Internal control for nucleic acid amplification

Abstract

Disclosed herein are methods, kits, and reaction mixtures suitable for use in monitoring amplification reactions. Some embodiments provide first and second quality control primers that are hybridizable to each other and each comprise a quenchable label or quencher.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 309,827, filed February 14, 2022, the entire contents of which are incorporated herein by reference for all purposes. Sequence Listing Reference This application is filed with a sequence listing in electronic format. The sequence listing is provided in file 68EB-317346-WO, created on February 13, 2023, and is 8.0 kilobytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. The present disclosure relates generally to molecular biology. More specifically, disclosed herein includes methods and compositions for monitoring nucleic acid amplification. [Background technology]

[0002] Two main strategies are used for internal controls ("IC") in nucleic acid amplification assays: competitive and non-competitive internal control approaches. The main difference between the two strategies is whether the internal control components share a common set of primers for the internal control and target amplification. When using competitive IC strategy, there is always some competition between target and IC, along with the simultaneous amplification of target and IC fragments flanked by the same primer. Competition by IC amplification can reduce the target amplification efficiency, thereby resulting in a lower detection limit. Therefore, competitive IC method requires more optimization of IC to achieve a high sensitivity detection limit. In non-competitive approach, target and IC are amplified using different primer sets, respectively. Although the reaction rate of each reaction is not affected by the competition for primers, IC amplification must be limited by controlled concentration of IC-specific primers and / or IC template to limit the competition between target and IC reaction for primers and DNA polymerase. Therefore, the nucleotide composition, copy number, and size of IC must be carefully considered. There is a need to overcome the challenges presented by competition for target amplification in current competitive and noncompetitive IC methods and to reduce the complexity in performing quality control for nucleic acid amplification. Summary of the Invention

[0003] Disclosed herein is a method for monitoring an amplification reaction. In some embodiments, the method includes the steps of: (a) providing a first quality control primer and a second quality control primer, each of which comprises a 3' overlap region capable of hybridizing with each other, each of which is 35 nucleotides long or less, and the first quality control primer comprises a quenchable label; (b) contacting the first quality control primer and the second quality control primer, thereby forming a duplex by hybridization between the 3' overlap regions of the first and second quality control primers; (c) subjecting the duplex to amplification conditions to generate an extended duplex; and (d) detecting a signal generated from the quenchable label of the first quality control primer during the amplification reaction to determine the generation of the extended duplex, a decrease in the signal during the amplification reaction indicating the generation of the extended duplex.

[0004] The quenchable label may be a fluorophore. The position of the quenchable label on the quality control primer may vary. In some embodiments, the quenchable label is outside the 3' overlap region of the first quality control primer. In some embodiments, the quenchable label is at the 5' end of the first quality control primer. In some embodiments, the quenchable label is in the 3' overlap region of the first quality control primer. The second quality control primer may include a quencher. In some embodiments, the quencher is outside the 3' overlap region of the second quality control primer. In some embodiments, the quencher is at the 5' end of the second quality control primer. In some embodiments, the quencher is in the 3' overlap region of the second quality control primer. In some embodiments, the second quality control primer does not include a quencher. In some embodiments, neither the first nor the second quality control primer includes a quencher.

[0005] The first quality control primer, the second quality control primer, or both may comprise one or more modified nucleotides. For example, the 3' overlap regions of the first and second quality control primers may each comprise one or more modified nucleotides. In some embodiments, the one or more modified nucleotides comprise a spacer, an abasic site, an unmethylated RNA base, a 2'-O-methylated nucleotide, and any combination thereof. In some embodiments, at least one of the one or more modified nucleotides is a 2'-O-methylated nucleotide. In some embodiments, the first quality control primer, the second quality control primer, or both may comprise one or more polymerase stoppers. In some embodiments, the 3' overlap regions of the first and second quality control primers each comprise one or more polymerase stoppers. In some embodiments, at least one of the one or more polymerase stoppers is a 2'-O-methylated nucleotide.

[0006] The 3' overlap region of the first quality control primer may be complementary to the 3' overlap region of the second quality control primer. In some embodiments, the 3' overlap region of the first quality control primer is fully complementary to the 3' overlap region of the second quality control primer. In some embodiments, the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer have the same length. In some embodiments, the 3' overlap region of the first quality control primer, the 3' overlap region of the second quality control primer, or both, are about 2 to about 10 nucleotides in length, e.g., 4 or 5 nucleotides in length.

[0007] In some embodiments, (b) contacting the first quality control primer and the second quality control primer is performed under amplification conditions. In some embodiments, (d) detecting a signal generated from the quenchable label of the first quality control primer during the amplification reaction comprises detecting the signal at two or more different time points during the amplification reaction. In some embodiments, the decrease in signal during the amplification reaction comprises a decrease over time during the amplification reaction. In some embodiments, the decrease in signal during the amplification reaction comprises a decrease over a period of about 10 minutes during the amplification reaction. In some embodiments, the period is about 3 minutes to about 12 minutes from the start of the amplification reaction. The method may comprise detecting the signal of the quenchable label of the first quality control primer before the amplification reaction, after the amplification reaction, or both.

[0008] The amplification reaction may be a real-time amplification reaction, including, for example, a PCR reaction. In some embodiments, the amplification reaction includes one or more of the following: Archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). The amplification reaction may be, for example, an isothermal amplification reaction. In some embodiments, the amplification conditions include one or more of an enzyme with hyperthermophile polymerase activity, dNTPs, and a buffer. In some embodiments, the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO:1, or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophile polymerase activity comprises the amino acid sequence of SEQ ID NO:1.

[0009] The isothermal amplification reaction may include a constant temperature of about 30° C. to about 72° C., including, but not limited to, a constant temperature of about 67° C. The isothermal amplification reaction can be carried out for a period of, for example, about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes. In some embodiments, the isothermal amplification reaction is carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions.

[0010] In some embodiments, detecting the signal generated from the quenchable label of the first quality control primer does not include the use of any probe. In some embodiments, (c) subjecting the duplex to amplification conditions includes subjecting the target nucleic acid and one or more additional primers and / or one or more probes specific to the target nucleic acid to amplification conditions. In some embodiments, the first quality control primer does not hybridize to the target nucleic acid under amplification conditions. In some embodiments, the second quality control primer does not hybridize to the target nucleic acid under amplification conditions. In some embodiments, the method does not include using a template nucleic acid capable of hybridizing to the first quality control primer. In some embodiments, the method does not include using a template nucleic acid capable of hybridizing to the second quality control primer. Disclosed herein is a kit for monitoring an amplification reaction. For example, the kit may include a first quality control primer and a second quality control primer, each of which includes a 3' overlap region capable of hybridizing to each other, each of which is 35 nucleotides long or less, and the first quality control primer includes a quenchable label.

[0011] The quenchable label may be a fluorophore. In some embodiments, the quenchable label is outside the 3' overlap region of the first quality control primer. In some embodiments, the quenchable label is at the 5' end of the first quality control primer. In some embodiments, the quenchable label is in the 3' overlap region of the first quality control primer. In some embodiments, the second quality control primer comprises a quencher. In some embodiments, the quencher is outside the 3' overlap region of the second quality control primer. In some embodiments, the quencher is at the 5' end of the second quality control primer. In some embodiments, the quencher is in the 3' overlap region of the second quality control primer.

[0012] In some embodiments, the first quality control primer, the second quality control primer, or both, comprise one or more modified nucleotides. In some embodiments, the 3' overlap region of the first and second quality control primers each comprise one or more modified nucleotides. In some embodiments, the one or more modified nucleotides comprise a spacer, an abasic site, an unmethylated RNA base, a 2'-O-methylated nucleotide, and any combination thereof. In some embodiments, at least one of the one or more modified nucleotides is a 2'-O-methylated nucleotide. In some embodiments, the first quality control primer, the second quality control primer, or both, comprise one or more polymerase stoppers. In some embodiments, the 3' overlap region of the first and second quality control primers each comprise one or more polymerase stoppers. In some embodiments, at least one of the one or more polymerase stoppers is a 2'-O-methylated nucleotide. In some embodiments, the 3' overlap region of the first quality control primer is complementary to the 3' overlap region of the second quality control primer. In some embodiments, the 3' overlap region of the first quality control primer is fully complementary to the 3' overlap region of the second quality control primer. In some embodiments, the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer have the same length. In some embodiments, one or more of the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer are about 2 to about 10 nucleotides in length. In some embodiments, the 3' overlap region of the first and second quality control primers are 4 or 5 nucleotides in length.

[0013] The kit may include one or more of an enzyme with hyperthermophile polymerase activity, dNTPs, and a buffer. In some embodiments, the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophile polymerase activity comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the first quality control primer and the second quality control primer are in lyophilized or freeze-dried form. In some embodiments, one or more of the enzyme with hyperthermophile polymerase activity, the dNTPs, and the buffer are in lyophilized or freeze-dried form. The kit may also include one or more additional primers and / or one or more probes specific for the target nucleic acid.

[0014] Disclosed herein is a reaction mixture. In some embodiments, the reaction mixture includes a first quality control primer and a second quality control primer, each of which includes a 3' overlap region that can hybridize with each other, each of which is 35 nucleotides long or less, and the first quality control primer includes a quenchable label; a target nucleic acid; and one or more additional primers and / or one or more probes specific to the target nucleic acid. The reaction mixture may include a duplex formed by hybridization between the 3' overlapping regions of the first and second quality control primers. The reaction mixture may include one or more of an enzyme with hyperthermophile polymerase activity, dNTPs, and a buffer. In some embodiments, the enzyme with hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% or at least 95% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophile polymerase activity comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the first quality control primer cannot hybridize to the target nucleic acid. In some embodiments, the second quality control primer cannot hybridize to the target nucleic acid. In some embodiments, the one or more additional primers and / or the one or more probes cannot hybridize to the first quality control primer, the second quality control primer, or both. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a non-limiting exemplary embodiment of a DIMER internal control (DIMER-IC) as disclosed herein formed by a forward IC primer (attached to a fluorophore at its 5' end) and a reverse IC primer (attached to a quencher at its 5' end) that can hybridize to each other at the 3' overlap region to form a dimer. [Diagram 2]FIG. 1 illustrates a non-limiting, exemplary embodiment of the DIMER-IC method disclosed herein in which fluorescence associated with the forward IC primer is quenched over time as the archaeal polymerase amplification (APA) reaction progresses to produce an extended duplex using the dimer formed by the forward and reverse IC primers as a template. [Figure 3A-3B] Figure 3 illustrates the inverse amplification curves produced by the DIMER-IC method described herein. Figure 3A illustrates the results using HEX fluorophores (FP1+RP2 contains a 4 bp 3' overlap, FP1+RP3 contains a 5 bp 3' overlap). Figure 3B illustrates the results using Syto61 intercalating dyes (FP1+RP2 contains a 4 bp 3' overlap, FP1+RP3 contains a 5 bp 3' overlap). [Figure 4] FIG. 1 illustrates a non-limiting, exemplary embodiment in which one or more nucleotides in the 3′ overlap region of the forward and / or reverse IC primers comprise a modification (e.g., 2′-O-methylation). [Figure 5A-5B] FIG. 1 shows how modified nucleotides in the 3′ overlap region of the forward and / or reverse IC primer in some embodiments can inhibit unintended primer extension. [Figure 6A-6C] Non-limiting diagram showing alternative locations for the quenchable label and quencher attached to the forward and / or reverse IC primer: both outside the overlap region (FIG. 6A), both inside the overlap region (FIG. 6B), or one (e.g., quencher) at the 5' end of the reverse primer and one (e.g., fluorophore) internal (FIG. 6C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, similar symbols typically identify similar components unless the context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments can be used and other changes can be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form part of the disclosure herein.

[0017] All priority applications, patents, published patent applications, other publications, and GenBank sequences and other databases of this application referenced herein are incorporated by reference in their entirety with respect to the relevant art. Disclosed herein are methods, compositions, kits, and reaction mixtures for assessing, monitoring, observing, and / or tracking the progress of nucleic acid amplification reactions.

[0018] Disclosed herein are methods for assessing, monitoring, observing, and / or tracking the progress of an amplification reaction. In some embodiments, the method includes the steps of: (a) providing a first quality control primer and a second quality control primer, each of which comprises a 3' overlap region capable of hybridizing to one another, the first quality control primer comprising a quenchable label; (b) contacting the first quality control primer and the second quality control primer to form a duplex by hybridization between the 3' overlap regions of the first and second quality control primers; (c) subjecting the duplex to amplification conditions to generate an extended duplex; and (d) detecting a signal generated from the quenchable label of the first quality control primer during the amplification reaction to determine the generation of the extended duplex, a decrease in the signal during the amplification reaction indicating the generation of the extended duplex. In some embodiments, each of the first quality control primer and the second quality control primer is 35 nucleotides in length or less.

[0019] Disclosed herein includes kits for assessing, monitoring, observing, and / or tracking the progress of an amplification reaction. In some embodiments, the kit includes a first quality control primer and a second quality control primer, each of which comprises a 3' overlap region capable of hybridizing to one another, each of the first quality control primer and the second quality control primer being 35 nucleotides in length or less, and the first quality control primer comprising a quenchable label. Disclosed herein is a reaction mixture. In some embodiments, the reaction mixture includes a first quality control primer and a second quality control primer, each of which includes a 3' overlap region that can hybridize with each other, each of which is 35 nucleotides long or less, and the first quality control primer includes a quenchable label; a target nucleic acid; and one or more additional primers and / or one or more probes specific to the target nucleic acid.

[0020] The methods, compositions and kits described herein have various advantages over currently available methods, compositions and kits for assessing, monitoring, observing and / or tracking the progress of nucleic acid amplification, including, but not limited to, lower oligonucleotide complexity possible for a universal IC system, no requirement for probes or target templates, direct control of the system by primer concentration and 3' overlap size (i.e., higher degree of compatibility), less inhibitory effects (e.g., no molecular beacon probes are required, thus less polymerase inhibition), and less likelihood of non-specific product formation (e.g., due to 2'-O-methyl (2'-OM) modifications of primers).

[0021] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below. As used herein, the term "primer" refers to an oligonucleotide that includes a nucleotide sequence that is capable of hybridizing or annealing to a target nucleic acid at or near (e.g., adjacent to) a particular region of interest. As used herein, the term "quenchable label" refers to a molecule capable of producing a detectable signal, which signal can be reduced by a quencher. As used herein, the term "quencher" may refer to a molecule that interferes with or absorbs the fluorescence emitted by a neighboring quenchable label, e.g., a fluorophore.

[0022] As used herein, the term "duplex" refers to regions in two substantially complementary or fully complementary polynucleotides (e.g., the 3' overlap regions of a first quality control primer and a second quality control primer) that base pair with each other, either by Watson-Crick base pairing or any other manner that allows for the formation of a stable duplex between the substantially complementary or fully complementary polynucleotide strands. For example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide strand of 21 nucleotide units such that the "duplex" has 19 base pairs, where only 19 bases on each of the two strands can form Watson-Crick base pairs (i.e., fully complementary). The remaining bases may be present, for example, as 5' and 3' overhangs. Furthermore, perfect (e.g., 100%) complementarity within a duplex is not required. For example, a mismatch in a duplex consisting of 19 base pairs results in 94.7% complementarity. In some embodiments, the duplex is formed by hybridization between the 3' overlap regions of the first and second quality control primers. In some embodiments, the complementarity comprises 4-5 nucleotides in the 3' overlap regions of the first and second quality control primers. Provided herein, in some embodiments, are methods, compositions, kits, and reaction mixtures that can use archaeal polymerase amplification ("APA") to isothermally amplify regions of interest within target nucleotide templates for real-time analyte detection. For example, disclosed herein include methods for monitoring or evaluating amplification reactions as an internal control ("IC") assay that utilizes, for example, a 3' overlap between quality control primers to initiate amplification (e.g., deliberate primer-dimer formation), along with probe-less "integrated" detection formats.

[0023] As disclosed herein, the quality control forward and reverse primers, in some embodiments, may contain complementarity (e.g., at least 4 (e.g., 4-6) base pair overlap) at their 3' ends and can consistently result in exponential amplification between these primers in the absence of other target templates. In some embodiments, the speed and robustness of this amplification correlates with the size of the 3' overlap region (longer base pair overlap correlates with increased amplification rate) as well as the overall concentration of these primers in solution (higher concentration correlates with increased amplification rate).

[0024] APA amplicons are short, in some embodiments, less than 35 base pairs (bp) in total length. This is also true for the dimer-based internal control structures described above. To reduce assay complexity, the need for a dedicated control probe has been advantageously eliminated in some embodiments described herein. For example, tagging one primer with a 5' quenchable label (e.g., a fluorophore) and tagging the other primer with a 5' quencher produced a signal response that correlated with amplification (as indicated by the inclusion of a DNA intercalating dye in the same APA reaction). Without being bound by any particular theory, the generation of a DIME-IC control amplicon (e.g., an extended duplex) may bring the fluorophore and quencher close enough to reduce the fluorescence output (and thus the observable amplification response). Note that this results in a "reverse" exponential amplification curve when compared to the standard intercalation curve response. One advantage of the cumulative system for monitoring amplification reactions using the methods described herein is the reduced assay complexity: signal is generated from only two primers (no probe or dedicated template is required). This reduced complexity can help improve APA performance when quality control primers and target-analyte assays are co-amplified in a common reaction vessel.

[0025] The methods, compositions, kits, and reaction mixtures for evaluating, monitoring, observing, and / or tracking an amplification reaction disclosed herein can be used, for example, to monitor the amplification efficiency of the reaction. As used herein, the term "amplification efficiency" shall have its normal meaning and also refers to the determination of the ability of an amplification reaction to synthesize nucleic acid. The presence of amplification inhibitors, missing or defective amplification reaction components, nuclease contamination, and defective equipment include non-limiting examples of causes of reduced amplification efficiency and can be detected by the methods, compositions, kits, and reaction mixtures disclosed herein. In some embodiments, the methods, compositions, kits, and reaction mixtures provided herein can be used for quantification, for example, the production of extended duplexes can be used as a quantitative standard in real-time quantitative PCR or isothermal amplification applications.

[0026] Quality Control Primer Provided herein are quality control primers (also referred to as "internal control primers" or "IC primers"). For example, a pair of quality control primers may include a first quality control primer (e.g., a forward primer) and a second quality control primer (e.g., a reverse primer), each of which includes a 3' overlap region capable of hybridizing to each other. As described herein, the first quality control primer may be a forward primer or a reverse primer, and similarly, the second quality control primer may be a forward primer or a reverse primer. In a primer pair, if the first quality control primer is a forward primer, the second quality control primer is a reverse primer, and vice versa. The length of the quality control primers may vary, for example, the quality control primers may be 35 nucleotides or less in length. In some embodiments, the quality control primer (e.g., a forward primer) includes a quenchable label. In some embodiments, the methods, compositions, kits, and reaction mixtures described herein include providing a first quality control primer and a second quality control primer, each of which comprises a 3' overlapping region capable of hybridizing to one another, wherein each of the first quality control primer and the second quality control primer is 35 nucleotides in length or less, and wherein the first quality control primer comprises a quenchable label.

[0027] The quenchable label may be, for example, a fluorophore. As used herein, the term "fluorophore" shall be given its ordinary meaning and also refers to any reporter group whose presence can be detected by its light emission properties. Non-limiting examples of fluorophores include Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), calcein (517), FITC (518), FluorX™ (519), Alexa™ (520), rhodamine 110 (520), Oregon Green™ 500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (533), rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY 530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY 558 / 568 (568), BODIPY 564 / 570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575), Phycoerythrin R&B (575), Rhodamine phalloidin (575), Calcium Orange™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), Texas Red (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), R-phycocyanin (642), C-phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660), DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610) (numbers in parentheses are maximum emission wavelengths in nanometers of the responsive fluorophore). In some embodiments, the fluorophore is Cy5™. In some embodiments, the fluorophore is hexachlorofluorescein (HEX). .

[0028] In different embodiments, the position of the quenchable label in the quality control primer may vary.For example, the quenchable label may be outside the 3' overlap region of the quality control primer (e.g., the first quality control primer).In some embodiments, the quenchable label is at the 5' end of the quality control primer.In some embodiments, the quenchable label is in the 3' overlap region of the quality control primer.

[0029] The second quality control primer may include a quencher. Quenching may be mediated by fluorescence resonance energy transfer (FRET). FRET is based on classical dipole-dipole interactions between the transition dipoles of the donor (e.g., fluorophore) and the acceptor (e.g., quencher) and depends on the donor-acceptor distance. FRET can typically occur over distances up to 100 Å. FRET also depends on the donor-acceptor spectral overlap and the relative orientation of the donor and acceptor transition dipole moments. Quenching of a fluorophore may also occur as a result of the formation of a non-fluorescent complex between the fluorophore and another fluorophore or a non-fluorescent molecule. This mechanism is known as "contact quenching", "static quenching" or "ground state complex formation". Without being bound by any particular theory, it is believed that a quencher moiety is not required in some embodiments of the methods disclosed herein to observe a detectable change in fluorescence, and that the adjacent base quenching effect is sufficient to cause a detectable shift in fluorescence that allows the nucleic acid amplification reaction to be evaluated, monitored, observed, and / or tracked. A quencher moiety is not required to cause fluorescence quenching in some embodiments disclosed herein. For example, fluorescence quenching can be achieved by attaching a fluorophore to a cytosine nucleotide (e.g., 5' cytosine) in a quality control primer (e.g., the first quality control primer) such that upon amplicon formation, a complementary guanine nucleotide is incorporated opposite the fluorophore-labeled C base, thus quenching the fluorescence signal by "adjacent G base" quenching.

[0030] Examples of quenchers include, but are not limited to, Iowa Black FQ, Iowa Black RQ, Black Hole Quencher-1 (BHQ-1), Black Hole Quencher-2 (BHQ-2), TMR, QSY-7, and Dabcyl. The location of the quencher in the quality control primer may vary. For example, the quencher may be outside the 3' overlap region of the quality control primer (e.g., the second quality control primer). In some embodiments, the quencher is at the 5' end of the quality control primer. In some embodiments, the quencher is in the 3' overlap region of the quality control primer.

[0031] The length of the quality control primers (e.g., the first and second quality control primers) can vary from about 5 to about 100 nucleotides, including, for example, about 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 nucleotides, or any value or range between any two of these values. In some embodiments, the first and second quality control primers each have a length of 10 to about 50 nucleotides, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, each of the first and second quality control primers is 35 nucleotides or less in length.

[0032] qualification The quality control primer may comprise or consist of modified nucleotides. The first quality control primer, the second quality control primer, or both may comprise one or more modified nucleotides. For example, the 3' overlap region of the first quality control primer, the 3' overlap region of the second quality control primer, or both may comprise one or more modified nucleotides. The position of the modification may be different in different embodiments. In some embodiments, the 3' overlap region of the first and second quality control primers each comprises one or more modified nucleotides.

[0033] The nucleotides (or bases) may be modified according to any modification described herein or known in the art. Modifications may include those made during primer synthesis and / or may include post-synthetic modifications. Modifications may include internal modifications, modifications at the 3' terminal region of the quality control primer, and / or modifications at the 5' terminal region of the quality control primer. In some embodiments, the quality control primer comprises a mixture of modified and unmodified nucleotides. In some embodiments, the quality control primer comprises unmodified nucleotides. In some embodiments, the quality control primer consists essentially of or consists of modified nucleotides.

[0034] Modifications and modified bases include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite™, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker™, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6dT, Uni-Link™ amino modifiers), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); spacer (C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer); modified base (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acid (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate linkage modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases groups, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases); and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, phosphorothioate linkages, 3' phosphate groups, spacer bases (e.g., C3 spacers or other spacer bases). The one or more modified nucleotides may include spacers, abasic sites, unmethylated RNA bases, 2'-O-methylated nucleotides, and any combination thereof.

[0035] Primers containing 2'-O-methyl RNA bases can be directly synthesized. 2'-O-methyl RNA bases can be included in primers, for example, to inhibit read-through by DNA polymerase. In some embodiments, the quality control primers contain one or more phosphorothioate (PS) linkages (e.g., phosphorothioate linkage modifications). PS linkages replace non-bridging oxygens in the phosphate backbone of the primer with sulfur atoms. This substitution typically renders the internucleotide linkage resistant to nuclease degradation. Phosphorothioate linkages can be introduced between about the last 3-5 nucleotides at the 5' or 3' end of the quality control primer, for example, to inhibit exonuclease degradation. In some embodiments, phosphorothioate linkages included throughout the primer can help reduce endonuclease attack. The quality control primers can contain, for example, a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases and can be used in certain cases to block extension by DNA polymerase. In some embodiments, the quality control primer comprises one or more spacer bases (e.g., one or more C3 spacers). A C3 spacer phosphoramidite can be incorporated internally or at the 5' end of the primer. Multiple C3 spacers can be added to either end of the primer to introduce long chain hydrophilic spacer arms for attaching, for example, fluorophores or other pendant groups. In some embodiments, at least one of the one or more modified nucleotides is C3 spacer, hexanediol, dSpacer, PC spacer, spacer 9, spacer 18, 2-aminopurine, 2,6-diaminopurine, dideoxycytidine, inverted dT, Iso-dG, Iso-dC, inverted dideoxy-T, and 5-nitroindole. At least one of the one or more modified nucleotides can be a 2'-O-methylated nucleotide. In some embodiments, the 2'-O methylated nucleotides include one or more of 2'-O-methyl uridine, 2'-O-methyl adenosine, 2'-O-methyl cytidine, and 2'-O-methyl guanosine hydrate.As shown in Figures 4-5B, nucleotide modifications in the 3' overlap region of the first and second quality control primers can, in some embodiments, inhibit unintended primer extension as a result of hybridization of the IC primer with a random oligonucleotide.

[0036] The quality control primers (e.g., the first quality control primer and / or the second quality control primer) may include one or more polymerase stoppers. The 3' overlap regions of the first and second quality control primers may each include one or more polymerase stoppers. As used herein, a "polymerase stopper" is a molecule (e.g., a modified nucleotide) that can terminate or inhibit polymerization. In some embodiments, at least one of the one or more polymerase stoppers is a 2'-O-methylated nucleotide. Non-limiting examples of 2'-O-methylated nucleotides include 2'-O-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methylguanosine.

[0037] Quality control primer overlap, duplex, and extended duplex In some embodiments, the methods, compositions, reaction mixtures, and kits provided herein include a first quality control primer and a second quality control primer, each of which comprises a 3' overlap region capable of hybridizing to each other. In some embodiments, the 3' overlap region of the first quality control primer is complementary to the 3' overlap region of the second quality control primer. The 3' overlap region of the first quality control primer may be fully complementary to the 3' overlap region of the second quality control primer.

[0038] Complementarity with respect to sequences generally refers to nucleotide sequences that will hybridize to each other. As used herein, the terms "annealing" or "hybridizing" may refer to the formation of a stable complex between two molecules. The stringency of the hybridization conditions can be varied to allow for different amounts of sequence mismatch. In some embodiments, the 3' overlap regions of the first and second quality control primers are at least 75% complementary to each other. For example, the 3' overlap regions of the first and second quality control primers may be or are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to each other.

[0039] Fully complementary generally refers to a nucleotide sequence of a first strand in which, for example, each base pairs in sequence (e.g., reading from 5' to 3') with the correspondingly ordered bases of a second strand, and there are no gaps, additional sequences, or unpaired bases within a sequence that is considered fully complementary. As used herein, fully complementary refers to all contiguous bases of a nucleotide sequence of a first strand (e.g., in the 3' overlap region of a first quality control primer) that are complementary to the corresponding contiguous bases of a nucleotide sequence of a second strand (e.g., in the 3' overlap region of a second quality control primer). A fully complementary sequence may be about 5 to about 25 contiguous bases long, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous bases long. In some embodiments, the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer have the same length. In some embodiments, the 3' overlap region of the first quality control primer, the 3' overlap region of the second quality control primer, or both are 2 to about 10 nucleotides in length (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). The 3' overlap region of the first and second quality control primers can be, for example, 4 or 5 nucleotides in length.

[0040] In some embodiments, the method includes one or more of the steps of contacting a first quality control primer and a second quality control primer, thereby forming a duplex by hybridization between the 3' overlapping regions of the first and second quality control primers; subjecting the duplex to amplification conditions, thereby generating an extended duplex; and performing the contacting of the first quality control primer and the second quality control primer under amplification conditions.

[0041] As described herein, quality control primers can be used in the methods, compositions, kits and reaction mixtures disclosed herein for evaluating, monitoring, observing and / or tracking nucleic acid amplification reactions. A nucleic acid amplification reaction may be or may include one or more amplification reactions including a target nucleic acid as a template and one or more primers specific to the target nucleic acid. In some embodiments, the methods disclosed herein (e.g., in the step of subjecting the duplex to amplification conditions) include subjecting the target nucleic acid and one or more additional primers and / or one or more probes specific to the target nucleic acid to amplification conditions. In some embodiments, it is advantageous that the first quality control primer, the second quality control primer, or both do not hybridize to the target nucleic acid under amplification conditions. In some embodiments, the method does not include using a nucleic acid (e.g., a template nucleic acid) that can hybridize to the first quality control primer, the second quality control primer, or both. For example, the method does not include using a template nucleic acid that is substantially complementary or fully complementary to the first control primer, the second control primer, or both, in some embodiments. In some embodiments, the first and second quality control primers are advantageously not complementary to the target nucleic acid and cannot prime the amplification reaction from the target nucleic acid sequence. In some embodiments, the 3' overlap region of the first and second quality control primers is not complementary to the target nucleic acid. In some embodiments, the 3' overlap region of the first and second quality control primers is not completely complementary to the target nucleic acid. For example, the first and second quality primers may contain no more than 2, 3, 4, or 5 complementary nucleotides to the target nucleic acid sequence.

[0042] Nucleic Acid Amplification Disclosed herein are methods for evaluating, monitoring, observing, and / or tracking an amplification reaction. The method may include the steps of: (a) providing a first quality control primer and a second quality control primer, each of which comprises a 3' overlap region capable of hybridizing to one another, where the first quality control primer comprises a quenchable label; (b) contacting the first quality control primer and the second quality control primer, thereby forming a duplex by hybridization between the 3' overlap regions of the first and second quality control primers. In some embodiments, the method includes subjecting the duplex to amplification conditions, thereby generating an extended duplex. In some embodiments, each of the first quality control primer and the second quality control primer is 35 nucleotides in length or less.

[0043] Disclosed herein includes a reaction mixture for nucleic acid amplification. In some embodiments, the reaction mixture includes a first quality control primer and a second quality control primer, each of which includes a 3' overlap region capable of hybridizing to one another, where the first quality control primer includes a quenchable label; a target nucleic acid; and one or more additional primers and / or one or more probes specific to the target nucleic acid. As used herein, a primer or probe specific to a target nucleic acid is a primer or probe that is complementary or fully complementary to at least a portion of the target nucleic acid or its complement or reverse complement. In some embodiments, each of the first quality control primer and the second quality control primer is 35 nucleotides or less in length. The reaction mixture may include a duplex formed by hybridization between the 3' overlap regions of the first and second quality control primers. The reaction mixture may include one or more of an enzyme having DNA polymerase activity (e.g., an enzyme having hyperthermophile polymerase activity), dNTPs, and a buffer. In some embodiments, the reaction mixture comprises a reverse transcriptase. The enzyme with hyperthermophile polymerase activity may have an amino acid sequence that is at least about 90% or at least 95% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophile polymerase activity comprises or consists of the amino acid sequence of SEQ ID NO:1. In some embodiments, the first quality control primer, the second quality control primer, or both, are unable to hybridize to the target nucleic acid. In some embodiments, the one or more additional primers and / or one or more probes are unable to hybridize to the first quality control primer, the second quality control primer, or both.

[0044] As used herein, nucleic acid amplification may refer to any known procedure for obtaining multiple copies of a target nucleic acid sequence, or its complement or fragments, using sequence-specific methods. Examples of nucleic acid amplification include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA) (e.g., multiple displacement amplification (MDA)), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), genomic exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA), as well as transcription-mediated amplification (TMA). In some embodiments, two or more of the above nucleic acid amplification methods can be performed sequentially. The term "isothermal amplification reaction" shall be given its ordinary meaning and shall include reactions in which the temperature does not change significantly during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10°C, e.g., by no more than 5°C and no more than 2°C, during the main enzymatic reaction step in which amplification occurs. Depending on the method of isothermal amplification of nucleic acids, different enzymes can be used for amplification. Exemplary isothermal amplification compositions and methods are described in WO2107176404, the contents of which are incorporated herein by reference in their entirety.

[0045] The amplification reaction may be a real-time amplification reaction, for example a real-time PCR reaction. PCR involves the amplification of a target sequence (e.g., a target nucleic acid) using two or more extendible sequence-specific oligonucleotide primers that flank the target sequence. A nucleic acid containing the target sequence of interest is subjected to a program of multiple rounds of temperature cycles (denaturation, annealing and extension) in the presence of primers, a thermostable DNA polymerase (e.g., Taq polymerase) and various dNTPs, resulting in the amplification of the target sequence. PCR uses multiple rounds of primer extension reactions in which complementary strands of defined regions of a DNA molecule are simultaneously synthesized by a thermostable DNA polymerase. At the end of each cycle, each newly synthesized DNA molecule acts as a template for the next cycle. During these repeated rounds of reactions, the number of newly synthesized DNA strands increases exponentially, and after, for example, 20-30 reaction cycles, the initial template DNA has been replicated thousands or millions of times. PCR can generate double-stranded amplification products suitable for post-amplification processing. The amplification products can be detected, for example, by visualization using agarose gel electrophoresis, enzyme immunoassay formats using probe-based colorimetric detection, fluorescence emission techniques, or other detection means known to those skilled in the art. Examples of PCR methods include, but are not limited to, real-time PCR, end-point PCR, amplified fragment length polymorphism PCR (AFLP-PCR), Alu-PCR, asymmetric PCR, colony PCR, DD-PCR, degenerate PCR, hot-start PCR, in situ PCR, inverse PCR, long PCR, multiplex PCR, nested PCR, PCR-ELISA, PCR-RFLP, PCR-single-strand conformation polymorphism (PCR-SSCP), quantitative competitive PCR (QC-PCR), rapid amplification of cDNA ends-PCR (RACE-PCR), random amplification of polymorphic DNA-PCR (RAPD-PCR), real-time PCR, repetitive extragenic palindrome-PCR (Rep-PCR), reverse transcriptase PCR (RT-PCR), TAIL-PCR, touchdown PCR, and vectorette PCR.

[0046] Real-time PCR, also called quantitative real-time polymerase chain reaction (QRT-PCR), can be used to simultaneously quantify and amplify specific portions of a given nucleic acid molecule. It can be used to determine whether a particular sequence is present in a sample and, if so, how many copies of the sequence are present. The term "real-time" may refer to periodic monitoring during PCR. Certain systems, such as the ABI7700 and 7900HT Sequence Detection Systems (Applied Biosystems, Foster City, Calif.), monitor during each thermal cycle at predefined or user-defined points. Real-time analysis of PCR using fluorescence resonance energy transfer (FRET) probes measures the change in fluorescent dye signal between cycles. The real-time procedure follows the general pattern of PCR, but the nucleic acid is quantified after each round of amplification. Two examples of quantification methods are the use of fluorescent dyes (e.g., SYBR Green) that intercalate into dsDNA and the use of modified DNA oligonucleotide probes that fluoresce when hybridized with complementary DNA. Intercalating agents have relatively low fluorescence when unbound and relatively high fluorescence when bound to double-stranded nucleic acids. Thus, intercalating agents can be used to monitor the accumulation of double-stranded nucleic acids during nucleic acid amplification reactions. Examples of such non-specific dyes useful in the embodiments disclosed herein include intercalating agents such as SYBR Green I (Thermo Fisher), propidium iodide, and ethidium bromide.

[0047] The terms "amplify", "amplification", "amplification reaction", or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification can also refer to an exponential increase in the target nucleic acid. However, "amplifying" can also refer to a linear increase in the number of target nucleic acids, but is distinct from a one-time, single primer extension step. In some embodiments, a limited amplification reaction, also known as preamplification, can be performed. Preamplification is a method in which a limited amount of amplification occurs because a small number of cycles, e.g., 10 cycles, are performed. Preamplification allows some amplification, but stops the amplification before the exponential phase, typically producing about 500 copies of the desired nucleotide sequence. Preamplification can be used to limit the inaccuracies associated with reactant exhaustion in a particular amplification reaction, and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a one-time primer extension is performed as a pre-step to linear or exponential amplification.

[0048] In some embodiments of the methods described herein, when a primer and its corresponding target nucleic acid are contacted, the complementary sequences anneal or hybridize to each other. The primer can anneal to the target nucleic acid at or near (e.g., adjacent, abutting, etc.) the sequence of interest. A primer annealed to a target may be referred to as a primer-target hybrid, hybridized primer-target, or primer-target duplex. The term "near" or "adjacent" when referring to a nucleotide sequence of interest refers to the distance (e.g., number of bases) or region between the end of the primer and one or more nucleotides (e.g., a nucleotide sequence) of the target. Generally, adjacent is in the range of about 1 nucleotide to about 50 nucleotides from the nucleotide or nucleotide sequence of interest, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values. In some embodiments, a set of primers (e.g., a pair of primers, a forward primer and a reverse primer, a first oligonucleotide and a second oligonucleotide) anneals within about 1-20 nucleotides of a nucleotide or nucleotide sequence of interest to generate an amplification product. In some embodiments, the primers anneal within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended along the target (i.e., the template strand) by a polymerase to generate a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplification product is generated. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step. In some embodiments of the methods described herein, the quality control primers are contacted and hybridized to form a duplex. Each of the quality control primers can serve as a template as well as a primer for the nucleic acid extension reaction.

[0049] Components of the amplification reaction may include, for example, one or more primers (e.g., quality control primers, including individual primers, primer pairs, primer sets, oligonucleotides, and multiple primer sets for multiplex amplification, additional primers, and / or probes), a nucleic acid target (e.g., a target nucleic acid from a sample), one or more polymerases, nucleotides (e.g., dNTPs, etc.), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, monovalent ions, and divalent ions). The amplification reaction may further include a reverse transcriptase and / or a reverse transcription primer. The amplification reaction may further include one or more detection agents, such as one or more of the detection agents described herein. In some embodiments, the one or more amplification reagents include, consist of, or consist essentially of primers, target nucleic acid, polymerase, nucleotides, and a suitable buffer, and optionally, a detection agent. In some embodiments, one or more amplification reagents comprise, consist of, or consist essentially of primers, target nucleic acid, polymerase, reverse transcriptase, reverse transcription primer, nucleotides, and suitable buffer, and optionally, detection agent and / or reverse transcription primer. Additional components or features that have no significant effect on amplification and / or are not necessary to generate detectable products may be included. For example, additional components or features that have no significant effect on the ability of the components and conditions herein to achieve amplification under isothermal conditions and generate detectable amplification products within about 20, 15, or 10 minutes or less may be included. Such additional components or features may be referred to as non-essential components and may include typical reaction components and / or common additives such as salts, buffers, detergents, ions, oils, proteins, polymers, etc.

[0050] Nucleic acid amplification can be performed in the presence of natural nucleotides, e.g., dNTPs, and / or derivatized nucleotides. Natural nucleotides generally refer to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid. Derivatized nucleotides generally are nucleotides other than natural nucleotides. Nucleotides are typically designated as follows: Ribonucleoside triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be designated herein as A, G, C, T, or U, without specific reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs can be used, such as analogs that include a detectable label (e.g., a fluorescent or colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, e.g., 32 P, 33 P, 125 I, or 35 It can be performed in the presence of a radioactive label such as S; an enzyme label such as alkaline phosphatase; a fluorescent label such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, or fluorescent dyes. In some embodiments, nucleic acid amplification is performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., CleanAmp™ dNTPs from TriLink).

[0051] The one or more amplification reagents may include non-enzymatic and enzymatic components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions; generally, they do not include proteins (e.g., nucleic acid binding proteins), enzymes, or proteins with enzymatic activity, such as polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. Enzymatic components, in some embodiments, exclude other proteins (e.g., nucleic acid binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, and the like. Amplification conditions may include enzymatic activity, such as enzymatic activity provided by a polymerase. In some embodiments, enzymatic activity is provided by a polymerase and a reverse transcriptase. In some embodiments, the enzymatic activity does not include enzymatic activity provided by other enzymes, e.g., helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, etc. The polymerase activity and reverse transcriptase activity may be provided by separate enzymes or separate enzyme types (e.g., a polymerase and a reverse transcriptase) or may be provided by a single enzyme or enzyme type (e.g., a polymerase).

[0052] Conventional nucleic acid amplification methods typically require a thermal cycling process, a protein (e.g., an enzyme) (e.g., a helicase, a recombinase) that promotes nucleic acid denaturation, strand unwinding, strand separation, and / or strand exchange, and / or an endonuclease agent (e.g., a restriction enzyme, a nicking enzyme), and often require a reaction time of at least 20 to 30 minutes. The nucleic acid amplification methods provided herein can be performed without thermal cycling, without thermal and / or enzymatic denaturation of the sample nucleic acid, without the addition of a protein (e.g., an enzyme) that promotes strand unwinding, strand separation, and / or strand exchange, without an endonuclease agent, and / or within a reaction time of about 10 to 15 minutes. In some embodiments of the methods described herein, the amplification reaction is an isothermal amplification reaction. In some embodiments, the amplification of the nucleic acid comprises a non-thermal cycling type of polymerase chain reaction (PCR). In some embodiments, the amplification of the nucleic acid comprises an isothermal amplification process. In some embodiments, the amplification of the nucleic acid comprises an isothermal polymerase chain reaction (iPCR).

[0053] Isothermal amplification conditions generally do not include a thermocycling (i.e., cycling between upper and lower temperature limits) component to the amplification process, and thus, during an isothermal amplification reaction, the temperature does not change significantly during the reaction (e.g., maintains essentially the same temperature during the reaction). For example, the temperature of an isothermal amplification reaction may not deviate by more than 10° C., e.g., by no more than 5° C. and no more than 2° C., during the main enzymatic reaction step in which amplification occurs. Depending on the method of isothermal amplification, different enzymes may be used for amplification. Nucleic acid amplification typically involves the enzymatic synthesis of nucleic acid amplicons (copies) that contain sequences complementary to the nucleotide sequence being amplified. The amplification method may be performed, for example, in a single vessel, a single chamber, and / or a single volume (i.e., in a continuous volume). In some embodiments, the amplification and detection (e.g., the detection steps / methods described herein) are performed (e.g., simultaneously) in a single vessel, a single chamber, and / or a single volume (i.e., in a continuous volume). The amplification conditions may include one or more of an enzyme having a hyperthermophilic polymerase activity, dNTPs, and a buffer. An enzyme with hyperthermophile polymerase activity may have an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof. An enzyme with hyperthermophile polymerase activity may include the amino acid sequence of SEQ ID NO:1.

[0054] Isothermal amplification reactions can be carried out at an essentially constant temperature. In some embodiments, the amplification reaction is maintained at exactly one temperature. In some embodiments, the isothermal amplification process may experience small variations in temperature (e.g., ±1-5°C, etc.), for example, due to environmental or equipment-based variables. In some embodiments, the entire reaction volume is kept at an essentially constant temperature, and isothermal reactions herein generally may advantageously not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel. In some embodiments, the isothermal amplification reaction is carried out at a temperature of about 30°C to about 75°C, including a temperature of about 55°C to about 75°C. For example, the isothermal amplification reaction can be carried out at a temperature of 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or 75°C, or at a temperature about these values, or a numerical value between any two of these values. In some embodiments, the isothermal amplification reaction is carried out at a temperature of about 55°C to about 65°C, e.g., at about 60°C or about 65°C. In some embodiments, the temperature element (e.g., heat source) is maintained at an essentially constant temperature, e.g., at about 75°C or below, e.g., at about 70°C or below, at about 65°C or below, or at about 60°C or below. The isothermal amplification reaction can be carried out for a period of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or any value or range between any two of these values. The isothermal amplification reaction can be carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions.

[0055] The amplification process described herein (e.g., isothermal amplification) can be carried out for a suitable duration. In some embodiments, the amplification process is carried out until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product can be detected by any suitable detection process and / or detection process described herein. In some embodiments, the amplification process is carried out for a length of time of about 30 minutes, about 20 minutes or less. For example, the amplification process can be carried out within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or within about these values. In some embodiments, the amplification produces a detectable nucleic acid amplification product within about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 minutes, or within a shorter time, or within a value or range between any two of these values.

[0056] In some embodiments, nucleic acid targets and duplexes (e.g., duplexes formed by hybridization between the 3' overlap regions of the first and second quality control primers) can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, nucleic acid targets and duplexes can be amplified without exposure to agents or conditions that promote strand separation during the amplification step (and / or other steps). In some embodiments, nucleic acid targets and duplexes can be amplified without exposure to agents or conditions that promote unwinding during the amplification step (and / or other steps). Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote unwinding include, but are not limited to, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, and proteins (e.g., enzymatic agents).

[0057] In some embodiments, the methods disclosed herein (e.g., the amplification step) do not include heat denaturation (e.g., heating a solution containing nucleic acid to an elevated temperature, such as, for example, above 75°C, above 80°C, above 90°C, above 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of the nucleic acid. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, a recombinase, an RNA replicase, and a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the methods do not include a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, a recombinase, an RNA replicase, and / or a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the methods do not include intercalating agents, alkylating agents, and / or chemicals such as formamide, glycerol, urea, dimethylsulfoxide (DMSO), and / or N,N,N-trimethylglycine (betaine). In some embodiments, the methods do not include contacting the nucleic acid with a denaturing agent (e.g., formamide). In some embodiments, the methods (e.g., amplification steps) and compositions disclosed herein do not include agents and / or conditions that denature the nucleic acid (e.g., promote strand separation and / or promote unwinding), or agents and / or conditions other than acid and / or low pH conditions. In some embodiments, the amplification step does not include agents and / or conditions that denature the nucleic acid (e.g., promote strand separation and / or promote unwinding), other than a polymerase (e.g., a hyperthermophile polymerase). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase) and / or low pH conditions (e.g., contact with acid).Compositions, kits, and methods for nucleic acid detection in which nucleic acid strands are dissociated under low pH conditions (e.g., by contact with an acidic lysis buffer) to facilitate subsequent rapid amplification and detection are described in PCT Patent Application No. PCT / US23 / 61978, entitled "METHOD FOR SEPARATING GENOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS," filed February 3, 2023, the contents of which are incorporated by reference herein in their entirety.

[0058] In some embodiments, the nucleic acid target and duplex (e.g., the duplex formed by hybridization between the 3' overlap regions of the first and second quality control primers) can be amplified without exposure to agents or conditions during the amplification step (and / or other steps) that promote strand separation and / or unwinding. For example, the nucleic acid can be amplified without exposure to a helicase. A helicase is an enzyme that can unwind and separate double-stranded nucleic acid into single strands. Amplification conditions that do not include the use of a helicase may be referred to herein as helicase-free amplification conditions.

[0059] The nucleic acid target (including the duplex formed by hybridization between the 3' overlapping regions of the first and second quality control primers) can be amplified without contacting or exposing to one or more of helicases, recombinases, nucleic acid binding proteins (e.g., single stranded binding proteins or single stranded DNA binding proteins (SSB)), ligases, RNA replicases, restriction enzymes, nicking enzymes, exonucleases, DNAses, RNAses, and topoisomerases prior to and / or during the amplification reaction. Non-limiting examples of recombinases include Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, T4 uvsX. In some embodiments, the nucleic acid can be amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY). In some embodiments, the nucleic acid is amplified without exposure to an SSB, e.g., T4 gp32. Non-limiting examples of exonucleases include DNAses, RNAses (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases.

[0060] Nucleic acid targets (including duplexes formed by hybridization between the 3' overlap regions of the first and second quality control primers) can be amplified with or without exposure to agents or conditions that destabilize the nucleic acid. As used herein, the term "destabilization" shall be given its ordinary meaning and refers to the disruption of the overall organization and geometric orientation of a nucleic acid molecule (e.g., double helix structure) by one or more of tilt, rotation, twist, slip, and flip effects. Destabilization does not generally refer to melting or separation (e.g., denaturation) of nucleic acid strands. Nucleic acid destabilization can be achieved, for example, by exposure to agents such as intercalating or alkylating agents, and / or chemicals such as formamide, urea, dimethylsulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents.

[0061] In some embodiments, the nucleic acid target and duplex (e.g., the duplex formed by hybridization between the 3' overlap regions of the first and second quality control primers) can be amplified as a template nucleic acid without cleavage or digestion. For example, amplification can be performed without prior exposure to one or more agents, and intact nucleic acid is amplified. In some embodiments, amplification can be performed without exposure to one or more cleavage agents during amplification. The term "cleavage agent" refers to an agent (e.g., a chemical or enzyme) that can cleave a nucleic acid at one or more specific or non-specific sites. A specific cleavage agent often specifically cleaves a specific site according to a specific nucleotide sequence. Cleavage agents include, but are not limited to, endonucleases (e.g., restriction enzymes, nicking enzymes, etc.); exonucleases (DNAses, RNAses (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases); and chemical cleavage agents.

[0062] Amplified nucleic acids (e.g., amplified target nucleic acids and / or extended duplexes) may be referred to herein as nucleic acid amplification products or amplicons. Amplified duplexes may be referred to as extended duplexes. Amplification products may include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and combinations thereof. Amplification products typically have a nucleotide sequence that is identical or substantially identical to that in the sequence of a sample nucleic acid (e.g., a target nucleic acid) or its complement. A "substantially identical" nucleotide sequence in an amplification product will generally have a high degree of sequence identity (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity) to the nucleotide sequence being amplified or its complement, although variations may be the result of poor polymerase fidelity or other variables.

[0063] In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is complementary or completely complementary to a target sequence in the sample nucleic acid. In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is partially complementary or completely complementary to a duplex. Fully complementary generally refers to, for example, a nucleotide sequence in a first strand where each base pairs in order (e.g., reading from 5' to 3') with a correspondingly ordered base in a second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered to be fully complementary. In other words, fully complementary refers to all consecutive bases of the nucleotide sequence of the first strand being complementary to the corresponding consecutive bases of the nucleotide sequence of the second strand. The nucleic acid amplification product may comprise a sequence that is fully complementary or complementary to one or more primers used in the amplification reaction. In some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is fully complementary to or identical to a first primer sequence, and a second nucleotide sequence that is fully complementary to or identical to a second primer sequence.

[0064] The nucleic acid amplification product may be at least 50 bases long. In some embodiments, the nucleic acid amplification product is about 15 to about 40 bases long, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases long, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases long. In some embodiments, the amplification product is about 20 to about 40 bases long, for example, about 20 to about 30 bases long. In some embodiments, the nucleic acid amplification products for a given target sequence have the same or substantially the same length (e.g., within 1-5 bases). Thus, the nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band in an electrophoretic gel) and generally do not produce multiple signals representing multiple lengths (e.g., ladders or smears in an electrophoretic gel). In a multiplex reaction, the nucleic acid amplification products of different target sequences may have different lengths.

[0065] The nucleic acid amplification product may include a spacer sequence. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is consecutively complementary or substantially identical to a portion of a target sequence in a sample nucleic acid, and is flanked by sequences in the amplification product that are complementary or substantially identical to one or more primers used in the amplification reaction. A spacer sequence flanked by sequences in an amplification product is generally located between a first sequence (complementary or substantially identical to a first primer) and a second sequence (complementary or substantially identical to a second primer). Thus, an amplification product typically includes a first sequence, followed by a spacer sequence, followed by a second sequence. The spacer sequence is generally neither complementary nor substantially identical to the sequence of the primer. The spacer sequence may be or include about 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists or consists essentially of a first nucleotide sequence that is contiguous complementary or identical to the first primer sequence, a second nucleotide sequence that is contiguous complementary or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence that is not contiguous complementary or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product) and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other additional sequence incorporated into the amplification product by other mechanisms. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguous complementary or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product) and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other additional sequence incorporated into the amplification product by other mechanisms.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or one extra base (e.g., at the 5' and / or 3' end, or within the product) introduced into the product, for example, by errors or promiscuity in the amplification process.

[0066] The methods and components described herein can be used to evaluate and / or monitor multiplex amplification, in which the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence) occurs. For example, multiplex amplification can refer to amplifying multiple sequences from the same sample, or amplifying one of several sequences in a sample. Multiplex amplification may also refer to the simultaneous or stepwise amplification of one or more sequences present in multiple samples. In some cases, an amplification reaction is arranged to detect at least two target sequences, although only one of the target sequences may be present in the sample being tested, so that both sequences are amplifiable, but only one sequence is amplified. In some cases, when two target sequences are present, the amplification reaction may result in the amplification of both target sequences. A multiplex amplification reaction with appropriate primers and enzymes may result in the amplification of one, some, or all of the target sequences. In some examples, an amplification reaction may be set up to detect two sequences using a pair of primers, one sequence being a target sequence and one sequence being a control sequence (e.g., a synthetic sequence that is amplifiable by the same primers as the target sequence but has spacer bases or a sequence different from the target). In some embodiments, the control sequence comprises a duplex formed by hybridization of a first and a second quality control primer. In some examples, an amplification reaction may be set up to detect multiple sets of sequences using corresponding primer pairs, each set comprising a target sequence and a control sequence.

[0067] Primer In some embodiments of the methods described herein, subjecting the duplex to amplification conditions may include subjecting the target nucleic acid and one or more additional primers and / or one or more probes specific for the target nucleic acid to amplification conditions.

[0068] Nucleic acid amplification is generally carried out in the presence of one or more primers. A primer (e.g., one or more additional primers) is generally characterized as an oligonucleotide that comprises a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid at or near (e.g., adjacent to) a specific region of interest (i.e., the target nucleic acid). A primer can, for example, allow for the specific determination of the nucleotide sequence of a target nucleic acid or the detection of a target nucleic acid or a feature thereof (e.g., the presence or absence of a sequence). A primer can be naturally occurring or synthetic. The term specific or specific generally refers to the binding or hybridization of one molecule, such as a primer to a target polynucleotide, with another molecule. That is, the term specific or specific refers to the recognition, contact, and formation of a stable complex between two molecules, compared to the substantially lesser recognition, contact, or complex formation of either of those two molecules with the other molecule. The term annealing or hybridization generally refers to the formation of a stable complex between two molecules. The terms primer, oligo, or oligonucleotide can be used interchangeably herein when referring to a primer.

[0069] Primers may be designed and synthesized using a suitable process and may be of any length suitable for hybridizing to a target sequence and performing the amplification process described herein. Primers are often designed according to the sequence of the target nucleic acid. Primers may, in some embodiments, be from about 5 bases to about 30 bases in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases in length. In some embodiments, primers are less than 28 bases in length, for example, from about 8 to about 16 bases in length or from about 10 to about 12 bases in length. Primers may be composed of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides) or mixtures thereof. Primers suitable for use with the methods described herein may be synthesized and labeled using any suitable technique. For example, primers can be chemically synthesized according to the solid-phase phosphoramidite triester method using an automated synthesizer. Purification of primers can be performed, for example, by native acrylamide gel electrophoresis or anion-exchange HPLC.

[0070] The primer may comprise or consist of modified nucleotides. The nucleotides (or bases) may be modified according to any modification described herein or known in the art. Modifications may include those made during primer synthesis and / or may include post-synthetic modifications. Modifications may include internal modifications, modifications at the 3' end of the primer, and / or modifications at the 5' end of the primer. In some embodiments, the primer comprises a mixture of modified and unmodified nucleotides. In some embodiments, the primer comprises unmodified nucleotides. Modifications and modified bases include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite™, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker™, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6dT, Uni-Link™ amino modifiers), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG), thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); fluorophores (e.g., Freedom™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO™ dyes, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy750 (NHS ester); Iowa Black® dark quencher modifications (e.g., Iowa Black® FQ, Iowa Black® RQ); dark quencher modifications (e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl);Spacers (C3 Spacer, PC Spacer, Hexanediol, Spacer 9, Spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA ), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases);and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, phosphorothioate linkages, 3' phosphate groups, spacer bases (such as C3 spacer or other spacer bases). For example, the primers may include one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is a post-transcriptional modification of RNA commonly found in tRNA and other small RNA molecules. Primers containing 2'-O-methyl RNA bases can be directly synthesized. This modification can, for example, increase the Tm of the RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in the primers to, for example, increase stability and binding affinity with the target sequence. In some embodiments, the primers may include one or more phosphorothioate linkages (e.g., phosphorothioate bond modifications). Phosphorothioate (PS) bonds replace non-bridging oxygens in the phosphate backbone of a primer with sulfur atoms. This modification typically renders the internucleotide bond resistant to nuclease degradation. Phosphorothioate bonds may be introduced between about the last 3-5 nucleotides at the 5' or 3' end of a primer, for example, to inhibit exonuclease degradation. In some embodiments, phosphorothioate bonds included throughout a primer can help reduce attack by endonucleases. A primer may, for example, include a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases and, in certain cases, can be used to block extension by DNA polymerase. In some embodiments, a primer includes one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated internally or at the 5' end of a primer. Multiple C3 spacers can be added to either end of the primer to introduce long hydrophilic spacer arms for attachment of, for example, fluorophores or other pendant groups;

[0071] A primer (e.g., a quality control primer or a target sequence specific primer) may comprise DNA bases, RNA bases, or both. In some embodiments, a primer comprises a mixture of DNA bases and RNA bases. The DNA bases and / or RNA bases may be modified or unmodified. In some embodiments, a primer consists of DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, a primer consists of RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, a primer does not comprise an RNA base. In some embodiments, a primer does not comprise an RNA base at the 3' end. In some embodiments, a primer comprises a DNA base (or a modified DNA base) at the 3' end. In some embodiments, a primer is not a chimeric primer. A chimeric primer is a primer that comprises DNA and RNA bases. In some embodiments, a primer is a homogenous primer (e.g., a homogenous DNA primer). A homogenous DNA primer may comprise unmodified DNA bases, modified DNA bases, or a mixture of modified and unmodified DNA bases, and does not comprise an RNA base.

[0072] In some embodiments, the primer does not include a cleavage agent recognition site. For example, the primer may not include a nicking enzyme recognition site. In some embodiments, the primer does not include a tail. In some embodiments, the primer does not include a tail that includes a nicking enzyme recognition site.

[0073] In some embodiments, all or part of the primer sequence may be complementary or completely complementary to the target nucleic acid. Complementarity with respect to sequences generally refers to nucleotide sequences that will hybridize with each other. The stringency of hybridization conditions can be varied to allow for different amounts of sequence mismatch. In some embodiments, the target and primer sequences are at least 75% complementary to each other. For example, the target and primer sequences may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to each other, or at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to each other. A primer that is complementary to a target nucleic acid sequence typically is also complementary to the complement (eg, the antisense strand) or is perfectly complementary to the target nucleic acid sequence.Primers that are complementary to the antisense strand of a target nucleic acid may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each other, or at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each other. 9% identical, may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or may be at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. Whether two nucleotide sequences are complementary can be determined by the percentage of identical nucleotide sequences shared.

[0074] As described herein, a pair of primers may include a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid). Due to complementarity at the 3' overlap region, a forward quality control primer can bind to a corresponding reverse quality primer, and the forward and reverse quality primers are a pair of quality control primers. In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, the amplification of a target sequence is performed using a pair of primers, and no additional primers or oligonucleotides are included in the amplification of the target sequence (e.g., the amplification reaction components do not include additional primer pairs for a given target sequence, nested primers, bumper primers, non-primer oligonucleotides, and probes, etc.). In some embodiments, the primers consist of a pair of primers, but in some embodiments, the amplification reaction may include additional primer pairs for amplifying different target sequences, such as in multiplex amplification. In some embodiments, the primers consist of a pair of primers, but in some embodiments, the amplification reaction may contain additional primers, oligonucleotides, or probes for the detection process that are not considered part of the amplification. In some embodiments, the primers are used in sets. An amplification primer set may contain a pair of forward and reverse primers for a given target sequence. In the case of multiplex amplification, the primers that amplify a first target sequence are considered to be a primer set, and the primers that amplify a second target sequence are considered to be a different primer set.

[0075] In some embodiments, the amplification reaction components include a first primer (first oligonucleotide) that is complementary to a target sequence (e.g., target nucleic acid) of a first strand (e.g., sense strand, forward strand) of a sample nucleic acid, and a second primer (second oligonucleotide) that is complementary to a target sequence of a second strand (e.g., antisense strand, reverse strand) of the sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) includes a first polynucleotide that is fully complementary to a target sequence of the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) includes a second polynucleotide that is fully complementary to a target sequence of the second strand of the sample nucleic acid. Fully complementary with respect to primer-target generally refers to a nucleotide sequence of a primer in which each base pairs in order with a corresponding base in the target sequence in the corresponding order, with no gaps, additional sequences, or unpaired bases in the sequence considered to be fully complementary. Alternatively stated, fully complementary generally refers to all contiguous bases of the nucleotide sequence in the primer being complementary to the corresponding contiguous bases of the nucleotide sequence in the target.

[0076] A primer may include one or more additional bases that do not add functional features to the primer (e.g., at the 5' and / or 3' end or within the primer). For example, additional sequences present in tail or loop primers generally add functional features and are not excluded from the primer in such embodiments. In some embodiments, a primer may include modifications such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifier that changes the binding properties of the primer. In some embodiments, a primer may include a detectable molecule or entity (e.g., fluorophores, radioisotopes, colorimetric reagents, particles, enzymes, etc.).

[0077] Polymerase In some embodiments, the amplification reaction components include one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides extending the 3' hydroxyl end of a primer molecule, such as an amplification primer described herein, to a nucleic acid target sequence (e.g., to which the primer anneals). The polymerase may include, for example, a thermophilic or hyperthermophilic polymerase that can exhibit activity at high reaction temperatures (e.g., greater than 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 80°C, 95°C, or 100°C). Hyperthermophilic polymerases are sometimes referred to as hyperthermophilic polymerases. The polymerase may or may not have strand displacement capabilities. In some embodiments, the polymerase is capable of incorporating from about 1 to about 50 nucleotides in a single synthesis, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range between any two of these values ​​in a single synthesis.

[0078] Amplification reaction conditions may include, but are not limited to, 9°N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA polymerase; Therminator™ II DNA polymerase; Therminator™ III DNA polymerase; Therminator™ gamma DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (e.g., E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo-); T4 DNA polymerase; T7 DNA polymerase; Deep VentR™ (exo-) DNA polymerase; Deep VentR™ DNA polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start The composition may comprise one or more DNA polymerases, including DNA polymerase; Phusion™ high fidelity DNA polymerase; VentR™ DNA polymerase; VentR™ (exo-) DNA polymerase; RepliPHI™ Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm™ DNA polymerase); MasterAmp™ AmpliTherm™ DNA polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase beta; and ThermoPhi DNA polymerase.

[0079] The DNA polymerase may be a hyperthermophilic DNA polymerase, e.g., a hyperthermophilic DNA polymerase that is thermostable at high temperatures, or a variant or functional fragment thereof. For example, the hyperthermophilic DNA polymerase may have a half-life of about 5-10 hours at 95° C. and a half-life of about 1-3 hours at 100° C. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Archaea, e.g., a DNA polymerase from Thermococcus (e.g., Thermococcaceaen archaean), Pyrococcus, Methanococcaceae, one or more species of Methanococcus, Thermus, or a combination thereof. In some embodiments, the amplification reaction conditions include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0080] A functional fragment of a hyperthermophile DNA polymerase generally retains one or more functions of the full-length polymerase, e.g., the ability to polymerize DNA (e.g., in an amplification reaction). A functional fragment can, for example, perform a function (e.g., polymerizing DNA in an amplification reaction) at a level of at least about 50%, 75%, 80%, 85%, 90%, or 95% of the functional level of the full-length polymerase. The level of polymerase activity can be assessed using a detectable nucleic acid amplification method, e.g., a method for monitoring an amplification reaction disclosed herein. In some embodiments, the hyperthermophile DNA polymerase comprises an amino acid sequence of SEQ ID NO:1 or a functional fragment of SEQ ID NO:1. In some embodiments, the hyperthermophile DNA polymerase comprises an amino acid sequence of SEQ ID NO:2 or a functional fragment of SEQ ID NO:2. In some embodiments, the amplification reaction conditions include a polymerase comprising an amino acid sequence that is at least about 90% identical, 95% identical, or 99% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the amplification reaction conditions include a polymerase comprising an amino acid sequence that is at least about 90% identical, 95% identical, or 99% identical to the amino acid sequence of SEQ ID NO:2, or a functional fragment thereof.

[0081] The polymerase may have reverse transcription capability. In such a case, the amplification reaction may, for example, amplify an RNA target in a single step without the use of a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase capability include Bst (large fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, and Therminator™ II. In some embodiments, the amplification reaction conditions include one or more separate reverse transcriptases. In some embodiments, more than one polymerase may be included in the amplification reaction. For example, the amplification reaction may include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity. In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases with no or low exonuclease activity are used during amplification, for example, the polymerase includes one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, a modified polymerase with low exonuclease activity may have 10% or less exonuclease activity compared to an unmodified polymerase. For example, a modified polymerase with low exonuclease activity may have less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase has no or low 5'-3' exonuclease activity and / or no or low 3'-5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand-dependent exonuclease activity and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of a polymerase include one or more amino acid substitutions at one or more of positions 141, 143, and 458 of SEQ ID NO:1. The amino acid positions corresponding to the positions in SEQ ID NO:1 can be identified, for example, by performing an amino acid sequence alignment. The modifications may include substitution of the natural amino acid at position 141 with alanine, e.g., D141A; substitution of the natural amino acid at position 143 with alanine, e.g., E143A; substitution of the natural amino acid at position 143 with aspartic acid, e.g., E143D; substitution of the natural amino acid at position 485 with leucine, e.g., A485L; or combinations thereof. In some embodiments, the modifications include one, two or more of D141A, E143A and A485L.

[0082] Detection and Quantification The methods disclosed herein may include detecting a signal generated from the quenchable label of the first quality control primer during the amplification reaction to determine the generation of an extended duplex, where a decrease in signal during the amplification reaction indicates the generation of an extended duplex. In some embodiments, detecting a signal generated from the quenchable label of the first quality control primer during the amplification reaction may include detecting a signal at two or more different time points during the amplification reaction. The decrease in signal during the amplification reaction may include a decrease over time during the amplification reaction. The decrease in signal during the amplification reaction may include a decrease over a period of about 10 minutes (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes, or a value or range between any two of these values) during the amplification reaction. This period may be about 3 minutes to about 12 minutes (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 minutes, or a value or range between any two of these values) from the start of the amplification reaction. The method may include detecting a signal of the quenchable label of the first quality control primer before the amplification reaction, after the amplification reaction, or both. In some embodiments, the detection of the signal generated from the quenchable label of the first quality control primer does not include the use of any probe. The methods described herein can advantageously provide reduced complexity in monitoring the amplification reaction without the need for additional primers and / or probes.

[0083] The methods described herein may include detecting and / or quantifying the amplification products (e.g., extended duplexes and / or target nucleic acid amplicons). The amplification products can be detected and / or quantified by any suitable detection and / or quantification method, including, for example, any of the detection or quantification methods described herein.Non-limiting examples of detection and / or quantification methods include: molecular beacons (e.g., real-time, end-point), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating / binding dyes, absorbance (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension (e.g., iPLEX™), Affymetrix molecular inversion probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, molecular spectrometry (MIP) analysis, and the like. Chilled specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, gene bit analysis (GBA), multiplex minisequencing, SnaPshot, GOOD assay, microarray miniseq, array primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed integration (TDI, incorporation), colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probes, invader assay, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips, and combinations thereof. In some embodiments, detection of the nucleic acid amplification products comprises the use of real-time detection methods (i.e., products are detected and / or continuously monitored during the amplification process).In some embodiments, detection of nucleic acid amplification products includes the use of end-point detection methods (i.e., products are detected after the amplification process is completed or stopped). Nucleic acid detection methods can also employ the use of labeled nucleotides that are directly incorporated into the target sequence or that are incorporated into a probe that contains a complementary sequence to the target. Such labels may be radioactive and / or fluorescent in nature and can be resolved in any of the manners discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, detection methods can be used in conjunction with measuring signal intensity and / or creating (or referencing) standard curves and / or look-up tables for quantification of nucleic acid amplification products.

[0084] Detection of nucleic acid amplification products (e.g., extended duplexes) may involve the use of fluorescence resonance energy transfer (FRET). FRET is an energy transfer mechanism between two chromophores: a donor molecule and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emission from the donor molecule as it returns to its ground state can transfer the excitation energy to the acceptor molecule through long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored, which is a function of the distance between the donor and acceptor, the overlap of the donor emission spectrum with the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be useful, for example, to quantify the molecular dynamics of DNA-DNA interactions, as described for molecular beacons. To monitor the production of a specific product, a probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Upon probe-target hybridization, the distance or orientation of the donor and acceptor changes and a FRET change is observed. In some embodiments, the quenchable label is the donor molecule. In some embodiments, the acceptor molecule may be a quencher.

[0085] Detection of nucleic acid amplification products (e.g., amplification products resulting from the amplification of a target nucleic acid) may involve the use of molecular beacons, e.g., hairpin-shaped oligonucleotides that contain a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin may contain a probe sequence that is complementary to the target sequence, and the stem is formed by annealing complementary arm sequences located on either side of the probe sequence. The fluorophore and quenching molecules may be covalently linked to the opposite ends of each arm. Under conditions that prevent the oligonucleotide from hybridizing to its complementary target, or when the molecular beacon is free in solution, the fluorescent molecule and the quenching molecule are in close proximity to each other, preventing fluorescence resonance energy transfer (FRET). When the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization may occur, and the loop structure is converted to a stable, more rigid conformation, causing separation of the fluorophore and quencher molecules, resulting in fluorescence. Because the probes are specific, the generation of fluorescence is generally only due to the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is identical or complementary to a sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is neither identical nor complementary to a sequence in the target nucleic acid (e.g., hybridizes to a tail amplification primer or a sequence added to the amplification product by ligation).

[0086] Detection of nucleic acid amplification products may include the use of fluorescence polarization (FP), surface capture, 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating and / or binding dyes, mass spectrometry, nucleic acid sequencing, digital amplification (e.g., digital PCR), and / or absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection of nucleic acid amplification products includes the use of dyes that specifically stain nucleic acids. For example, intercalating dyes exhibit enhanced fluorescence upon binding to DNA or RNA. Dyes include DNA or RNA intercalating fluorophores, and may include, for example, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Dyes provide an opportunity to increase the sensitivity of nucleic acid detection when used with various detection methods. For example, ethidium bromide can be used to stain DNA in agarose gels after gel electrophoresis; propidium iodide and Hoechst 33258 can be used in flow cytometry to determine the DNA ploidy of cells; SYBR® Green 1 can be used in the analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection; PicoGreen® can be used to enhance the detection of double-stranded DNA after matched ion-pair polynucleotide chromatography.

[0087] Provided herein are methods, kits, and reaction mixtures for evaluating, monitoring, observing, or tracking the progress of an amplification reaction. In another aspect, the methods, kits, and reaction mixtures disclosed herein can be used to determine the amplification efficiency of a reaction. Non-limiting examples of factors that can interfere with amplification efficiency can include amplification inhibitors (e.g., hemoglobin, humic acid, fulvic acid, divalent cations, chelating molecules), the absence and / or deficiency of amplification reaction components, and enzymes and other proteins (e.g., nucleases).

[0088] The methods, kits, and reaction mixtures described herein can be used to determine the absence of an amplification reaction component and / or the presence of defects (e.g., quality) of an amplification reaction component. The quality of a component can vary from batch to batch or over time. Thus, the quality of a component can be controlled by evaluating the performance of the component in each batch using the methods described herein, or by evaluating the performance of the component in a batch over time. For example, the performance of two polymerases produced in a reference batch and a sample batch can be evaluated using the disclosed methods. The performance of the polymerase in the sample batch can be compared to that of the polymerase in the reference batch to determine whether the polymerase in the sample batch is suitable for use. Alternatively, the activity of the polymerase can be compared before and after the passage of a predetermined time to determine whether the activity of the polymerase decreases over time. In some embodiments, the disclosed methods can determine the shelf life of one or more amplification components. For example, the activity of the polymerase is evaluated at predetermined time intervals from initial production. If the activity decreases below a predetermined level after a certain period of time, that time can be determined as the shelf life of the polymerase.

[0089] In some embodiments, the methods, kits, and reaction mixtures disclosed herein can be used (e.g., as an internal control) to monitor the amplification efficiency, for example, the presence of an amplification inhibitor that interferes with the amplification efficiency. In general, an internal control is used to monitor the inhibitory activity of a sample (e.g., the presence of an amplification inhibitor). The internal control may include (i) a template that is unrelated to the target nucleic acid sequence suspected to be contained in the sample, and (ii) primers and probes for amplifying and detecting the template. Advantageously, the amplification efficiency of the internal control is not affected by the amplification of the target nucleic acid sequence contained in the sample, and vice versa. Amplification of the internal control indicates that the sample does not have an inhibitory activity against the amplification reaction, i.e., the sample does not contain a substance that inhibits the amplification reaction.

[0090] The first and second quality control primers described herein can replace the components of conventional internal control, i.e., template, primer pair and probe. The first and second quality control primers described herein can be further added to the amplification reaction of the target nucleic acid sequence in the sample, so that the amplification of the duplex formed by hybridization between the 3' overlapping regions of the first and second quality control primers can occur simultaneously with the amplification of the target nucleic acid sequence. The production of the extended duplex can allow the determination of whether an inhibitor to the amplification reaction exists in the sample.

[0091] The method, kit and reaction mixture described herein can be used as quantification standard.For example, in quantitative real-time PCR reaction, the quantification of target nucleic acid sequence is achieved by carrying out amplification reaction using a dilution series of known standard to obtain a standard curve in which the logarithmic value of the initial amount of target nucleic acid sequence is plotted against Ct value, and then the Ct value obtained from unknown sample is compared with the standard curve to calculate the amount of target nucleic acid sequence in the sample.The first and second quality control primers can advantageously be used instead of standard.

[0092] target nucleic acid As used herein, a target nucleic acid is a nucleic acid of interest that is subjected to amplification in an amplification reaction that is evaluated and / or monitored using the quality control methods and compositions disclosed herein. The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein. The term refers to nucleic acids of any composition, such as DNA (e.g., complementary DNA (cDNA) and genomic DNA (gDNA)), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., including base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single-stranded or double-stranded form and may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides, unless otherwise limited. Nucleic acids may be present in plasmids, phages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or in vivo. It may be or be derived from other nucleic acids that are capable of being replicated or replicated in vitro or in a host cell, a cell, a cell nucleus, mitochondria, or the cytoplasm of a cell. Unless otherwise limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0093] A target nucleic acid may be referred to as a target sequence, a target polynucleotide, and / or a target polynucleotide sequence, and may include double-stranded and single-stranded nucleic acid molecules. A target nucleic acid may be, for example, DNA or RNA. A target sequence may refer to either the sense or antisense strand of a nucleic acid sequence, and may refer to a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence may be a subsequence within a larger polynucleotide. For example, a target sequence may be a short sequence (e.g., 20-50 bases) within a nucleic acid fragment, chromosome, or plasmid that is targeted for amplification. In some embodiments, a target sequence refers to a sequence in a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence may refer to the entire sequence that is targeted for amplification, or may refer to a subsequence in a target nucleic acid to which an oligonucleotide binds. An amplification product may be a larger molecule that includes the target sequence as well as at least one other sequence, or other nucleotides. In some embodiments, an amplification product is approximately the same length as the target sequence, or exactly the same length as the target sequence. In some embodiments, the amplification product comprises the target sequence. In some embodiments, the amplification product consists of the target sequence.

[0094] The target nucleic acid may include, for example, genomic nucleic acid, plasmid nucleic acid, mitochondrial nucleic acid, cellular nucleic acid, extracellular nucleic acid, bacterial nucleic acid, and viral nucleic acid. In some embodiments, the target nucleic acid may include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, genes, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA, or synthetic oligonucleotides. The genomic nucleic acid may include any nucleic acid derived from any genome, for example, an animal genome, a plant genome, an insect genome, a viral genome, and a bacterial genome, for example, a genome present in a spore. In some embodiments, the genomic target nucleic acid may be present within a specific genomic locus or multiple genomic loci. The genomic locus may include any or a combination of open reading frame DNA, non-transcribed DNA, intronic sequences, exonic sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequences considered to be associated with a given genomic locus. The target nucleic acid may include a microRNA (miRNA) or a small interfering RNA (siRNA).

[0095] Nucleic acids can be obtained from any suitable biological specimen or sample, and are often isolated from samples obtained from subjects. A subject may be any living or non-living organism, including but not limited to humans, non-human animals, plants, bacteria, fungi, viruses, or protists. Any human or non-human animal can be selected, including but not limited to mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), porcines (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), ursidae (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female, and the subject can be of any age (eg, embryo, fetus, infant, child, adult).

[0096] The sample or test sample may be any specimen isolated or obtained from a subject or a portion thereof, including, but not limited to, blood or blood products (such as, for example, serum or plasma), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy sample, intestinal puncture sample, cells (e.g., blood cells) or portions thereof (e.g., mitochondria, nuclei, or extracts), female reproductive tract washings, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, mammary fluid, hard tissue (e.g., liver, spleen, kidney, lung, or ovary), or combinations thereof. The term blood, as conventionally defined, includes whole blood, blood products, or any fraction of blood such as serum, plasma, or buffy coat. Plasma refers to the fraction of whole blood obtained from centrifugation of blood that has been treated with an anticoagulant. Serum refers to the watery part of the fluid that remains after a blood sample has clotted. Fluid or tissue samples are often collected according to standard protocols commonly followed by hospitals or clinics.

[0097] The sample or test sample may include a sample containing nucleic acid from a spore, a virus, a cell, a prokaryote or a eukaryote, or any free nucleic acid. For example, the methods described herein can be used to detect nucleic acid outside of a spore (e.g., without the need for lysis). The sample can be isolated from any material suspected of containing the target sequence, such as from a subject as described above. In some embodiments, the target sequence is present in air, plants, soil, or other material suspected of containing a biological organism. Any suitable method for isolating, extracting and / or purifying nucleic acids from a biological sample can be used, including, but not limited to, DNA preparation methods in the art and various commercially available reagents or kits such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wis.), and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, N.J.), or the like, or combinations thereof.

[0098] A sample may include sample nucleic acids (e.g., multiple types of sample nucleic acids). The term "multiple types" is used herein to mean two or more. Thus, in some embodiments, a sample includes two or more (e.g., three or more, five or more, ten or more, twenty or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, or five thousand or more) sample nucleic acids (e.g., DNA / RNA). The method of the present disclosure can be used as a highly sensitive method for detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, a sample includes five, ten, twenty, twenty-five, fifty, one hundred, five hundred, ten ... 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 Seeds or 10 7The sample may contain DNA / RNA from 10, 50, or more species that differ in sequence from each other. In some embodiments, the sample contains DNA / RNA from a cell (e.g., a eukaryotic cell, a mammalian cell, or a human cell) or a cell lysate (e.g., a eukaryotic cell lysate, a mammalian cell lysate, a human cell lysate, a prokaryotic cell lysate, or a plant cell lysate, etc.).

[0099] The term "sample" as used herein shall be given its ordinary meaning and shall include any sample containing RNA and / or DNA (e.g., for determining whether target DNA and / or target RNA is present in a population of RNA and / or DNA). A sample may be from any source, e.g., a sample may be a synthetic combination of purified DNA and / or RNA; a sample may be a cell lysate, a DNA / RNA enriched cell lysate, or DNA / RNA isolated and / or purified from a cell lysate. A sample may be from a patient (e.g., for diagnostic purposes). A sample may be from permeabilized cells, crosslinked cells, tissue sections, or combinations thereof. A sample may be from tissue prepared by crosslinking followed by delipidation and adjustment to a uniform refractive index. A sample may include a target nucleic acid (e.g., target DNA / RNA) and multiple species of non-target DNA / RNA. In some embodiments, the target DNA / RNA may be 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 Present in the sample at 1 copy per non-target DNA / RNA.

[0100] Samples related to patients include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, as well as samples that have been manipulated in some way after procurement (such as treatment with a reagent); washed; or enriched for a particular cell population (e.g., cancer cells) or a particular type of molecule (e.g., RNA). Samples may include or may be biological samples including, but not limited to, clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, and bone marrow. Biological samples may also include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), such as samples containing RNA obtained from such cells (e.g., cell lysates or other cell extracts containing RNA).

[0101] The source of the sample may be a diseased (or suspected) cell, fluid, tissue, or organ, or may be a normal (non-diseased) cell, fluid, tissue, or organ. In some embodiments, the source of the sample is a cell, tissue, or organ infected (or suspected) with a pathogen. For example, the source of the sample may be an individual, which may or may not be infected, and the sample may be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, fecal sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., buccal swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal secretion, tears, buffy coat, mucosal sample, epithelial cell sample (e.g., epithelial cell scraping), etc.). The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens may be viruses, fungi, protozoans, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites, etc. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny head worms, and cestodes (Taenia). Protozoan infections include infections with Giardia spp., Trichomonas spp., African trypanosomiasis, amebic dysentery, babesiosis, balantidiosis, Chagas disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens, such as parasitic / protozoan pathogens, include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans, and the like.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza viruses, dengue fever, West Nile virus, herpes viruses, yellow fever viruses, hepatitis C viruses, hepatitis A viruses, hepatitis B viruses, and papilloma viruses. Pathogenic viruses include, but are not limited to, papovaviruses (e.g., HPV, polyomaviruses); hepadnaviruses; herpes viruses (e.g., HSV (e.g., HSV I, HSV II), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasis rosea, and the like. Rosea), Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; variola virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; and Phycodnaviridae. Non-limiting examples of pathogens include Mycobacterium tuberculosis and Streptococcus agalactiae, Methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Haemophilus influenzae type B, and the like.B), Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Rabies virus, Human serum bulbo-like virus, Respiratory syncytial virus, Measles virus, Adenovirus, Human T-cell leukemia virus, Murine leukemia virus, Mumps virus, Vesicular stomatitis virus, Sindbis virus, Lymphocytic choriomeningitis virus, Wart virus, Bluetongue virus, Sendai virus, Feline leukemia virus, Reovirus, Poliovirus, Simian virus 40, Mouse mammary tumor virus, Dengue virus, Rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis arthritidis, M.hyorhinis, M.orale, M.arginini, Acholeplasma laidlowiilaidlawii, M. salivarium, and M. pneumoniae.

[0102] kit Disclosed herein is a kit for assessing, monitoring, observing, or tracking the progress of an amplification reaction. The kit may include a first quality control primer and a second quality control primer, each of which comprises a 3' overlap region capable of hybridizing to each other, where the first quality control primer comprises a quenchable label. In some embodiments, each of the first quality control primer and the second quality control primer is 35 nucleotides in length or less.

[0103] The quenchable label may be a fluorophore. The quenchable label may be outside the 3' overlap region of the first quality control primer. The quenchable label may be at the 5' end of the first quality control primer. The quenchable label may be in the 3' overlap region of the first quality control primer. The second quality control primer may include a quencher. The quencher may be outside the 3' overlap region of the second quality control primer. The quencher may be at the 5' end of the second quality control primer. The quencher may be in the 3' overlap region of the second quality control primer.

[0104] The first quality control primer, the second quality control primer, or both may comprise one or more modified nucleotides. The 3' overlap region of the first and second quality control primers may each comprise one or more modified nucleotides. The one or more modified nucleotides may comprise a spacer, an abasic site, an unmethylated RNA base, a 2'-O-methylated nucleotide, and any combination thereof. At least one of the one or more modified nucleotides may be a 2'-O-methylated nucleotide. The first quality control primer, the second quality control primer, or both may comprise one or more polymerase stoppers. In some embodiments, the 3' overlap region of the first and second quality control primers may each comprise one or more polymerase stoppers. In some embodiments, at least one of the one or more polymerase stoppers may be a 2'-O-methylated nucleotide.

[0105] The 3' overlap region of the first quality control primer may be complementary to the 3' overlap region of the second quality control primer. The 3' overlap region of the first quality control primer may be fully complementary to the 3' overlap region of the second quality control primer. In some embodiments, the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer have the same length. One or more of the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer may be about 2 to about 10 nucleotides in length. The 3' overlap regions of the first and second quality control primers may be 4 or 5 nucleotides in length.

[0106] The kit may include one or more of an enzyme with hyperthermophile polymerase activity, dNTPs, and a buffer. The enzyme with hyperthermophile polymerase activity has an amino acid sequence that may be at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. The enzyme with hyperthermophile polymerase activity may include the amino acid sequence of SEQ ID NO: 1. The kit may further include, for example, modified nucleotides used in the reaction, vessels, cuvettes, or other containers, or vials of water or buffer for rehydrating lyophilized or heat-dried components. The buffer used may, for example, be suitable for both polymerase activity and primer annealing activity. The kit may include one or more additional primers and / or one or more probes specific for the target nucleic acid.

[0107] The kit may also include one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates, and the like, or the components may be combined in various combinations in such containers. The components of the kit may, for example, be present in one or more containers. In some embodiments, all of the components are provided in one container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a separate container from the primers. The components may, for example, be lyophilized, heat-dried, freeze-dried, or present in a stable buffer. In some embodiments, the polymerase and / or reverse transcriptase are present in a single container in lyophilized or heat-dried form, and the primers are either lyophilized, heat-dried, freeze-dried, or present in a buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase and the primers are present in a single container in lyophilized or heat-dried form.

[0108] The compositions described herein (e.g., dry compositions) can be provided in a "dry form" or in a form not suspended in a liquid medium. A "dry form" of a composition can include a dry powder, a lyophilized composition, a spray-dried composition, or a precipitated composition. A "dry form" composition can include one or more lyoprotectants such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine and histidine; lyotropic salts, such as MgSO4; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or polypropylene glycol; and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethylcellulose. As used herein, the terms "lyophilization," "lyophilized," and "freeze-drying" refer to a process in which the material to be dried is first frozen and then the ice or freezing solvent is removed by sublimation in a vacuum environment. "Lyophilisate" refers to a material that has been freeze-dried. As disclosed herein, the dry composition may include one or more additives and one or more amplification reagents.

[0109] The dry composition may be frozen or lyophilized or spray dried. The dry composition may be heat dried. The dry composition may include one or more additives (e.g., polymers, sugars, or sugar alcohols). The sugar or sugar alcohol may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The one or more additives may include one or more amino acids. The one or more additives may include Tween 80, Tween 20, and / or Triton X-100. In some embodiments, the one or more additives aid in the lyophilization of the reaction composition and / or dissolution of the dried pellet. The one or more additives may include a non-ionic detergent at a concentration of about 0.01% in the dry composition (eg, dry pellets). The frozen or lyophilized or spray-dried or heat-dried composition, or the aqueous composition for preparing the frozen or lyophilized or spray-dried composition, may include one or more of the following: (i) non-aqueous solvents such as ethylene glycol, glycerol, dimethylsulfoxide, and dimethylformamide. (ii) surfactants such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic F127 (polyoxyethylene-polyoxypropylene copolymer), also known as poloxamer, poloxamine, and SDS. (iii) disaccharides such as trehalose, sucrose, lactose, and maltose. (iv) polymers (which may have different MW) such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, and albumin. (v) amino acids such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, gamma-aminobutyric acid. The first quality control primer and the second quality control primer may be in lyophilized or freeze-dried form. One or more of the enzyme having hyperthermophile polymerase activity, dNTPs, and buffer may be in lyophilized or freeze-dried form.

[0110] The kit may include instructions for carrying out one or more of the methods described herein and / or instructions for one or more of the components described herein. The instructions and / or instructions may be in printed form or may be included in a kit insert. The kit may also include a written description of an internet location that provides such instructions or instructions. In some embodiments, the kit includes reagents used in a detection method, such as reagents used for FRET, lateral flow devices, dipsticks, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. EXAMPLES

[0111] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0112] Example 1 DIMER Internal Control This example describes the monitoring of an amplification reaction. Forward and reverse IC primers were used in the amplification reaction. The forward IC primer has a fluorophore attached to its 5' end, and the reverse IC primer has a quencher attached to its 5' end (Figure 1). At the start of the reaction, the forward and reverse IC primers are individually in solution, so the fluorophore on the forward IC primer is fully emitting (producing a high signal) (Figure 2). As the archaeal polymerase amplification (APA) reaction progresses, the Dimer-IC amplicon (e.g., extended duplex) brings the fluorophore and quencher into close proximity such that FRET quenching can occur, causing a drop in signal. Thus, an "inverted" amplification curve is eventually produced, as shown in Figure 2.

[0113] 3A-3B show inversion amplification curves observed using the Dimer-IC system disclosed herein: Forward primer (FP1) + reverse primer (RP3) (5 bp overlap), Forward primer (FP1) + reverse primer (RP2) (4 bp overlap), Forward primer only control (FP1 only) (no amplification expected). The intercalating dye signal (run in the same reaction) indicated that amplification had occurred as intended. FIG. 4 shows a pair of quality control primers: (1) forward quality control primer FP3 having the sequence of SEQ ID NO:3 and an internal 2'OM modification at nucleotide position 10 and (2) reverse quality control primer RP4 having the sequence of SEQ ID NO:5 and an internal 2'OM modification at nucleotide position 9.

[0114] In some cases, unintended primer extension by quality primers with other oligos can be prevented by inclusion of one or more nucleotides with 2'OM modifications in the quality control primer. As shown in FIG. 5A, nucleic acid extension from unintended hybridization occurring at nucleotide positions 12-13 of the forward quality control primer (FP3) with a non-IC primer oligonucleotide is halted because the DNA polymerase cannot read through the 2'-OM modified nucleotide at position 10 in FP3. In some embodiments, unintended primer extension (e.g., on human gDNA) and subsequent amplification by unintended reverse priming (by one of the other primers present in the reaction) can be similarly prevented (or significantly reduced) by inclusion of modified nucleotides (e.g., nucleotides with 2'OM modifications) at one or more positions in the quality control primer. As shown in FIG. 5B, the inability of the DNA polymerase to read through nucleotide position 10 in the depicted forward quality control primer FP3 makes this quality control primer much more resistant to nonspecific amplification.

[0115] As shown in Figures 6A-6C, the quenchable label and quencher can be placed in various regions of the quality control primers, for example, outside and / or inside the 3' overlap region of the first and second quality control primers. Without being bound by any particular theory, it is expected that placing the quenchable label in the 3' overlap region of the forward quality control primer and the quencher in the 3' overlap region of the reverse quality control primer may result in contact quenching (as opposed to FRET quenching), resulting in a larger change in signal (Figure 6B). In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0116] With respect to the use of substantially any plural and / or singular terminology herein, one of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless otherwise indicated.

[0117] Those skilled in the art will understand that the terms used in this specification, generally, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Moreover, those skilled in the art will understand that if a specific number is intended in the introduced claim language, such intent will be expressly recited in the claim, and that in the absence of such recitation, no such intent exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim language. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim containing such an introduced claim recitation to an embodiment containing only one such recitation, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. In addition, even if a specific number is explicitly recited in an introduced claim recitation, a person skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the basic recitation of "two recitations" means at least two recitations, or two or more recitations, in the absence of other modifiers).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, those skilled in the art will appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either such terms, or both terms.

[0118] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of every individual member or subgroup of members of the Markush group. As will be appreciated by those of skill in the art, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as being capable of dividing the same range into at least two, three, four, five, ten, etc., as fully described. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Also, as will be appreciated by those of skill in the art, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the numerical values ​​recited and that can be subsequently divided into subranges as discussed above. Finally, as will be appreciated by those of skill in the art, ranges include each individual member. Thus, for example, a group containing 1-3 items refers to a group containing 1, 2, or 3 items. Similarly, a group containing 1-5 items refers to a group containing 1, 2, 3, 4, or 5 items, etc.

[0119] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for monitoring an amplification reaction, comprising: (a) providing a first quality control primer and a second quality control primer, each comprising a 3′ overlap region capable of hybridizing to one another, wherein each of the first quality control primer and the second quality control primer is 35 nucleotides in length or less, the first quality control primer comprising a quenchable label, the quenchable label being a fluorophore, and the second quality control primer comprising a quencher; (b) contacting the first quality control primer and the second quality control primer, thereby forming a duplex by hybridization between the 3' overlap regions of the first and second quality control primers; (c) subjecting the duplex to amplification conditions, thereby producing an extended duplex; and (d) detecting a signal generated from the quenchable label of the first quality control primer during the amplification reaction to determine the formation of the extended duplex, wherein a decrease in the signal during the amplification reaction indicates the formation of the extended duplex. A method comprising:

2. A method according to claim 1, a. the quenchable label comprises: i. outside the 3' overlap region of the first quality control primer, optionally at the 5' end of the first quality control primer; or ii. in the 3' overlap region of the first quality control primer; and / or b. The quencher is i. outside the 3' overlap region of the second quality control primer, optionally at the 5' end of the second quality control primer; or ii. in the 3' overlap region of the second quality control primer.

3. A method according to claim 1, a. the first quality control primer, the second quality control primer, or both, i. comprises one or more modified nucleotides; Optionally, 1. The 3' overlap regions of the first and second quality control primers each optionally contain one or more modified nucleotides; and / or 2. The one or more modified nucleotides may include a spacer, an abasic site, an unmethylated RNA base, a 2'-O-methylated nucleotide, and any combination thereof; and / or 3. At least one of the one or more modified nucleotides may be a 2'-O-methylated nucleotide; or ii. comprises one or more polymerase stoppers; Optionally, 1. The 3' overlap regions of the first and second quality control primers each optionally contain one or more polymerase stoppers; and / or 2. At least one of the one or more modified nucleotides may be a 2'-O-methylated nucleotide, and / or b. the 3' overlap region of the first quality control primer is complementary to the 3' overlap region of the second quality control primer, and optionally the 3' overlap region of the first quality control primer may be fully complementary to the 3' overlap region of the second quality control primer; and / or c. the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer have the same length; and / or d. one or more of the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer are from about 2 to about 10 nucleotides in length; and / or e. The method of any preceding claim, wherein the 3' overlap region of the first and second quality control primers is 4 or 5 nucleotides in length.

4. The method of claim 1, (b) the step of contacting the first quality control primer and the second quality control primer is performed under the amplification conditions; and / or (d) detecting the signal generated from the quenchable label of the first quality control primer during the amplification reaction comprises detecting the signal at two or more different time points during the amplification reaction; and / or c. the decrease in the signal during the amplification reaction comprises a decrease over time during the amplification reaction; Optionally, the decrease in the signal during the amplification reaction is i. may include a decrease over a period of about 10 minutes during the amplification reaction; or ii. The decrease may occur over a period of about 3 minutes to about 12 minutes from the start of the amplification reaction; and / or d. The method, wherein the method comprises detecting the signal of the quenchable label of the first quality control primer before the amplification reaction, after the amplification reaction, or both.

5. The method of claim 1, a. the amplification reaction is a real-time amplification reaction, and / or b. The amplification reaction i. a PCR reaction, or ii. comprising one or more of the following: Archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA); or iii. An isothermal amplification reaction, Optionally, 1. The amplification conditions may include one or more of an enzyme with hyperthermophilic polymerase activity, dNTPs, and a buffer, and optionally, the enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof, and optionally, the enzyme with hyperthermophilic polymerase activity may comprise the amino acid sequence of SEQ ID NO: 1; and / or 2. The isothermal amplification reaction may comprise a constant temperature of about 30°C to about 72°C, and optionally, the isothermal amplification reaction may comprise a constant temperature of about 67°C; and / or 3. The isothermal amplification reaction may be carried out for a period of about 5 minutes to about 60 minutes; and / or 4. The method as described above, wherein the isothermal amplification reaction may be carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions.

6. The method of claim 1, a. detection of the signal generated from the quenchable label of the first quality control primer does not involve the use of any probe; and / or (c) subjecting the duplex to said amplification conditions comprises subjecting a target nucleic acid and one or more additional primers and / or one or more probes specific to the target nucleic acid to said amplification conditions; Optionally, i. the first quality control primer may not hybridize to the target nucleic acid under the amplification conditions, and / or ii. The method of claim 1, wherein the second quality control primer does not hybridize to the target nucleic acid under the amplification conditions.

7. The method according to claim 1, wherein the method comprises: a. the method does not involve using a template nucleic acid capable of hybridizing to the first quality control primer, and / or b. The method of any of the preceding claims, wherein the method does not include using a template nucleic acid capable of hybridizing to the second quality control primer.

8. 1. A kit for monitoring an amplification reaction, comprising: a first quality control primer and a second quality control primer, each comprising a 3' overlap region capable of hybridizing to one another, wherein the first quality control primer and the second quality control primer are each 35 nucleotides in length or less, the first quality control primer comprising a quenchable label, and the second quality control primer comprising a quencher; The above kit.

9. The kit according to claim 8, a. the quenchable label is a fluorophore, and / or b. the quenchable label is i. outside the 3' overlap region of the first quality control primer, and optionally at the 5' end of the first quality control primer; or ii. in the 3' overlap region of the first quality control primer; and / or c. the quencher is i. outside the 3' overlap region of the second quality control primer, and optionally at the 5' end of the second quality control primer; or ii. in the 3' overlap region of the second quality control primer.

10. The kit according to claim 8, a. the first quality control primer, the second quality control primer, or both, i. comprises one or more modified nucleotides; arbitrarily 1. The 3' overlap regions of the first and second quality control primers each optionally contain one or more modified nucleotides; and / or 2. The one or more modified nucleotides may comprise a spacer, an abasic site, an unmethylated RNA base, a 2'-O-methylated nucleotide, and any combination thereof; or ii. comprises one or more polymerase stoppers; arbitrarily 1. The 3' overlap regions of the first and second quality control primers each optionally contain one or more polymerase stoppers; and / or 2. At least one of the one or more polymerase stoppers may be a 2'-O-methylated nucleotide; and / or b. the 3' overlap region of the first quality control primer is complementary to the 3' overlap region of the second quality control primer, and optionally the 3' overlap region of the first quality control primer may be fully complementary to the 3' overlap region of the second quality control primer; and / or c. the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer have the same length; and / or d. one or more of the 3' overlap region of the first quality control primer and the 3' overlap region of the second quality control primer are from about 2 to about 10 nucleotides in length; and / or e. The above kit, wherein the 3' overlap region of the first and second quality control primers is 4 or 5 nucleotides in length.

11. The kit of claim 8, The method comprises one or more of an enzyme having hyperthermophilic polymerase activity, dNTPs, and a buffer; Optionally, the enzyme with hyperthermophile polymerase activity may have an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof, and optionally, the enzyme with hyperthermophile polymerase activity may comprise the amino acid sequence of SEQ ID NO:

1.

12. The kit according to any one of claims 8 to 11, a. the first quality control primer and the second quality control primer are in lyophilized or freeze-dried form, and optionally the one or more of the enzyme with hyperthermophilic polymerase activity, dNTPs, and buffering agents may also be in lyophilized or freeze-dried form; and / or b. The kit as described above, further comprising one or more additional primers and / or one or more probes specific for the target nucleic acid.

13. a first quality control primer and a second quality control primer, each comprising a 3' overlap region capable of hybridizing to one another, wherein each of the first quality control primer and the second quality control primer is 35 nucleotides in length or less, the first quality control primer comprising a quenchable label, and the second quality control primer comprising a quencher; a target nucleic acid; and one or more additional primers and / or one or more probes specific to said target nucleic acid; A reaction mixture comprising:

14. The reaction mixture of claim 13, a. the reaction mixture comprises a duplex formed by hybridization between the 3' overlap regions of the first and second quality control primers, and / or b. The reaction mixture comprises one or more of an enzyme having polymerase activity, dNTPs, and a buffer, wherein optionally the enzyme is an enzyme having hyperthermophilic polymerase activity, and optionally the enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof, and optionally the enzyme having hyperthermophilic polymerase activity comprises the amino acid sequence of SEQ ID NO:

1.

15. The reaction mixture of claim 13 or 14, a. the first quality control primer and the second quality control primer are incapable of hybridizing to the target nucleic acid; and / or b. The reaction mixture above, wherein the one or more additional primers and / or one or more probes are unable to hybridize to the first quality control primer, the second quality control primer, or both.

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