[0199] In another embodiment, the present compositions and methods can be assembled into kits for use in reverse transcription or amplification of a nucleic acid molecule. Kits according to this embodiment can comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, plates, bottles and the like, wherein a first container means contains one or more polypeptides of the present disclosure having reverse transcriptase activity and one or more DNA polymerases. When more than one reverse transcriptases or DNA polymerases are used, they can be in a singlecontainer as mixtures of two or more (e.g., 2, 3, 4, 5, etc) reverse transcriptases or DNA polymerases, or in separate containers. The kits provided herein can also comprise (in the same or separate containers), a suitable buffer, one or more nucleotides, one or more stabilizing agents, one or more qPCR inhibitor blocking agents, one or more probes, or one or more primers. In some preferable embodiments, the reverse transcriptase(s), DNA polymerase(s), stabilizing agent(s), qPCR inhibitor blocking agent(s), nucleotides, and a suitable buffer are combined in a single tube or container.

[0200] In a specific embodiment, the reverse transcription and amplification kits can comprise one or more components (in mixtures or separately) including one or more polypeptides having reverse transcriptase activity and one or more DNA polymerases. Such reverse transcription and amplification kits can further comprise one or more nucleotides needed for synthesis of a nucleic acid molecule, one or more probes or one or more primers (e.g., oligo(dT) for reverse transcription). Preferred polypeptides having reverse transcriptase activity, DNA polymerases, nucleotides, probes, primers and other components suitable for use in the reverse transcription and amplification kits provided herein include those described above. The kits encompassed by this embodiment can further comprise additional reagents and compounds necessary for carrying out standard nucleic acid reverse transcription and / or amplification protocols. Such polypeptides having reverse transcriptase activity, DNA polymerases, stabilizing agents, qPCR inhibitor blocking agents, nucleotides, probes, primers, and additional reagents, components or compounds can be contained in one or more containers, and can be contained in such containers in a mixture of two or more of the above-noted components or can be contained in the present kits in separate containers. Those of skill in the art will understand that other components, either in the same tube or in separate tubes, may also be included in the kit to further facilitate or enhance reverse transcription or amplification. Such components or additives can include for example, Mg2+, uracil DNA glycosylase, a passive reference control to minimize sample-to-sample or well-to-well variations in quantitative real-time DNA-detection assays (e.g., dyes such as ROX) and various hot start components (e.g., antibodies, oligonucleotides, beads, etc).

[0201] In another embodiment, the present kits can comprise compositions for use in nucleic acid synthesis (e.g., qPCR or RT-qPCR). Such compositions can be formulated as concentrated stock solutions (e.g., 2X, 3X, 4X, 5X, 6X, etc). In some embodiments, the compositions can be formulated as concentrated stock solutions in a single tube or container, comprising one or morepolypeptides having reverse transcriptase activity and one or more DNA polymerases. In some preferred embodiments, such concentrated stock compositions can further comprise one or more stabilizing agents, one more qPCR inhibitor blocking agents, one or more nucleotides, one or more host start components, one or more passive reference controls, or one or more RNase inhibitor proteins (RIP) in a buffered solution. In some additional preferred embodiments, such buffer solutions can comprise glycerol, DMSO, Mg2+, or a detergent (such as TWEEN 20 or NP-40). Collectively, the components of the present composition can be formulated together to create a master mix.

[0202] Typically master mixes for use in nucleic acid synthesis or amplification methods are stored at freezing temperatures to maintain enzyme stability (for example, of the reverse transcriptases or DNA polymerases) and are then thawed and diluted for subsequent assembly into final reactions mixtures. However, repeated master mix freeze-thaw cycles over time can lead to degradation of the enzymes resulting in decreased stability or functionality. In one aspect, the compositions, kits, or master mixes included in the kits described herein can be stored at about -16 °C, about -18 °C, about -20 °C, about -22 °C, about -24 °C, about -26 °C, about -28 °C, about -30 °C without freezing. In some preferable embodiments, the compositions of the kits are stored at about -20 °C without freezing. In some embodiments, the present composition can be stored at freezing temperatures (e.g., below 0 °C, below -5 °C, below -20 °C, below, -30 °C, below -40 °C, etc.) without having to be thawed prior to use. As used herein, the term “thaw,” “thawing” or “thawed” refers to the process whereby heat changes something from a solid (e.g., frozen) to a gel or liquid. In some embodiments, the present compositions can be a liquid or a gel (or viscous liquid) at freezing temperatures. In some embodiments, such compositions, especially if in gel form, can be incubated at about 4 °C prior to subsequent use to ensure proper mixing, but do not require thawing per se.Experimental Data

[0203] FIGS. 2A and 2B are amplification plots at t=0 and / =24 hrs of a PCR reaction performed using a first exemplary composition.

[0204] Referring to FIGS. 2A and 2B, a PCR assay was run on a QuantStudio Flex with a 384- well block using 10-pl reaction mixtures including bacteriophage MS2 as a target and ABY as a reporter dye. Cl shows amplification of amplification products prepared using an exemplarycomposition including a RT aptamer, a stabilizing agent and qPCR inhibitor blocking agents. C2 shows amplification of amplification products prepared using a conventional composition that does not include a RT aptamer, the stabilizing agent or qPCR inhibitor blocking agents. FIG. 2A shows the amplification at the start of the run (t = 0), and FIG. 2B shows the amplification at t = 24hrs at 24°C.

[0205] As shown in FIG. 2B, the amplification of the amplification products prepared using an exemplary composition, Cl, is significantly greater after 24 hours than that of the amplification products prepared using a conventional composition, C2. Thus, the exemplary composition, Cl, demonstrated enhanced benchtop stability compared to the conventional composition, C2.

[0206] FIGS. 3A and 3B are amplification plots at t=Q and Z=24 hrs of a PCR reaction performed using a second exemplary composition.

[0207] Referring to FIGS. 3 A and 3B, a PCR assay was run on a QuantStudio Flex with a 384- well block using 10-pl reaction mixtures including MOB kinase activator IB as a target and FAM as a reporter dye. Cl shows amplification of amplification products prepared using an exemplary composition including a RT aptamer, a stabilizing agent and qPCR inhibitor blocking agents. C2 shows amplification of amplification products prepared using a conventional composition that does not include a RT aptamer, the stabilizing agent or qPCR inhibitor blocking agents. FIG. 2A shows the amplification at the start of the run (t = 0), and FIG. 2B shows the amplification at t = 24hrs at 24°C.

[0208] As shown in FIG. 3B, the amplification of the amplification products prepared using an exemplary composition, Cl, is greater after 24 hours than that of the amplification products prepared using a conventional composition, C2. Thus, the exemplary composition, Cl, demonstrated enhanced benchtop stability compared to the conventional composition, C2.

[0209] FIG. 4 shows amplification plots of different targets using an exemplary composition.

[0210] Referring to FIG. 4, a 6-plex gene assay was run on a sample including 6 different targets in a single well.

[0211] The target genes, the reporter dyes and PCR efficiencies are shown in Table 3.TABLE 3

[0212] As shown in FIG. 4 and Table 3, the exemplary composition has a wide dynamic range and high sensitivity for targets at low copy numbers in a 6-plex performed in a single well.

[0213] FIG. 5 is a graph showing tolerance of exemplary compositions including an inhibitor blocking agent to the inhibitor heparin.

[0214] As show in FIG. 5, higher concentrations of an inhibitor blocking agent in the exemplary compositions resulted in lower dCq. Thus, the exemplary compositions exhibited better tolerance to heparin as the concentration of inhibitor blocking agent was increased.

[0215] FIG. 6 is a graph showing tolerance of exemplary compositions including a polyoxyethylene surfactant to the inhibitor heparin.

[0216] As show in FIG. 6, higher concentrations of a polyoxyethylene surfactant in the exemplary compositions resulted in lower dCq. Thus, the exemplary compositions exhibited better tolerance to heparin as the concentration of polyoxyethylene surfactant was increased.

[0217] In a first aspect, a composition, comprises a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

[0218] In an embodiment of the first aspect, said reverse transcriptase comprises a thermostable reverse transcriptase.

[0219] In an embodiment of the first aspect, said thermostable reverse transcriptase comprises an Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or a mutant, variant, or derivative thereof.

[0220] In an embodiment of the first aspect, said M-MLV reverse transcriptase comprises one or more mutations selected from the group consisting of: Y64, R116, D124, H126, Y133, K152, Q190, T197, H204, V223, M289, T306, and F309.

[0221] In an embodiment of the first aspect, said DNA polymerase comprises a thermostable DNA polymerase.

[0222] In an embodiment of the first aspect, said thermostable DNA polymerase comprises Taq DNA polymerase or a mutant, variant, or derivative thereof.

[0223] In an embodiment of the first aspect, said thermostable DNA polymerase comprises a recombinant Taq DNA polymerase or a mutant, variant, or derivative thereof.

[0224] In an embodiment of the first aspect, said at least one stabilizing agent comprises an RNA aptamer.

[0225] In an embodiment of the first aspect, said RNA aptamer interacts with the reverse transcriptase.

[0226] In an embodiment of the first aspect, said at least one stabilizing agent further comprises a sulfate additive.

[0227] In an embodiment of the first aspect, said sulfate additive is at least one selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0228] In an embodiment of the first aspect, said sulfate additive is at least one selected from the group consisting of magnesium sulfate and potassium sulfate.

[0229] In an embodiment of the first aspect, the composition further comprises at least one qPCR inhibitor blocking agent.

[0230] In an embodiment of the first aspect, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0231] In an embodiment of the first aspect, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0232] In an embodiment of the first aspect, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0233] In an embodiment of the first aspect, the composition further comprises a silicone-based emulsifier.

[0234] In an embodiment of the first aspect, the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0235] In an embodiment of the first aspect, the composition further comprises a hot-start component that reversibly inactivates the at least one DNA polymerase.

[0236] In an embodiment of the first aspect, the composition excludes gelatin or fish gelatin.

[0237] In an embodiment of the first aspect, the composition excludes uracil-DNA glycosylase (UDG) or uracil-N-glycosylase (UNG).

[0238] In an embodiment of the first aspect, the composition further comprises a passive reference control.

[0239] In an embodiment of the first aspect, said passive reference control comprises a ROX dye.

[0240] In an embodiment of the first aspect, said composition is for use in nucleic acid synthesis, nucleic acid amplification, a quantitative polymerase chain reaction (qPCR), or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR).

[0241] In a second aspect, a method of performing a quantitative polymerase chain reaction (qPCR) or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) on a nucleic acid sample, comprises mixing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or said RT-qPCR, with the nucleic acid sample; at least one primer pair having specificity for a nucleic acid in the nucleic acid sample; at least one labeled probe having specificity for amplicons complementary to all or a portion of the nucleic acid; and performing said qPCR or said RT-qPCR on said nucleic acid sample.

[0242] In an embodiment of the second aspect, said at least one stabilizing agent comprises an RNA aptamer that interacts with the reverse transcriptase to increase the stability of the assembled reactions.

[0243] In an embodiment of the second aspect, said at least one stabilizing agent further comprises a sulfate additive.

[0244] In an embodiment of the second aspect, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0245] In an embodiment of the second aspect, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0246] In an embodiment of the second aspect, said composition further comprises at least one qPCR inhibitor blocking agent.

[0247] In an embodiment of the second aspect, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0248] In an embodiment of the second aspect, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0249] In an embodiment of the second aspect, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0250] In an embodiment of the second aspect, the composition further comprises an non-ionic silicone emulsifier.

[0251] In an embodiment of the second aspect, said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0252] In an embodiment of the second aspect, said labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.

[0253] In an embodiment of the second aspect, said qPCR or said RT-qPCR is performed in a single tube or a single reaction.

[0254] In an embodiment of the second aspect, said performing said qPCR or said RT-qPCR comprises thermal cycling or fast thermal cycling.

[0255] In an embodiment of the second aspect, said quantitative polymerase chain reaction (qPCR) or said quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is a multiplex reaction capable of detecting up to six target nucleic acids.

[0256] In a third aspect, a method for amplifying a nucleic acid comprises mixing a nucleic acid template with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incubating said reaction mixture under conditions sufficient to amplify the nucleic acid, the nucleic acid being complementary to all or a portion of said nucleic acid template.

[0257] In an embodiment of the third aspect, said nucleic acid template is a template having RNA.

[0258] In an embodiment of the third aspect, said nucleic acid is DNA.

[0259] In an embodiment of the third aspect, said at least one stabilizing agent comprises anRNA aptamer.

[0260] In an embodiment of the third aspect, said RNA aptamer interacts with the reverse transcriptase.

[0261] In an embodiment of the third aspect, said at least one stabilizing agent further comprises a sulfate additive.

[0262] In an embodiment of the third aspect, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0263] In an embodiment of the third aspect, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0264] In an embodiment of the third aspect, said composition further comprises at least one qPCR inhibitor blocking agent.

[0265] In an embodiment of the third aspect, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0266] In an embodiment of the third aspect, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0267] In an embodiment of the third aspect, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0268] In an embodiment of the third aspect, the composition further comprises an non-ionic silicone emulsifier.

[0269] In an embodiment of the third aspect, the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0270] In a fourth aspect, a method for nucleic acid synthesis comprises mixing a sample having first nucleic acid with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; and incubating said mixture under conditions sufficient to produce a second nucleic acid that is complementary to all or a portion of said first nucleic acid.

[0271] In an embodiment of the fourth aspect, said first nucleic acid is RNA.

[0272] In an embodiment of the fourth aspect, said second nucleic acid is DNA.

[0273] In an embodiment of the fourth aspect, said at least one stabilizing agent comprises anRNA aptamer.

[0274] In an embodiment of the fourth aspect, said RNA aptamer interacts with the reverse transcriptase.

[0275] In an embodiment of the fourth aspect, said at least one stabilizing agent further comprises a sulfate additive.

[0276] In an embodiment of the fourth aspect, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0277] In an embodiment of the fourth aspect, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0278] In an embodiment of the fourth aspect, said composition further comprises at least one qPCR inhibitor blocking agent.

[0279] In an embodiment of the fourth aspect, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0280] In an embodiment of the fourth aspect, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0281] In an embodiment of the fourth aspect, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0282] In an embodiment of the fourth aspect, the composition further comprises an non-ionic silicone emulsifier.

[0283] In an embodiment of the fourth aspect, said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0284] In a fifth aspect, a reaction mixture comprises a master mix composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; at least one primer having a sequence specific to a target nucleic acid in a sample mixed with said reaction mixture in the assembled polymerase chain reaction; and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

[0285] In an embodiment of the fifth aspect, said at least one labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.

[0286] In an embodiment of the fifth aspect, said target nucleic acid is RNA, and the amplicons are DNA.

[0287] In an embodiment of the fifth aspect, said at least one stabilizing agent comprises an RNA aptamer.

[0288] In an embodiment of the fifth aspect, said RNA aptamer interacts with said at least one reverse transcriptase.

[0289] In an embodiment of the fifth aspect, said at least one stabilizing agent further comprises a sulfate additive.

[0290] In an embodiment of the fifth aspect, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0291] In an embodiment of the fifth aspect, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0292] In an embodiment of the fifth aspect, said composition further comprises at least one qPCR inhibitor blocking agent.

[0293] In an embodiment of the fifth aspect, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0294] In an embodiment of the fifth aspect, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0295] In an embodiment of the fifth aspect, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0296] In an embodiment of the fifth aspect, wherein the master mix composition further comprises an non-ionic silicone emulsifier.

[0297] In an embodiment of the fifth aspect, said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0298] In a sixth aspect, a kit comprises a single container containing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

[0299] In an embodiment of the sixth aspect, said kit is used for qPCR or RT-qPCR.

[0300] In an embodiment of the sixth aspect, said kit is used for nucleic acid synthesis.

[0301] In an embodiment of the sixth aspect, said kit is used for nucleic amplification.

[0302] In an embodiment of the sixth aspect, the kit further comprises another container containing a second composition including at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction, and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

[0303] In an embodiment of the sixth aspect, the kit further comprises another container containing at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to the assembled polymerase chain reaction; and a further container containing at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

[0304] In an embodiment of the sixth aspect, said at least one probe is a dual-labeled probe with a reporter dye and a quencher dye.

[0305] In an embodiment of the sixth aspect, said reverse transcriptase is a Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or any mutant, variant or derivative thereof having reverse transcriptase activity.

[0306] In an embodiment of the sixth aspect, said polymerase is Taq DNA polymerase or any mutant, variant or derivative thereof having DNA polymerase activity.

[0307] In an embodiment of the sixth aspect, said at least one stabilizing agent comprises an RNA aptamer.

[0308] In an embodiment of the sixth aspect, said RNA aptamer interacts with said reverse transcriptase.

[0309] In an embodiment of the sixth aspect, said at least one stabilizing agent further comprises a sulfate additive.

[0310] In an embodiment of the sixth aspect, said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

[0311] In an embodiment of the sixth aspect, said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

[0312] In an embodiment of the sixth aspect, said composition further comprises at least one qPCR inhibitor blocking agent.

[0313] In an embodiment of the sixth aspect, said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

[0314] In an embodiment of the sixth aspect, said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

[0315] In an embodiment of the sixth aspect, said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

[0316] In an embodiment of the sixth aspect, the composition further comprises an non-ionic silicone emulsifier.

[0317] In an embodiment of the sixth aspect, the silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

[0318] Components of the kit other than the compositions disclosed herein can be provided in individual containers or in a single container, as appropriate. Instructions and protocols for using the kit advantageously can be provided.

[0319] It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein may be made without departing from the scope of the present disclosure or any embodiment thereof. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMSWhat is claimed:

1. A composition, comprising: a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

2. The composition of claim 1, wherein said reverse transcriptase comprises a thermostable reverse transcriptase.

3. The composition of claims 1 or 2, wherein said thermostable reverse transcriptase comprises an Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or a mutant, variant, or derivative thereof.

4. The composition of claims 1-3, wherein said M-MLV reverse transcriptase comprises one or more mutations selected from the group consisting of: Y64, R116, D124, H126, Y133, K152, Q190, T197, H204, V223, M289, T306, and F309.

5. The composition of claims 1-4, wherein said DNA polymerase comprises a thermostable DNA polymerase.

6. The composition of claim 5, wherein said thermostable DNA polymerase comprises Taq DNA polymerase or a mutant, variant, or derivative thereof.

7. The composition of claim 5, wherein said thermostable DNA polymerase comprises a recombinant Taq DNA polymerase or a mutant, variant, or derivative thereof.

8. The composition of claims 1-7, wherein said at least one stabilizing agent comprises an RNA aptamer.

9. The composition of claim 8, wherein said RNA aptamer interacts with said reverse transcriptase.

10. The composition of claims 1-9, wherein said at least one stabilizing agent further comprises a sulfate additive.

11. The composition of claim 10, wherein said sulfate additive is at least one selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

12. The composition of claim 11, wherein said sulfate additive is at least one selected from the group consisting of magnesium sulfate and potassium sulfate.

13. The composition of claims 1-12, further comprising at least one qPCR inhibitor blocking agent.

14. The composition of claim 13, wherein said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

15. The composition of claims 13-14, wherein said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

16. The composition of claim 15, wherein said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

17. The composition of claims 15 or 16, further comprising: a silicone-based emulsifier.

18. The composition of claim 17, wherein said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

19. The composition of claims 1-18, further comprising: a hot-start component that reversibly inactivates said at least one DNA polymerase.

20. The composition of claims 1-19 excluding gelatin or fish gelatin.

21. The composition of claims 1-20 excluding uracil-DNA glycosylase (UDG) or uracil-N- glycosylase (UNG).

22. The composition of claims 1-21, further comprising: a passive reference control.

23. The composition of claim 22, wherein said passive reference control comprises a ROX dye.

24. The composition of claims 1-23, wherein said composition is for use in nucleic acid synthesis, nucleic acid amplification, a quantitative polymerase chain reaction (qPCR), or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR).

25. A method of performing a quantitative polymerase chain reaction (qPCR) or a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) on a nucleic acid sample, comprising: mixing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said stabilizing agent increases stability of assembled reactions of said qPCR or said RT-qPCR, with said nucleic acid sample; at least one primer pair having specificity for a nucleic acid in said nucleic acid sample;at least one labeled probe having specificity for amplicons complementary to all or a portion of said nucleic acid; and performing said qPCR or said RT-qPCR on said nucleic acid sample.

26. The method of claim 25, wherein said at least one stabilizing agent comprises an RNA aptamer that interacts with said reverse transcriptase to increase the stability of the assembled reactions.

27. The method of claims 25-26, wherein said at least one stabilizing agent further comprises a sulfate additive.

28. The method of claim 27, wherein said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

29. The method of claim 27-28, wherein said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

30. The method of claims 25-29, wherein said composition further comprises at least one qPCR inhibitor blocking agent.

31. The method of claim 30, wherein said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

32. The method of claims 30-31, wherein said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

33. The method of claims 32, wherein said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

34. The method of claims 32-33, wherein said composition further comprises an non-ionic silicone emulsifier.

35. The method of claim 34, wherein said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

36. The method of claims 25-35, wherein said labeled probe is a dual-labeled probe with a reporter dye and a quencher dye.

37. The method of claims 25-36, wherein said qPCR or said RT-qPCR is performed in a single tube or a single reaction.

38. The method of claims 25-37, wherein said performing said qPCR or said RT-qPCR comprises thermal cycling or fast thermal cycling.

39. The method of claims 25-38, wherein said quantitative polymerase chain reaction (qPCR) or said quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) is a multiplex reaction capable of detecting up to six target nucleic acids.

40. A method for amplifying a nucleic acid, said method comprising: mixing a nucleic acid template with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction, to form a reaction mixture; and incubating said reaction mixture under conditions sufficient to amplify said nucleic acid, the nucleic acid being complementary to all or a portion of said nucleic acid template.

41. The method of claim 40, wherein said nucleic acid template is a template having RNA.

42. The method of claims 40-41, wherein said nucleic acid is DNA.

43. The method of claims 40-42, wherein said at least one stabilizing agent comprises an RNA aptamer.

44. The method of claim 43, wherein said RNA aptamer interacts with said reverse transcriptase.

45. The method of claims 40-44, wherein said at least one stabilizing agent further comprises a sulfate additive.

46. The method of claim 45, wherein said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

47. The method of claims 45-46, wherein said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

48. The method of claims 40-47, wherein said composition further comprises at least one qPCR inhibitor blocking agent.

49. The method of claim 48, wherein said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

50. The method of claims 48-49, wherein said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

51. The method of claim 50, wherein said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and poly oxy ethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

52. The method of claims 50-51, wherein said composition further comprises an non-ionic silicone emulsifier.

53. The method of claim 52, wherein said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

54. A method for nucleic acid synthesis, said method comprising: mixing a sample having first nucleic acid with a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; and incubating said mixture under conditions sufficient to produce a second nucleic acid that is complementary to all or a portion of said first nucleic acid.

55. The method of claim 54, wherein said first nucleic acid is RNA.

56. The method of claims 54-55, wherein said second nucleic acid is DNA.

57. The method of claims 54-56, wherein said at least one stabilizing agent comprises an RNA aptamer.

58. The method of claim 57, wherein said RNA aptamer interacts with said reverse transcriptase.

59. The method of claims 54-58, wherein said at least one stabilizing agent further comprises a sulfate additive.

60. The method of claim 59, wherein said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

61. The method of claims 59-60, wherein said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

62. The method of claims 54-61, wherein said composition further comprises at least one qPCR inhibitor blocking agent.

63. The method of claim 62, wherein said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

64. The method of claims 62-63, wherein said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

65. The method of claim 64, wherein said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

66. The method of claims 64-65, wherein said composition further comprises an non-ionic silicone emulsifier.

67. The method of claim 66, wherein said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

68. A reaction mixture, comprising: a master mix composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction; at least one primer having a sequence specific to a target nucleic acid in a sample mixed with said reaction mixture in said assembled polymerase chain reaction; and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

69. The reaction mixture of claim 68, wherein said at least one labeled probe is a duallabeled probe with a reporter dye and a quencher dye.

70. The reaction mixture of claims 68-69, wherein said target nucleic acid is RNA, and said amplicons are DNA.

71. The reaction mixture of claims 68-70, wherein said at least one stabilizing agent comprises an RNA aptamer.

72. The reaction mixture of claim 71, wherein said RNA aptamer interacts with said at least one reverse transcriptase.

73. The reaction mixture of claims 68-72, wherein said at least one stabilizing agent further comprises a sulfate additive.

74. The reaction mixture of claim 73, wherein said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

75. The reaction mixture of claims 73-74, wherein said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

76. The reaction mixture of claims 68-75, wherein said composition further comprises at least one qPCR inhibitor blocking agent.

77. The reaction mixture of claim 76, wherein said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

78. The reaction mixture of claims 76-77, wherein said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

79. The reaction mixture of claim 78, wherein said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

80. The reaction mixture of claims 78-79, wherein said reaction mixture further comprises an non-ionic silicone emulsifier.

81. The reaction mixture of claim 80, wherein said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

82. A kit, comprising: a single container containing a composition including a reverse transcriptase, a DNA polymerase, at least one dNTP selected from the group consisting of dTTP, dATP, dCTP, dGTP and dUTP, and at least one stabilizing agent, wherein said at least one stabilizing agent increases stability of an assembled polymerase chain reaction.

83. The kit of claim 82, wherein said kit is used for qPCR or RT-qPCR.

84. The kit of claims 82-83, wherein said kit is used for nucleic acid synthesis.

85. The kit of claims 82-83, wherein said kit is used for nucleic amplification.

86. The kit of claims 82-85, further comprising: another container containing a second composition including at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to said assembled polymerase chain reaction, and at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

87. The kit of claims 82-85, further comprising: another container containing at least one primer having a sequence specific to a target nucleic acid in a sample to be subjected to said assembled polymerase chain reaction; and a further container containing at least one labeled probe that hybridizes to amplicons complementary to all or a portion of said target nucleic acid.

88. The kit of claims 86-87, wherein said at least one probe is a dual-labeled probe with a reporter dye and a quencher dye.

89. The kit of claims 82-88, wherein said reverse transcriptase is a Moloney Murine Leukemia virus (M-MLV) reverse transcriptase or any mutant, variant or derivative thereof having reverse transcriptase activity.

90. The kit of claims 82-88, wherein said polymerase is Taq DNA polymerase or any mutant, variant or derivative thereof having DNA polymerase activity.

91. The kit of claims 82-90, wherein said at least one stabilizing agent comprises an RNA aptamer.

92. The kit of claim 91, wherein said RNA aptamer interacts with said reverse transcriptase.

93. The kit of claims 82-92, wherein said at least one stabilizing agent further comprises a sulfate additive.

94. The kit of claim 93, wherein said sulfate additive is selected from the group consisting of magnesium sulfate, ammonium sulfate and potassium sulfate.

95. The kit of claims 93-94, wherein said sulfate additive is selected from the group consisting of magnesium sulfate and potassium sulfate.

96. The kit of claims 82-95, wherein said composition further comprises at least one qPCR inhibitor blocking agent.

97. The kit of claim 96, wherein said at least one qPCR inhibitor blocking agent comprises at least one selected from ethylene glycol, betaine and 1,2-propanediol.

98. The kit of claims 96-97, wherein said at least one qPCR inhibitor blocking agent comprises a nonionic polyoxyethylene surfactant.

99. The kit of claim 98, wherein said nonionic polyoxyethylene surfactant comprises at least one selected from polyoxyethylene octyl phenyl ether, polyoxyethylene lauryl ether, and polyoxyethylene(23)cetyl ether, and polyethylene glycol sorbitan monolaurate.

100. The kit of claims 98-99, wherein said composition further comprises an non-ionic silicone emulsifier.

101. The kit of claim 100, wherein said silicone-based emulsifier includes antifoam A, antifoam B, antifoam C, antifoam Y-30, and antifoam SE-15.

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