Hairpin internal standards for isothermal nucleic acid amplification

Abstract

Disclosed herein are methods, compositions, and kits for monitoring an amplification reaction. Some embodiments include a quality control template comprising a 5' subdomain, a 3' subdomain, and a loop domain between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. The quality control primer can be capable of hybridizing to at least a portion of the 3' subdomain. Subjecting the quality control template and the quality control primer to an amplification reaction can produce a first quality control product. The method can include detecting the first quality control product.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 374,835, filed September 7, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Sequence Listing Reference This application is filed with an electronic Sequence Listing. The Sequence Listing is provided in file number 68EB-317350-WO, created September 6, 2023, and is 23,044 bytes 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 methods and compositions for amplifying (eg, isothermal amplification) nucleic acids. [Background technology]

[0002] Nucleic acid-based diagnostics can be useful for the rapid detection of infections, diseases, and / or genetic mutations. For example, identifying bacterial or viral nucleic acids in a sample can be useful for diagnosing certain types of infectious diseases. Other examples include identifying single nucleotide polymorphisms for disease control or forensics, and identifying genetic mutations indicative of genetically modified foods. Nucleic acid-based diagnostic assays often require the amplification of specific portions of nucleic acids in a sample. A common technique for nucleic acid amplification is polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermal cycling) to proceed through the following steps: denaturation (e.g., separating the strands of a double-stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short strands of complementary DNA sequence), and extension of the primers along their complementary targets by a polymerase. Such thermal cycling can be a time-consuming process that generally requires specialized machinery. Therefore, there is a need for faster nucleic acid amplification methods that can be performed without thermal cycling. Such methods can be useful, for example, for on-site testing and point-of-care diagnostics. In particular, there is a need for compositions and methods for more rapid nucleic acid detection in which internal standard(s) ("IC") do not interfere with target amplification efficiency. Two main IC strategies have been used in nucleic acid detection assays: competitive IC approach and non-competitive IC approach. The difference between the two lies in whether the IC shares a common set of primers for IC and target amplification. With competitive IC strategy, there is always some competition between the target and IC due to the simultaneous amplification of target and IC fragments flanked by the same primers. Competition due to IC amplification can reduce target amplification efficiency, thereby resulting in a lower detection limit. Therefore, competitive IC methods require further IC optimization to achieve a highly sensitive detection limit. In the non-competitive approach, the target and IC are amplified using different primer sets, respectively.The kinetics of each reaction is not affected by competition for primers, but IC amplification must be limited by controlled concentrations of IC-specific primers and / or IC templates to limit competition between the target and IC reactions for primers and DNA polymerase. Thus, the nucleotide composition, copy number, and size of the IC must be carefully considered with this approach. There is a need for compositions and methods for monitoring amplification reactions with reduced assay complexity and reduced undesired interactions between target amplification and IC. Summary of the Invention

[0003] Disclosed herein is a method for monitoring an amplification reaction. In some embodiments, the method includes providing a quality control template comprising a 5' subdomain; a 3' subdomain; and a loop domain between the 5' subdomain and the 3' subdomain, wherein intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, the method includes providing a quality control primer capable of hybridizing to at least a portion of the 3' subdomain. In some embodiments, the method includes subjecting the quality control template and the quality control primer to an amplification reaction capable of generating a first quality control product. In some embodiments, the method includes detecting the first quality control product. In some embodiments, the amplification reaction is performed in an amplification reaction mixture under amplification conditions (e.g., isothermal amplification conditions). In some embodiments, subjecting the quality control template and the quality control primer to an amplification reaction capable of generating a first quality control product includes amplifying the quality control template using the quality control primer in the amplification reaction mixture under amplification conditions, thereby generating the first quality control product. In some embodiments, the amplification reaction comprises a reverse transcription reaction.

[0004] In some embodiments, the method further comprises providing an enzyme having polymerase activity (e.g., an enzyme having hyperthermophile polymerase activity); and / or providing a reverse transcriptase. In some embodiments, the enzyme having hyperthermophile polymerase activity has reverse transcriptase activity. In some embodiments, the amplification reaction comprises contacting a quality control primer with the quality control template for hybridization and extending the quality control primer hybridized to the quality control template with the enzyme having polymerase activity, thereby generating a first quality control product. In some embodiments, the amplification reaction comprises contacting a quality control primer with the first quality control product for hybridization and extending the quality control primer hybridized to the first quality control product with the enzyme having polymerase activity, thereby generating a second quality control product. In some embodiments, the amplification reaction includes contacting a quality control primer with a second quality control product for hybridization and extending the quality control primer hybridized to the second quality control product with an enzyme having polymerase activity, thereby generating a first quality control product. In some embodiments, the first quality control product and the second quality control product comprise a 5' subdomain and a 3' subdomain capable of forming a paired stem domain. In some embodiments, the first quality control product and the second quality control product have the same stem domain. In some embodiments, the first quality control product and the second quality control product comprise complementary loop domains. In some embodiments, the amplification reaction includes linear and / or exponential amplification of the first quality control product and the second quality control product. In some embodiments, the 5' subdomain comprises at least a portion of the sequence of the quality control primer. In some embodiments, both the first quality control product and the second quality control product are capable of forming a hairpin structure. In some embodiments, the quality control template comprises a 5'-terminal domain that is 5' of the 5' subdomain and / or the quality control template comprises a 3'-terminal domain that is 3' of the 3' subdomain, hi some embodiments, the 5'-terminal domain of the quality control template comprises at least a portion of the sequence of the quality control primer.In some embodiments, the combined sequence of the 5' terminal domain and the 5' subdomain comprises the entire sequence of a quality control primer.

[0005] Detecting the first quality control product can include detecting the first quality control product with a signal-generating oligonucleotide. In some embodiments, the signal-generating oligonucleotide is capable of hybridizing to the first quality control product. In some embodiments, the detecting step includes contacting the first quality control product with the signal-generating oligonucleotide for hybridization. In some embodiments, the signal-generating oligonucleotide includes a quencher, a label, or both. In some embodiments, the label includes a quenchable label (e.g., a fluorophore). In some embodiments, the signal-generating oligonucleotide includes a quencher. In some embodiments, the quencher is capable of quenching the label. In some embodiments, the detecting step includes contacting the first quality control product with the signal-generating oligonucleotide for hybridization. In some embodiments, the label is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the first quality control product. In some embodiments, the label generates a signal after the signal-generating oligonucleotide hybridizes to the first quality control product. In some embodiments, the signal is fluorescent. In some embodiments, detecting the first quality control product comprises detecting a signal generated by a label of the signal-generating oligonucleotide. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, detecting comprises detecting the signal of the label before, during, or after the amplification reaction, or any combination thereof.

[0006] In some embodiments, the method further comprises providing a signal-generating oligonucleotide; subjecting the signal-generating oligonucleotide to an amplification reaction; and detecting a first quality control product using the signal-generating oligonucleotide. In some embodiments, the quality control template is a signal-generating oligonucleotide. In some embodiments, the quality control template is (i) a template for synthesis of the first quality control product and (ii) a means for detecting the first quality control product. In some embodiments, the signal-generating oligonucleotide is capable of (i) detecting the first quality control product and (ii) serving as a template for quality control primer-driven synthesis of the first quality control product.

[0007] In some embodiments, the 5'-terminal domain of the quality control template comprises one or more RNA nucleotides; and / or at least a portion of the sequence of a quality control primer. In some embodiments, the quality control template does not comprise a 3'-terminal domain; and / or the 3'-end of the quality control template is complementary to the 5'-end of the 5' subdomain of the quality control template. In some embodiments, a reverse transcriptase can use one or more RNA nucleotides of the 5'-terminal domain of the quality control template as a template to extend the 3'-end of the quality control template, thereby generating an extended quality control template. In some embodiments, the 3'-end of the extended quality control template comprises a sequence complementary to at least a portion of the quality control primer. In some embodiments, the amplification reaction comprises contacting a reverse transcriptase with the quality control template, thereby generating an extended quality control template. In some embodiments, the extended quality control template comprises cDNA. In some embodiments, the amplification reaction includes contacting a quality control primer with the 3' end of the extended quality control template for hybridization, and extending the quality control primer hybridized to the 3' end of the extended quality control template using an enzyme having reverse transcriptase and / or polymerase activity, thereby generating a first quality control product.

[0008] In some embodiments, the quality control template is a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide comprises a label, and the loop domain comprises one or more RNA nucleotides. In some embodiments, the label comprises a quenchable label (e.g., a fluorophore). In some embodiments, the signal-generating oligonucleotide comprises a quencher. In some embodiments, the label is in the 3'-terminal domain and the quencher is in the 5'-terminal domain, and / or the label is in the 5'-terminal domain and the quencher is in the 3'-terminal domain. In some embodiments, the amplification reaction includes contacting a quality control primer with the quality control template for hybridization and extending the quality control primer hybridized to the quality control template with a reverse transcriptase, thereby generating a first quality control product. In some embodiments, the reverse transcriptase comprises RNase H activity. In some embodiments, the reverse transcriptase cleaves the quality control template at one or more RNA nucleotides during generation of the first quality control product, thereby generating a first cleavage product and a second cleavage product comprising the label. In some embodiments, detecting the first quality control product comprises detecting a signal generated by the first cleavage product comprising a label. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, the method further comprises providing an auxiliary quality control primer; and subjecting the auxiliary quality control primer to an amplification reaction.

[0009] In some embodiments, the signal-generating oligonucleotide is about 10 to about 100 nucleotides in length; the quality control template is about 10 to about 100 nucleotides in length; the quality control primer and / or auxiliary quality control primer is about 5 to about 25 nucleotides in length; and / or the 5' subdomain, 3' subdomain, loop domain, 5' terminal domain, and / or 3' terminal domain are about 1 to about 25 nucleotides in length. In some embodiments, the signal-generating oligonucleotide, quality control template, and / or quality control primer comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids (LNAs). In some embodiments, the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. The signal-generating oligonucleotide can comprise one or more LNAs. In some embodiments, the one or more LNAs are in the loop domain (e.g., the one or more LNAs enhance the detectability of the first quality control product) and / or the stem loop. In some embodiments, the signal-generating oligonucleotide is configured (e.g., configured with one or more LNAs in the loop domain) so that the melting temperature (Tm) of the first quality control product / signal-generating oligonucleotide duplex is equal to or exceeds the melting temperature (Tm) of the paired stem domain of the signal-generating oligonucleotide.

[0010] In some embodiments, the signal-generating oligonucleotide does not include a dye capable of quenching the label. In some embodiments, the signal-generating oligonucleotide does not include a moiety capable of quenching the label other than the nucleotides of the signal-generating oligonucleotide. In some embodiments, the 5'-terminal domain of the quality control template and / or the signal-generating oligonucleotide includes a label. In some embodiments, the 5'-terminal domain and / or the 5' subdomain of the quality control template and / or the signal-generating oligonucleotide includes one or more cytosine bases. In some embodiments, the 3'-terminal domain and / or the 3' subdomain of the quality control template and / or the signal-generating oligonucleotide includes one or more guanine and / or adenine bases. In some embodiments, the one or more guanine and / or adenine bases are capable of quenching the label after the quality control template and / or the signal-generating oligonucleotide forms a hairpin structure. In some embodiments, the 3'-terminal domain of the quality control template and / or the signal-generating oligonucleotide includes a label. In some embodiments, the 3'-terminal domain and / or 3'-subdomain of the quality control template and / or signal-generating oligonucleotide comprises one or more cytosine bases. In some embodiments, the 5'-terminal domain and / or 5'-subdomain of the quality control template and / or signal-generating oligonucleotide comprises one or more guanine and / or adenine bases. In some embodiments, the one or more guanine and / or adenine bases can quench the label after the quality control template and / or signal-generating oligonucleotide forms a hairpin structure.

[0011] In some embodiments, detecting the first quality control product comprises detecting a decrease in the amount of signal generated by the label of the quality control primer. In some embodiments, the label is a fluorophore and the signal is fluorescent. In some embodiments, the generation of the first quality control product and the second quality control product correlates with the attenuation of the detected signal. In some embodiments, the 5' end of the quality control primer comprises a label. In some embodiments, the quality control primer comprises one or more pyrimidine bases adjacent to the label. In some embodiments, the quality control primer comprises one or more cytosine bases adjacent to the label. In some embodiments, the 3' terminal domain and / or 3' subdomain of the quality control template, the first quality control product, and / or the second quality control product comprises one or more guanine and / or adenine bases. In some embodiments, after the quality control primer binds to the quality control template and / or the second quality control product and is extended by the enzyme having polymerase activity to generate a first quality control product, one or more guanine and / or adenine bases present in the 3'-terminal domain and / or 3' subdomain of the first quality control product are capable of quenching the label after the first quality control product forms a hairpin structure. In some embodiments, after the quality control primer binds to the first quality control product and is extended by the enzyme having polymerase activity to generate a second quality control product, one or more guanine and / or adenine bases present in the 3'-terminal domain and / or 3' subdomain of the second quality control product are capable of quenching the label after the second quality control product forms a hairpin structure. In some embodiments, detecting the first quality control product comprises contacting the first quality control product with a fluorescent dye.

[0012] In some embodiments, providing quality control primers, quality control templates, and / or signal-generating oligonucleotides comprises providing a reagent composition comprising the quality control primers, quality control templates, and / or signal-generating oligonucleotides. In some embodiments, subjecting the quality control primers, quality control templates, and / or signal-generating oligonucleotides to an amplification reaction comprises contacting the reagent composition with the processed sample to produce an amplification reaction mixture. In some embodiments, the method further comprises detecting a target nucleic acid sequence in the sample. In some embodiments, the method comprises subjecting the target nucleic acid sequence to an amplification reaction capable of producing a nucleic acid amplification product. In some embodiments, the method comprises detecting the nucleic acid amplification product using a target signal-generating oligonucleotide, the target signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product. In some embodiments, subjecting the target nucleic acid sequence to an amplification reaction capable of producing a nucleic acid amplification product comprises amplifying the target nucleic acid sequence in the amplification reaction mixture under amplification conditions, thereby producing the nucleic acid amplification product.

[0013] The method can include contacting a sample containing the biological entities with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence. The method can also include contacting a reagent composition with the processed sample to produce an amplification reaction mixture, the reagent composition comprising one or more amplification reagents. In some embodiments, the one or more amplification reagents comprise a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity (e.g., an enzyme having hyperthermophilic polymerase activity and reverse transcriptase activity); a forward primer; a reverse primer; a reverse transcription primer; and / or dNTPs. In some embodiments, the sample nucleic acid comprises a nucleic acid comprising the target nucleic acid sequence. In some embodiments, amplifying a target nucleic acid sequence includes amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, wherein the amplifying step includes contacting a nucleic acid comprising the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the nucleic acid is double-stranded DNA; and / or the product of a reverse transcription reaction. In some embodiments, the nucleic acid is the product of a reverse transcription reaction generated from a sample ribonucleic acid (e.g., the amplifying step includes generating a nucleic acid by a reverse transcription reaction).

[0014] In some embodiments, the amplification reaction is carried out for a period of about 5 minutes to about 60 minutes. In some embodiments, amplifying the quality control template comprises generating a first quality control product and / or a second quality control product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes. In some embodiments, amplifying the target nucleic acid sequence comprises generating a nucleic acid amplification product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes. In some embodiments, the detecting step is carried out for less than about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, or about 2 minutes from the time of contacting the reagent composition with the treated sample. In some embodiments, the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. In some embodiments, the pH of the lysis buffer is about 1.0 to about 10.0. In some embodiments, the pH of the lysis buffer is about 2.2. In some embodiments, the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid. In some embodiments, the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof.

[0015] In some embodiments, the reagent composition is lyophilized, heat-dried, and / or comprises one or more additives, where the one or more additives comprise Tween 20, Triton X-100, and / or Tween 80; an amino acid; a sugar or sugar alcohol (e.g., sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof); and / or a polymer (e.g., polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof). In some embodiments, contacting the reagent composition with the processed sample comprises dissolving the reagent composition in the processed sample. In some embodiments, the one or more lysis reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) (e.g., about 0.2% (wt / vol)) of the processed sample; and / or include a detergent (e.g., a cationic surfactant, an anionic surfactant, a nonionic surfactant, an amphoteric surfactant). In some embodiments, the method is performed in a single reaction vessel; does not include the use of any enzymes other than the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity; does not include the use of any enzymes other than the enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid and / or quality control template during the amplification step; and / or does not include a step of contacting the nucleic acid and / or quality control template with a signal-stranded DNA binding protein.

[0016] In some embodiments, the target nucleic acid sequence comprises a length of about 20 nucleotides or less to about 90 nucleotides or less. In some embodiments, the target nucleic acid sequence comprises a length of about 30 nucleotides. In some embodiments, the forward primer, reverse primer, and / or reverse transcription primer are about 8 to 16 bases in length. In some embodiments, the nucleic acid amplification product is about 20 to 40 bases in length. In some embodiments, the spacer sequence comprises a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1 to 10 bases in length. In some embodiments, the isothermal amplification conditions comprise a constant temperature of about 30°C to about 72°C (e.g., about 55°C to about 75°C, about 56°C to about 67°C). In some embodiments, the amplifying step is performed for a period of about 5 minutes to about 60 minutes (e.g., about 15 minutes); and / or under helicase-, signal strand binding protein-, cleavage agent-, and recombinase-free isothermal amplification conditions.

[0017] In some embodiments, the enzyme with 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. In some embodiments, the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase and the enzyme with hyperthermophilic polymerase activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, and the forward and reverse primers. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

[0018] In some embodiments, the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the biological entity comprises a virus, a bacterium, a fungus, a protozoan, a portion thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoan. In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a wastewater sample, a saltwater sample, a sample exposed to air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is or is derived from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

[0019] In some embodiments, the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences, and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. In some embodiments, the two or more target nucleic acid sequences are specific to two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C). In some embodiments, the amplifying step comprises one or more of 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). In some embodiments, the amplifying step does not comprise loop-mediated isothermal amplification (LAMP). In some embodiments, the methods do not include one or more of: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture.

[0020] In some embodiments, the methods, reagent compositions and / or amplification reaction mixtures do not include a template capable of generating a first quality control product other than a quality control template; a probe capable of detecting the first quality control product other than a signal-generating oligonucleotide; a double-stranded template capable of generating a first quality control product; a linear template capable of generating a first quality control product; and / or a primer other than a quality control primer that is capable of hybridizing to the quality control template, the first quality control product and / or the second quality control product. In some embodiments, the method includes determining the presence, absence, and / or amount of a first quality control product. In some embodiments, the presence, absence, and / or amount of a signal indicates the presence, absence, and / or amount of the first quality control product. In some embodiments, the presence, absence, and / or amount of a signal indicates the presence, absence, and / or amount of one or more interfering components in the amplification reaction mixture. In some embodiments, the presence, absence, and / or amount of a signal indicates (i) the integrity of one or more amplification reagents in the amplification reaction mixture; (ii) a failure of the apparatus on which the amplification reaction is performed; and / or (iii) sample-derived inhibition of the amplification reaction. In some embodiments, sample-derived inhibition includes matrix-derived inhibition. In some embodiments, the presence, absence, and / or amount of a signal indicates the extent to which amplification of a target nucleic acid sequence is inhibited in the amplification reaction.

[0021] In some embodiments, the methods, reagent compositions, and / or amplification reaction mixtures include at least about 50,000 to about 500,000 copies of the quality control template. In some embodiments, comparable methods of monitoring amplification reactions use about 20 to about 100 copies of the internal standard template. In some embodiments, the methods, reagent compositions, and / or amplification reaction mixtures include at least about 1.1-fold more copies of the quality control template and / or quality control primers than comparable methods of monitoring amplification reactions that do not include the quality control template and / or quality control primers. In some embodiments, the comparable methods include an internal standard template that is not capable of forming a hairpin structure. In some embodiments, comparable methods of monitoring amplification reactions that do not include the quality control template and / or quality control primers inhibit amplification of the target nucleic acid sequence and / or detection of the nucleic acid amplification product by at least about 1.1-fold more than the methods disclosed herein. In some embodiments, the quality control template and / or quality control primers are not capable of hybridizing to the target nucleic acid sequence. In some embodiments, the presence of a quality control template and / or quality control primers does not inhibit amplification of a target nucleic acid sequence and / or detection of a nucleic acid amplification product. In some embodiments, the presence of a quality control template and / or quality control primers in an amplification reaction mixture improves amplification of a target nucleic acid sequence and / or detection of a nucleic acid amplification product by at least about 1.1-fold compared to a comparable method in which the quality control template and / or quality control primers are absent from the amplification reaction mixture. In some embodiments, the number of false priming events and / or generation of primer-dimers is reduced by at least about 1.1-fold compared to a comparable method of monitoring an amplification reaction that does not include a quality control template and / or quality control primers.

[0022] Disclosed herein is a kit for monitoring an amplification reaction. In some embodiments, the kit includes a quality control template disclosed herein; a quality control primer disclosed herein; a signal-generating oligonucleotide disclosed herein; and / or an auxiliary quality control primer disclosed herein. The kit can include a lysis buffer containing one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence. In some embodiments, the one or more lysis agents include a detergent, wherein the detergent includes one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit can also include a reagent composition containing one or more amplification reagents containing one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the one or more components for amplification include (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a first strand sequence of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a second strand sequence of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of generating a nucleic acid amplification product. In some embodiments, the enzyme with 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. In some embodiments, the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the quality control template, signal generating oligonucleotide, quality control primer, auxiliary quality control primer, and / or one or more components for amplification are in lyophilized or freeze-dried form and / or are present in a reagent composition.

[0023] Disclosed herein includes a reaction mixture. In some embodiments, the reaction mixture includes a quality control template disclosed herein; a quality control primer disclosed herein; a signal-generating oligonucleotide disclosed herein; an auxiliary quality control primer disclosed herein; a target nucleic acid sequence; and / or one or more additional primers and / or one or more probes specific to the target nucleic acid sequence.

[0024] In some embodiments, the one or more additional primers and / or one or more probes specific to the target nucleic acid sequence include a forward primer and / or a reverse primer, where the forward primer is capable of hybridizing to a first strand sequence of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a second strand sequence of the target nucleic acid sequence; and / or a target signal-generating oligonucleotide capable of hybridizing to a nucleic acid amplification product generated by amplifying the target nucleic acid sequence. The reaction mixture can include one or more of an enzyme having polymerase activity, dNTPs, and a buffer. In some embodiments, the enzyme is an enzyme having hyperthermophile polymerase activity. In some embodiments, the enzyme having hyperthermophile polymerase activity has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. [Brief explanation of the drawings]

[0025] [Figure 1] 1 depicts a non-limiting, exemplary embodiment of a DNA hairpin internal standard assay disclosed herein. IC primer, internal standard primer; HpIC1 MB2, hairpin internal standard molecular beacon; HpIC1 P1, hairpin internal standard product 1; HpIC1 P2, hairpin internal standard product 2. [Figure 2] FIG. 2 depicts a non-limiting, exemplary embodiment of a thermal setup for the hairpin IC (HPIC) isothermal amplification reaction described herein. [Figure 3A-3B]Figures 3A-B show data for target Neisseria gonorrhoeae (Ng) (ROX) detection and hairpin IC detection (HEX) in a clean sample (Figure 3A) compared to a sample containing 10% normal urine (Figure 3B). NTC, no target control; cp, copy. Molecular beacon IC (HPIC) is detected with the HEX fluorophore, and the target (Ng) is detected with the ROX fluorophore. [Figures 4A-4D] Figures 4A-4D show data for target Neisseria gonorrhoeae (Ng) (ROX) detection and hairpin IC detection (HEX). Figure 4A shows the Ng-only reaction, while Figures 4B-4D depict reactions with increasing levels of IC template (ICT): 0 ICT (Figure 4B), 50k ICT (Figure 4C), and 500k ICT (Figure 4D). The asterisk in Figure 4D indicates the presence of an air bubble in the pipette tip for that sample. NTC, no target control; cp, copy. Molecular beacon IC (HPIC) is detected with the HEX fluorophore, and the target (Ng) is detected with the ROX fluorophore. [Figures 5A-5C] Figures 5A-5C show data for target Neisseria gonorrhoeae (Ng) (ROX) detection and hairpin IC detection (HEX) in samples with increasing levels of inhibitory urine: 10% (Figure 5A), 20% (Figure 5B), and 30% (Figure 5C). NTC, no target control; cp, copy. Molecular beacon IC (HPIC) is detected by the HEX fluorophore, and the target (Ng) is detected by the ROX fluorophore. [Figure 6] Figure 6A depicts data for Ng detection in normal urine in the absence of an internal standard. The Ng ROX probe was used for target detection, and Syto 82 intercalating dye was used for amplification product detection. Figure 6B depicts data for Ng / hairpin IC detection in normal urine, where the hairpin IC probe (HPIC) is detected with the HEX fluorophore and the target (Ng) is detected with the ROX fluorophore. [Figures 7A-7B]Figures 7A-7B depict data showing a comparison of target Neisseria gonorrhoeae (Ng) (ROX) detection and hairpin IC detection (HEX) in reactions using hairpin IC from different lots: 9.14 (Figure 7A) and 10.12 (Figure 7B). NTC, no target control; cp, copy. Molecular beacon IC (HPIC) is detected with the HEX fluorophore, and target (Ng) is detected with the ROX fluorophore. [Figure 8A-8B] 8A-8B show non-limiting, exemplary schematics of isothermal amplification reactions provided herein. [Figures 9A-9F] Figures 9A-9F depict data on the detection of amplified internal standards by molecular beacons HpIC1b MB1 and HpIC1b MB2 (Figures 9A-9C) and intercalating dyes (Figures 9D-9F). The hairpin-shaped internal standard target was amplified in an APA reaction for 10 minutes, with simultaneous detection by a detection probe (Figure 9A) and an intercalating dye (Figure 9D). The hairpin-shaped internal standard was labeled with HEX at the 5' end and IBFQ at the 3' end. In some such embodiments, it serves as both the internal standard target and the detection probe. Following the reaction, the temperature of the reaction was immediately ramped from the assay temperature to 90°C for melting curve analysis (Figures 9B and 9E) and melting derivative evaluation (Figures 9C and 9F). DETAILED DESCRIPTION OF THE INVENTION

[0026] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments set forth in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may 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 this disclosure. All patents, published patent applications, other publications, and GenBank sequences and other databases referenced herein are incorporated by reference in their entirety with respect to the relevant art.

[0027] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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. Disclosed herein is a method for monitoring an amplification reaction. In some embodiments, the method includes providing a quality control template comprising a 5' subdomain; a 3' subdomain; and a loop domain between the 5' subdomain and the 3' subdomain, wherein intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, the method includes providing a quality control primer capable of hybridizing to at least a portion of the 3' subdomain. In some embodiments, the method includes subjecting the quality control template and the quality control primer to an amplification reaction capable of generating a first quality control product. In some embodiments, the method includes detecting the first quality control product. In some embodiments, the amplification reaction is performed in an amplification reaction mixture under amplification conditions (e.g., isothermal amplification conditions). In some embodiments, subjecting the quality control template and the quality control primer to an amplification reaction capable of generating a first quality control product includes amplifying the quality control template using the quality control primer in the amplification reaction mixture under amplification conditions, thereby generating the first quality control product. In some embodiments, the amplification reaction comprises a reverse transcription reaction.

[0028] Disclosed herein is a kit for monitoring an amplification reaction. In some embodiments, the kit includes a quality control template disclosed herein; a quality control primer disclosed herein; a signal-generating oligonucleotide disclosed herein; and / or an auxiliary quality control primer disclosed herein. Disclosed herein includes a reaction mixture. In some embodiments, the reaction mixture includes a quality control template disclosed herein; a quality control primer disclosed herein; a signal-generating oligonucleotide disclosed herein; an auxiliary quality control primer disclosed herein; a target nucleic acid sequence; and / or one or more additional primers and / or one or more probes specific to the target nucleic acid sequence.

[0029] Hairpin internal standard Provided herein, in some embodiments, are methods, compositions, kits, and reaction mixtures that can use archaeal polymerase amplification ("APA") to isothermally amplify a region of interest within a target nucleotide template for real-time analyte detection while simultaneously monitoring or evaluating the amplification reaction (e.g., an internal control ("IC") assay). The methods, compositions, reaction mixtures, and kits provided herein can address the aforementioned need in the art by leveraging the stability of hairpin structures to overcome challenges posed by competing target amplifications and reduce nonspecific interactions with the primary target amplification. Advantages of the hairpin IC systems, methods, compositions, reaction mixtures, and kits provided herein can include reduced assay complexity and reduced undesired interactions with the target amplification. Unexpectedly, in some embodiments, target amplifications, when duplexed with hairpin IC, exhibit improved low-copy detection compared to target-only assays. Disclosed herein are methods, compositions, reaction mixtures, and kits for internal standard assays that can be duplexed with specific target assays and report core reagent integrity, instrument failure, and / or sample inhibition in the absence of a specific signal from target amplification. In some embodiments, the internal standard assays are designed to utilize APA to simultaneously amplify a DNA target and an internal standard template for real-time detection under isothermal conditions.

[0030] The disclosed internal standard approach can enable simultaneous amplification of specific target(s) and an internal standard (IC) by leveraging the characteristic structural stability of stem-loop hairpins. Unlike currently available IC methods in which linear DNA is used as the IC template, some embodiments of the methods and compositions provided herein use a hairpin-shaped template (e.g., a quality control template). Due to the complementary nature of the hairpin stem, in some embodiments, only a single primer complementary to the 3' end of the stem is required for amplification of the hairpin IC target. The IC primer (e.g., a quality control primer) can extend on the hairpin template to generate a first-round hairpin product (e.g., a first quality control product) complementary to the IC template. Subsequent iterative extension of the IC template driven by the single IC primer can generate two hairpin products with stem structures and complementary loop sequences. The generated IC products can be detected by signal-generating oligonucleotides (e.g., probes, molecular beacons). In some embodiments, the signal-generating oligonucleotide (e.g., a molecular beacon) is modified with LNA to enhance the detectability of the hairpin product. In some embodiments, a hairpin template can be labeled with a signal-generating moiety and serve as both a template and a detection probe. In some embodiments, a DNA internal standard is provided that includes an IC primer and a hairpin probe that also serves as an IC template.

[0031] The IC approach described herein can be extended to RNA IC assays that include IC primers, signal-generating oligonucleotides (e.g., molecular beacons), and / or hairpin-shaped IC templates. The IC template can contain an RNA segment at the 5' end and a hairpin-shaped DNA segment with an RT primer sequence at the 3' end in the stem region. In the presence of reverse transcriptase, the RT primer in the stem region of the hairpin can extend across the RNA segment of the IC template and generate cDNA that is also complementary to the IC primer. Subsequent amplification driven by a single IC primer can generate a hairpin-shaped product that is complementary in the loop region, which can be detected by a signal-generating oligonucleotide (e.g., molecular beacon). In some embodiments, the signal-generating oligonucleotide (e.g., molecular beacon) is modified with LNA.

[0032] The methods, compositions, reaction mixtures, and kits disclosed herein advantageously use a hairpin-based IC approach, which can enable strong internal control amplification without the risk of competition with target amplification. In some embodiments, only a single IC primer is required. In some embodiments, high concentrations of IC primers can be used without affecting target amplification. In some embodiments, high IC template copy numbers can be used without interfering with target amplification, e.g., 50,000-500,000 copies of IC target, compared to the 20-100 copies typically used in other IC systems to reduce competition with target amplification. In some embodiments provided herein, where a hairpin IC probe is also used as a quality control template, the copy number can be even higher (e.g., in the 25-50 nanomolar range or >10 12(copy). In some embodiments, only a single primer and a hairpin oligonucleotide labeled with a signal-generating moiety that functions as both a template and a detection probe are required. High concentrations of hairpin oligonucleotides can be used without affecting target amplification. Without being bound by any particular theory, the use of a single primer and hairpin-shaped template and product may result in less primer dimerization and false priming.

[0033] In some embodiments, the internal standard assay comprises a single primer, a hairpin-shaped internal standard template, and / or a signal-generating oligonucleotide (e.g., a molecular beacon) for detection of the amplified hairpin product. In some embodiments, the internal standard assay comprises a single primer and a signal-generating oligonucleotide (e.g., a molecular beacon) that functions as both a template and a detector. In some embodiments, the signal-generating oligonucleotide (e.g., a molecular beacon) contains a fluorophore at the 5' end and a quencher at the 3' end. In some embodiments, the signal-generating oligonucleotide (e.g., a molecular beacon) can be replaced with a hairpin-shaped DNA probe that does not contain a quencher to detect hybridization to the hairpin amplification product. In the absence of IC amplification, the fluorescence of the hairpin probe due to the fluorophore attached to the 5' end can be quenched by guanine and / or adenine bases in the complementary stem. Without being bound by any particular theory, after hybridization, a conformational rearrangement may occur, resulting in an increase in fluorescence intensity.

[0034] The underlying technical principles used in the design of the hairpin-based internal standards provided herein can include nearest neighbor (NN) theory for predicting DNA thermodynamics and secondary structure dynamics of DNA hairpins using energy values. Without being bound by any particular theory, the mechanism of hairpin amplification and its advantages as an internal standard can be related to the thermodynamics of the hairpin stem-loop structure. Additional embodiments of the methods and compositions provided herein include probe-free versions of hairpin-based internal standards. In this approach, a single IC primer labeled with a fluorophore at its 5' end and a hairpin template may be the only two components required for IC amplification and detection. The 5' end of the primer may contain one or more cytosine bases adjacent to the fluorophore. The labeled primer may exponentially copy the hairpin template, resulting in a hairpin product in which the 5'-terminal fluorophore is quenched by the proximity of the guanine base(s) via photoinduced electron transfer. In some embodiments, a single IC primer labeled with a fluorophore at its 5' end may be the only component required for IC amplification and detection. The 5' end of the primer may contain one or more cytosine bases adjacent to the fluorophore. The 3' end of the labeled primer is self-complementary and can be exponentially copied, resulting in a palindromic hairpin product in which the 5' terminal fluorophore is quenched by the proximity of the guanine base(s) via photoinduced electron transfer. In this one-component DNA IC assay, (i) the quality control template, (ii) the quality control primer, and (iii) the signal-generating oligonucleotide are present in the same molecule.

[0035] Three-component IC assay In some embodiments, an RNA hairpin IC assay includes an IC primer, a signal-generating oligonucleotide (e.g., a molecular beacon), and / or a hairpin-shaped IC template. The IC template can include an RNA segment at the 5' end and a hairpin-shaped DNA segment with an RT primer sequence at the 3' end in the stem region. In the presence of reverse transcriptase, the RT primer in the stem region of the hairpin can extend across the RNA segment of the IC template and generate a cDNA that is also complementary to the IC primer. Without being bound by any particular theory, subsequent amplification can be driven entirely by a single IC primer to form a pan-handle-shaped product. To facilitate amplification and detection, the product hybridization melting temperature (Tm) can be designed to exceed or equal the product hairpin Tm. In some embodiments, a signal-generating oligonucleotide (e.g., a molecular beacon) modified with a locked nucleic acid (LNA) in the spacer region can be used for IC product detection. In some embodiments, a fluorescent dye can be used for detecting the amplified IC product.

[0036] A DNA hairpin IC assay can include a hairpin-shaped IC template, a single IC primer, and / or a beacon. In some embodiments, the IC primer can extend on the IC template to generate two "panhandle"-shaped products that are complementary in the loop (spacer) region. Without being bound by any particular theory, subsequent iterative extension of the IC primer on the panhandle products can generate exponential amplification. To facilitate amplification and detection, the product hybridization Tm can be designed to exceed or equal the product hairpin Tm. LNA-modified signal-generating oligonucleotides (e.g., molecular beacons) can be used for IC product detection in some embodiments. In some embodiments, fluorescent dyes can be used for amplified IC product detection. The disclosed hairpin IC methods, in some embodiments, can provide reduced primer-related interactions with the target being assayed. In some embodiments, only three IC amplification components are required: primer, template, and probe (e.g., molecular beacon) for either DNA or RNA IC assays. In the RNA IC assays provided herein, the RT primer can be embedded in a chimeric IC template. In some embodiments, the combination of low IC primer concentration and the formation of a "panhandle" structure can further reduce nonspecific interactions with the target being assayed.

[0037] Two-component IC assay In some embodiments, the hairpin IC method involves a single IC primer and a signal-generating oligonucleotide (e.g., a molecular beacon) for both IC amplification and detection (Figure 1). The sequence of the signal-generating oligonucleotide (e.g., a molecular beacon) can include a partial or entire IC primer, a spacer region of the IC template, and 4-5 nucleotides adjacent to the IC spacer and complementary to the 3' end of the IC primer. The IC primer can hybridize to the beacon and generate a first-round extension product whose 3' end is complementary to the IC primer. Subsequent extension of the IC primer can generate an internal control product in the shape of a "panhandle." Without being bound by any particular theory, subsequent iterative extension of the IC primer on the panhandle product can be entirely driven by a single IC primer. The generated IC product can be detected by an IC beacon modified, for example, with LNA, to enhance the detectability of the panhandle product. The two-component hairpin-based IC approach described herein can be extended to RNA IC assays. In this case, the signal-generating oligonucleotide (e.g., a molecular beacon) can contain several RNA bases in the loop region. The RT primer can extend along the RNA bases in the beacon to generate cDNA. The RNA bases in the loop region can be degraded by the RNase H activity of reverse transcriptase during cDNA synthesis, resulting in fluorescent signal emission. Depending on the beacon design, in some embodiments, IC primers can be used to improve exponential amplification.

[0038] Advantageously, this approach can have minimal primer-related interactions with the target being assayed compared to alternative methods of assay monitoring. In some embodiments, there are only two IC components in a DNA IC assay: a primer and a hairpin probe (e.g., a beacon) for both IC amplification and detection as an internal standard. For the two-component RNA IC assay provided herein, the minimum components can be an RT primer and a signal-generating oligonucleotide (e.g., a molecular beacon) containing an RNA stretch in the loop region, and in some embodiments, an optional IC primer is used.

[0039] Methods for monitoring amplification reactions Disclosed herein is a method for monitoring an amplification reaction. In some embodiments, the method includes providing a quality control template comprising a 5' subdomain; a 3' subdomain; and a loop domain between the 5' subdomain and the 3' subdomain, wherein intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, the method includes providing a quality control primer capable of hybridizing to at least a portion of the 3' subdomain. In some embodiments, the method includes subjecting the quality control template and the quality control primer to an amplification reaction capable of generating a first quality control product. In some embodiments, the method includes detecting the first quality control product. The amplification reaction can be performed in an amplification reaction mixture under amplification conditions (e.g., isothermal amplification conditions). In some embodiments, subjecting the quality control template and the quality control primer to an amplification reaction capable of generating a first quality control product includes amplifying the quality control template using the quality control primer in the amplification reaction mixture under amplification conditions, thereby generating the first quality control product. The amplification reaction can include a reverse transcription reaction.

[0040] The method can include providing an enzyme having polymerase activity (e.g., an enzyme having hyperthermophile polymerase activity). The method can include providing a reverse transcriptase. In some embodiments, the enzyme having hyperthermophile polymerase activity has reverse transcriptase activity. The amplification reaction can include contacting a quality control primer with a quality control template for hybridization and extending the quality control primer hybridized to the quality control template with an enzyme having polymerase activity, thereby generating a first quality control product. The amplification reaction can include contacting a quality control primer with the first quality control product for hybridization and extending the quality control primer hybridized to the first quality control product with an enzyme having polymerase activity, thereby generating a second quality control product. The amplification reaction can include contacting a quality control primer with a second quality control product for hybridization and extending the quality control primer hybridized to the second quality control product with an enzyme having polymerase activity, thereby generating the first quality control product. The first quality control product and the second quality control product can comprise a 5' subdomain and a 3' subdomain capable of forming a paired stem domain. In some embodiments, the first quality control product and the second quality control product have the same stem domain. The first quality control product and the second quality control product can comprise loop domains that are complementary to each other. The amplification reaction can comprise linear and / or exponential amplification of the first quality control product and the second quality control product. The 5' subdomain can comprise at least a partial sequence of the quality control primer. Both the first quality control product and the second quality control product can be capable of forming a hairpin structure. As used herein, the term "hairpin structure" is given its ordinary meaning and also refers to a double helix region formed by base pairing between adjacent, inverted, complementary sequences in a single strand of RNA or DNA.The quality control template can include a 5' terminal domain that is 5' of the 5' subdomain, and / or the quality control template can include a 3' terminal domain that is 3' of the 3' subdomain. The 5' terminal domain of the quality control template can include at least a portion of the sequence of the quality control primer. The combined sequence of the 5' terminal domain and the 5' subdomain can include the entire sequence of the quality control primer.

[0041] Detecting the first quality control product can include detecting the first quality control product using a signal-generating oligonucleotide. The signal-generating oligonucleotide can be capable of hybridizing to the first quality control product. The detecting step can include contacting the first quality control product with the signal-generating oligonucleotide for hybridization. The signal-generating oligonucleotide can include a quencher, a label, or both. The label can include a quenchable label (e.g., a fluorophore). The quenchable label can be, for example, a fluorophore. As used herein, the term "fluorophore" is given its ordinary meaning and also refers to any reporter group whose presence can be detected by its light-emitting 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™ (527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY558 / 568 (568), BODIPY564 / 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) (the number in parentheses is the maximum emission wavelength in nanometers for the corresponding fluorophore). In some embodiments, the fluorophore is Cy5™. In some embodiments, the fluorophore is hexachlorofluorescein (HEX).

[0042] The signal-generating oligonucleotide can include a quencher. The quencher can be capable of quenching the label. Quenching can be mediated by fluorescence resonance energy transfer (FRET). FRET is based on classical dipole-dipole interactions between the transition dipoles of a donor (e.g., a fluorophore) and an acceptor (e.g., a quencher) and depends on the donor-acceptor distance. FRET can typically occur over distances of up to 100 Å. FRET also depends on donor-acceptor spectral overlap and the relative orientation of the donor and acceptor transition dipole moments. Fluorophore quenching can 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, in some embodiments of the methods disclosed herein, a quencher moiety is not required to observe a detectable change in fluorescence, and it is believed that a proximal-base quenching effect is sufficient to produce a detectable shift in fluorescence that allows for the nucleic acid amplification reaction to be assessed, monitored, observed, and / or tracked. 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.

[0043] The detecting step can include contacting the first quality control product with a signal-generating oligonucleotide for hybridization. The label can be capable of generating a signal after the signal-generating oligonucleotide hybridizes to the first quality control product. In some embodiments, the label generates a signal after the signal-generating oligonucleotide hybridizes to the first quality control product. The signal can be fluorescent. The detecting step can include detecting a signal generated by the label of the signal-generating oligonucleotide. The label can be a fluorophore, and the signal can be fluorescent. The detecting step can include detecting the signal of the label before the amplification reaction, during the amplification reaction, after the amplification reaction, or any combination thereof.

[0044] The method can include providing a signal-generating oligonucleotide; subjecting the signal-generating oligonucleotide to an amplification reaction; and detecting a first quality control product using the signal-generating oligonucleotide. The quality control template can be the signal-generating oligonucleotide. The quality control template can be (i) a template for synthesis of the first quality control product and (ii) a means for detecting the first quality control product. The signal-generating oligonucleotide can be capable of (i) detecting the first quality control product and (ii) serving as a template for quality control primer-driven synthesis of the first quality control product.

[0045] In some embodiments, the 5'-terminal domain of the quality control template comprises one or more RNA nucleotides; and / or at least a portion of the sequence of the quality control primer. In some embodiments, the quality control template does not comprise a 3'-terminal domain. The 3'-end of the quality control template may be complementary to the 5'-end of the 5' subdomain of the quality control template. In some embodiments, a reverse transcriptase may use one or more RNA nucleotides of the 5'-terminal domain of the quality control template as a template to extend the 3'-end of the quality control template, thereby generating an extended quality control template. The 3'-end of the extended quality control template may comprise a sequence complementary to at least a portion of the quality control primer. The amplification reaction may include contacting the reverse transcriptase with the quality control template, thereby generating an extended quality control template. The extended quality control template may comprise cDNA. In some embodiments, the amplification reaction includes contacting a quality control primer with the 3' end of the extended quality control template for hybridization, and extending the quality control primer hybridized to the 3' end of the extended quality control template using an enzyme having reverse transcriptase and / or polymerase activity, thereby generating a first quality control product.

[0046] The quality control template can be a signal-generating oligonucleotide. The signal-generating oligonucleotide can include a label. The loop domain can include one or more RNA nucleotides. The label can include a quenchable label (e.g., a fluorophore). The signal-generating oligonucleotide can include a quencher. The label can be located in the 3'-terminal domain and the quencher can be located in the 5'-terminal domain, and / or the label can be located in the 5'-terminal domain and the quencher can be located in the 3'-terminal domain. The amplification reaction can include contacting a quality control primer with the quality control template for hybridization and extending the quality control primer hybridized to the quality control template using a reverse transcriptase, thereby generating a first quality control product. The reverse transcriptase can include RNase H activity. In some embodiments, the reverse transcriptase cleaves the quality control template at one or more RNA nucleotides during generation of the first quality control product, thereby generating a first cleavage product and a second cleavage product comprising the label. Detecting the first quality control product can include detecting a signal generated by the first cleavage product, which includes a label. Detecting the first quality control product can include detecting a signal of the label. The first cleavage product can include a label. The amount of the detected signal can indicate the absence, presence, or amount of the first cleavage product. The absence, presence, or amount of the first cleavage product can indicate the absence, presence, or amount of the first quality control product. The label can be a fluorophore, and the signal can be fluorescent. The method can include providing auxiliary quality control primers; and subjecting the auxiliary quality control primers to an amplification reaction.

[0047] The signal-generating oligonucleotide can be about 10 to about 100 nucleotides in length. The quality control template can be about 10 to about 100 nucleotides in length. The quality control primer and / or auxiliary quality control primer can be about 5 to about 25 nucleotides in length. The 5' subdomain, 3' subdomain, loop domain, 5' terminal domain, and / or 3' terminal domain can be about 1 to about 25 nucleotides in length. The signal-generating oligonucleotide, quality control template, and / or quality control primer can comprise one or more phosphorothioate linkages and / or one or more locked nucleic acids. The signal-generating oligonucleotide can be a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide.

[0048] The signal-generating oligonucleotide can contain one or more LNAs. The one or more LNAs can be located within the loop domain (e.g., the one or more LNAs enhance the detectability of the first quality control product). The signal-generating oligonucleotide can be configured so that the melting temperature (Tm) of the first quality control product / signal-generating oligonucleotide duplex is equal to or exceeds the melting temperature (Tm) of the paired stem domain of the signal-generating oligonucleotide (e.g., it can be configured with one or more modifications and / or modified bases, such as an LNA in the loop domain). The LNA can be located in the stem domain. In some embodiments, an LNA is also located in the stem region to enhance hairpin stability and enhance detectability. Additionally, and without being bound by any particular theory, in some embodiments, when an archaeal polymerase is used, the 3'-terminal base is LNA-modified (or otherwise modified) to block the 3'-to-5' exonuclease activity of the polymerase. Stem LNAs may be most effective in configuring the Tm of the probe above the assay temperature, thereby (i) stabilizing the hairpin and reducing the baseline signal, and / or (ii) increasing the Tm of the quality control product / signal-generating oligonucleotide duplex, thereby enhancing the detectability of the quality control product. In some embodiments, and without being bound by any particular theory, there is a difference between LNAs in the 5' stem and the 3' stem. In some embodiments, when in the stem at the 3' end, LNAs enhance priming of the IC primer and increase the Tm of the quality control product / signal-generating oligonucleotide duplex. In some embodiments, when in the 5' stem, LNAs can slow or inhibit IC product generation, even if they can enhance the Tm of the quality control product / signal-generating oligonucleotide duplex.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 modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link™ amino modifier), alkyne (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 C3 SS, dithiol, thiol modifier C6 SS); spacer (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, Super T®, Super Modifications and modified bases may include G®, locked nucleic acids (LNA's), 5-nitroindole, 2'-O-methyl RNA bases, hydroxmethyl 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 linkage modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases); and click chemistry modifications. In some embodiments, 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 can include a spacer, an abasic site, an unmethylated RNA base, a 2'-O-methylated nucleotide, and any combination thereof.

[0049] In some embodiments, the signal-generating oligonucleotide does not include a dye capable of quenching the label. In some embodiments, the signal-generating oligonucleotide does not include a moiety capable of quenching the label other than the nucleotides of the signal-generating oligonucleotide. The 5'-terminal domain of the quality control template and / or the signal-generating oligonucleotide can include a label. The 5'-terminal domain and / or the 5' subdomain of the quality control template and / or the signal-generating oligonucleotide can include one or more cytosine bases. The 3'-terminal domain and / or the 3' subdomain of the quality control template and / or the signal-generating oligonucleotide can include one or more guanine and / or adenine bases. The one or more guanine and / or adenine bases can quench the label after the quality control template and / or the signal-generating oligonucleotide forms a hairpin structure. The 3'-terminal domain of the quality control template and / or the signal-generating oligonucleotide can include a label. The 3'-terminal domain and / or the 3' subdomain of the quality control template and / or the signal-generating oligonucleotide can include one or more cytosine bases. The 5'-terminal domain and / or 5'-subdomain of the quality control template and / or signal-generating oligonucleotide may contain one or more guanine and / or adenine bases, which may be capable of quenching the label after the quality control template and / or signal-generating oligonucleotide forms a hairpin structure.

[0050] Detecting the first quality control product can include detecting a decrease in the amount of signal generated by the label of the quality control primer. The label can be a fluorophore, and the signal can be fluorescent. The generation of the first quality control product and the second quality control product can be correlated with the attenuation of the detected signal. The 5' end of the quality control primer can include a label, and the quality control primer can include one or more cytosine bases adjacent to the label. The 3'-terminal domain and / or 3' subdomain of the quality control template, the first quality control product, and / or the second quality control product can include one or more guanine and / or adenine bases. After the quality control primer binds to the quality control template and / or the second quality control product and is extended by an enzyme with polymerase activity to generate the first quality control product, the one or more guanine and / or adenine bases present in the 3'-terminal domain and / or 3' subdomain of the first quality control product can quench the label after the first quality control product forms a hairpin structure. After the quality control primer binds to the first quality control product and is extended by the enzyme having polymerase activity to generate the second quality control product, one or more guanine and / or adenine bases present in the 3'-terminal domain and / or 3' subdomain of the second quality control product can be capable of quenching the label after the second quality control product forms a hairpin structure. Detecting the first quality control product can include contacting the first quality control product with a fluorescent dye.

[0051] The step of providing quality control primers, quality control templates, and / or signal-generating oligonucleotides can include providing a reagent composition including the quality control primers, quality control templates, and / or signal-generating oligonucleotides. Subjecting the quality control primers, quality control templates, and / or signal-generating oligonucleotides to an amplification reaction can include contacting the reagent composition with the processed sample to produce an amplification reaction mixture. The method can include detecting a target nucleic acid sequence in the sample. The method can include subjecting the target nucleic acid sequence to an amplification reaction capable of producing a nucleic acid amplification product. The method can include detecting the nucleic acid amplification product using a target signal-generating oligonucleotide, the target signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product. Subjecting the target nucleic acid sequence to an amplification reaction capable of producing a nucleic acid amplification product can include amplifying the target nucleic acid sequence in the amplification reaction mixture under amplification conditions, thereby producing the nucleic acid amplification product. The method can include contacting a sample containing the biological entities with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids suspected of containing a target nucleic acid sequence. The method can include contacting a reagent composition with the processed sample to produce an amplification reaction mixture, the reagent composition comprising one or more amplification reagents.

[0052] The one or more amplification reagents may include a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity (e.g., an enzyme having hyperthermophilic polymerase activity and reverse transcriptase activity); a forward primer; a reverse primer; a reverse transcription primer; and / or dNTPs. The sample nucleic acid may include a nucleic acid comprising a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence includes amplifying the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, the amplifying step including contacting the nucleic acid comprising the target nucleic acid sequence with i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the nucleic acid is dsDNA and / or a product of a reverse transcription reaction. The nucleic acid can be a product of a reverse transcription reaction generated from the sample ribonucleic acid (e.g., the amplifying step includes generating the nucleic acid by a reverse transcription reaction). The amplification reaction can be carried out for a period of about 5 minutes to about 60 minutes. In some embodiments, amplifying the quality control template can include generating a first quality control product and / or a second quality control product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes.

[0053] In some embodiments, the method is performed in a single reaction vessel; does not include the use of any enzymes other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include the use of any enzymes other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid and / or quality control template during the amplification step; and / or does not include a step of contacting the nucleic acid and / or quality control template with a signal-stranded DNA binding protein.

[0054] In some embodiments, the methods, reagent compositions and / or amplification reaction mixtures do not include a template capable of generating a first quality control product other than a quality control template; a probe capable of detecting the first quality control product other than a signal-generating oligonucleotide; a double-stranded template capable of generating a first quality control product; a linear template capable of generating a first quality control product; and / or a primer other than a quality control primer that is capable of hybridizing to the quality control template, the first quality control product and / or the second quality control product.

[0055] The method can include determining the presence, absence, and / or amount of a first quality control product. The presence, absence, and / or amount of a signal can indicate the presence, absence, and / or amount of the first quality control product. The presence, absence, and / or amount of a signal can indicate the presence, absence, and / or amount of one or more interfering components in the amplification reaction mixture. The presence, absence, and / or amount of a signal can indicate (i) the integrity of one or more amplification reagents in the amplification reaction mixture; (ii) a failure of the equipment on which the amplification reaction is performed; and / or (iii) sample-derived inhibition of the amplification reaction (e.g., matrix-derived inhibition). The presence, absence, and / or amount of a signal can indicate the extent to which amplification of a target nucleic acid sequence is inhibited in the amplification reaction.

[0056] The methods, reagent compositions and / or amplification reaction mixtures can include at least about 50,000 copies to about 500,000 copies of quality control templates (e.g., in a three-component assay). 12A concentration of about 25 nM to 50 nM of IC template / probe, which can serve as an IC template for a number of copies (e.g., in a two-component assay), can be included. Comparable methods of monitoring amplification reactions can use about 20 to about 100 copies of an internal control template. In some embodiments, the methods, reagent compositions, and / or amplification reaction mixtures include at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any of these values) more copies of the quality control template and / or quality control primers than a comparable method of monitoring an amplification reaction that does not include the quality control template and / or quality control primers. In some embodiments, the comparable method includes an internal control template that is not capable of forming a hairpin structure. In some embodiments, a comparable method of monitoring an amplification reaction that does not include a quality control template and / or quality control primers inhibits amplification of the target nucleic acid sequence and / or detection of nucleic acid amplification products by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any of these values) more than the methods disclosed herein. In some embodiments, the quality control template and / or quality control primers are not capable of hybridizing to the target nucleic acid sequence. In some embodiments, the presence of the quality control template and / or quality control primers does not inhibit amplification of the target nucleic acid sequence and / or detection of nucleic acid amplification products. The presence of a quality control template and / or quality control primers in an amplification reaction mixture can improve amplification of a target nucleic acid sequence and / or detection of a nucleic acid amplification product by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any of these values) compared to a comparable method in which the quality control template and / or quality control primers are absent from the amplification reaction mixture.The number of false priming events and / or generation of primer dimers may be reduced by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any of these values) compared to a comparable method of monitoring an amplification reaction that does not include a quality control template and / or quality control primers.

[0057] Disclosed herein is a reaction mixture. In some embodiments, the reaction mixture includes a quality control template disclosed herein; a quality control primer disclosed herein; a signal-generating oligonucleotide disclosed herein; an auxiliary quality control primer disclosed herein; a target nucleic acid sequence; and / or one or more additional primers and / or one or more probes specific to the target nucleic acid sequence. The reaction mixture can include one or more of an enzyme having polymerase activity, dNTPs, and a buffer. Amplifying the target nucleic acid sequence can include producing a nucleic acid amplification product and / or quality control product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes. The detecting step can be performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition is contacted with the processed sample.

[0058] The lysis buffer may contain one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. The pH of the lysis buffer may be about 1.0 to about 10.0 (e.g., about 2.2). The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample nucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, the one or more amplification reagents include a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity; and / or dNTPs. In some embodiments, the one or more amplification reagents include an enzyme having reverse transcriptase activity, an enzyme having hyperthermophilic polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer. The reagent composition may be lyophilized, heat-dried, and / or include one or more additives. In some embodiments, the one or more additives include Tween 20, Triton X-100, and / or Tween 80; an amino acid; a sugar or sugar alcohol; and / or a polymer. The sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof. Contacting the reagent composition with the processed sample can include dissolving the reagent composition in the processed sample. In some embodiments, the one or more solubility reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) of the processed sample (e.g., about 0.2% (wt / vol) of the processed sample); and / or include a detergent (e.g., one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant).

[0059] In some embodiments, the method is performed in a single reaction vessel; does not include the use of any enzymes other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include the use of any enzymes other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein. The target nucleic acid sequence can have a length of about 20 nucleotides or less to about 90 nucleotides or less (e.g., about 30 nucleotides). The forward primer, reverse primer, and / or reverse transcription primer can be about 8 to 16 bases long. The nucleic acid amplification product can be about 20 to 40 bases long. The spacer sequence can comprise a portion of the target nucleic acid sequence. The spacer sequence can be 1 to 10 bases long. Isothermal amplification conditions can include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, and optionally about 56°C to about 67°C. The amplifying step can be carried out for a period of about 5 minutes to about 60 minutes (e.g., a period of about 15 minutes). The amplifying step can be carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions. The amplifying step can be carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). The PCR can be real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0060] The enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that may be at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. The enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that may be at least about 95% identical to the amino acid sequence of SEQ ID NO: 1. The enzyme with hyperthermophilic polymerase activity may be a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase and the enzyme with hyperthermophilic polymerase activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, and the forward and reverse primers. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

[0061] In some embodiments, quality control primers are provided. The quality control primers can include a sequence that is at least 80% identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to SEQ ID NO:3 or SEQ ID NO:9. The quality control primers can include a sequence that has 1, 2, 3, 4, or more mismatched or universal nucleotides compared to SEQ ID NO:3 or SEQ ID NO:9. In some embodiments, a quality control template (e.g., a hairpin internal standard (HPIC) molecular beacon) is provided. The quality control template can comprise a sequence that is at least 80% identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:11. The quality control template can comprise a sequence with 1, 2, 3, 4, or more mismatched or universal nucleotides compared to SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:11. The quality control primer and quality control template can comprise one or more modifications (e.g., phosphorothioated DNA bases, LNA). The quality control template can comprise a 5' modification (e.g., 5HEX) and / or a 3' modification (e.g., 3IABkFQ). The IC assay components provided herein can be used in multiplex assays (e.g., in concert with assays to detect C. trachomatis and / or N. gonorrhea).

[0062] In some embodiments, the amplifying step includes and / or does not include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplifying step does not include LAMP. In some embodiments, the methods do not include one or more of the following: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture.

[0063] 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., not more than 5°C or not 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. Isothermal amplification compositions and methods are described in PCT application published as WO2017176404, the entire contents of which are incorporated herein by reference. In some embodiments, the methods and compositions described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer is resistant to the formation of precipitates over a period of time under storage conditions (e.g., a storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer resists precipitation are described in International Application No. PCT / US23 / 61980, filed February 3, 2023, entitled "NON-OPAQUE LYTIC BUFFER COMPOSITION FORMULATIONS," the entire contents of which are incorporated herein by reference. Some embodiments of 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 acid and / or low pH conditions. 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 International Application No. PCT / US23 / 61978, filed February 3, 2023, entitled "METHOD FOR SEPARATING GEOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS," the entire contents of which are incorporated herein by reference.

[0064] In some embodiments, the methods and compositions described herein can include a lysis buffer and / or reagent composition. A lysis buffer comprising a lysis agent and a reducing agent, and a reagent composition comprising one or more protectants (e.g., cyclodextrin compounds) capable of sequestering the amplification agent and the lysis agent are described in International Application No. PCT / US22 / 21015, filed March 18, 2022, entitled "ISOTHERMAL AMPLIFICATION OF PATHOGENS," the entire contents of which are incorporated herein by reference. In some embodiments, the methods and compositions described herein can include a signal-generating oligonucleotide (e.g., a protected signal-generating oligonucleotide) that includes one or more polymerase stoppers. Compositions, kits, and methods for nucleic acid detection, in which protected signal-generating oligonucleotides allow for reduced nonspecific product formation and / or fewer false positives, are described in U.S. Provisional Patent Application No. 63 / 374,772, filed September 7, 2022, entitled "MODIFIED MOLECULAR BEACONS FOR IMPROVED DETECTION SPECIFICITY," the entire contents of which are incorporated herein by reference.

[0065] Some embodiments of the methods and compositions described herein can include probe(s) that melt at a temperature different from the optimal APA reaction temperature to allow for multiplexing of target and / or internal standard(s). Compositions, kits, and methods for multiplexed nucleic acid detection are described in U.S. Provisional Patent Application No. 63 / 374,831, filed September 7, 2022, entitled "ARCHEAL POLYMERASE AMPLIFICATION," the entire contents of which are incorporated herein by reference.

[0066] Some embodiments of the methods and compositions described herein can, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits for detecting pathogens described in U.S. Provisional Patent Application No. 63 / 374,774, filed September 7, 2022, entitled "METHODS AND COMPOSITIONS FOR PATHOGEN DETECTION," the entire contents of which are incorporated herein by reference.

[0067] Nucleic acids, subjects, samples and nucleic acid processing Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein. This term refers to nucleic acids of any composition, including 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., containing base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single- or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be found 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 can be replicated or replicated in vitro or in a host cell, cell, cell nucleus, mitochondria, or cell cytoplasm. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.The term can also include nucleotide analogs, single-stranded polynucleotides ("sense" or "antisense," "plus" or "minus" strand, "forward" or "reverse" reading frame, "forward" or "reverse" strand), and RNA or DNA equivalents, derivatives, variants, and analogs synthesized from double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain and generally includes regions preceding and following the coding region (leader and trailer) involved in transcription / translation of the gene product and regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). In RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed as the number of bases.

[0068] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The 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. The target nucleic acid may be, for example, DNA or RNA. If the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may include a single-stranded target sequence. If the target nucleic acid is double-stranded, the target nucleic acid generally comprises a first strand and a second strand. The first strand and the second strand may be referred to as the forward strand and the reverse strand, and are generally complementary to each other. If the target nucleic acid is single-stranded, the complementary strand may be generated, for example, by polymerization and / or reverse transcription, to make the target nucleic acid double-stranded and have a first / forward strand and a second / reverse strand.

[0069] A target sequence can refer to either the sense or antisense strand of a nucleic acid sequence, a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence can also be a subsequence within a larger polynucleotide. For example, a target sequence can be a short sequence (e.g., 20-50 bases) within a nucleic acid fragment, chromosome, or plasmid targeted for amplification. In some embodiments, a target sequence can refer to a sequence within a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence can refer to the entire sequence targeted for amplification, or to a subsequence within the target nucleic acid to which the oligonucleotide binds. An amplification product can be a larger molecule containing the target sequence as well as at least one other sequence or other nucleotide. An amplification product can be approximately the same length as the target sequence, e.g., exactly the same length as the target sequence. An amplification product can comprise or consist of the target sequence. The target sequence length and / or guanine cytosine (GC) concentration (percentage) may depend, in part, on the temperature at which the amplification reaction is performed, which may in turn depend, in part, on the stability of the polymerase used in the reaction. Trial assays may be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, if the polymerase is stable up to 60°C-65°C, the target sequence may be, for example, 19-50 nucleotides in length, or, for example, about 40-50, 20-45, 20-40, or 20-30 nucleotides in length. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0070] Target nucleic acids may include, for example, genomic nucleic acids, plasmid nucleic acids, mitochondrial nucleic acids, cellular nucleic acids, extracellular nucleic acids, bacterial nucleic acids, and viral nucleic acids. In some embodiments, target nucleic acids 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. Genomic nucleic acids may include any nucleic acid derived from any genome, for example, animal genomes, plant genomes, insect genomes, viral genomes, and bacterial genomes (e.g., genomes present in spores). In some embodiments, genomic target nucleic acids are present within a specific genomic locus or multiple genomic loci. A 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.

[0071] The target sequence may comprise one or more types of repetitive elements (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as a repetitive element (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellite repeats, minisatellite repeats, etc.). For example, the target sequence may occur multiple times as a repetitive element, and one, some, or all occurrences of the target sequence within the repetitive element can be amplified (e.g., using a single pair of primers) using the methods described herein. In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as duplicates and / or paralogs. The target nucleic acid may include a microRNA. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21-23 nucleotides in length), single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNAs and are partially complementary to them. The target nucleic acid may also include microRNA precursors, such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop RNAs that are further processed into miRNAs. The target nucleic acid may also include small interfering RNAs (siRNAs), which are short (e.g., about 20-25 nucleotides in length), at least partially double-stranded RNA molecules involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0072] Nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, isolated from a sample obtained from a subject. The subject can 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), suidae (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).

[0073] A sample or test sample may be any specimen isolated or obtained from a subject or a portion thereof. Non-limiting examples of specimens include fluids or tissues derived from a subject, including, but not limited to, blood or blood products (such as 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), serum, plasma, urine, aspirate, 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 the like, 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 by centrifugation of blood that has been treated with an anticoagulant. Serum refers to the aqueous portion 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. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 milliliters) is often collected and can be stored according to standard procedures before or after preparation.

[0074] The sample can include spores, viruses, cells, nucleic acids, and / or samples containing any free nucleic acid from prokaryotes or eukaryotes. For example, the methods described herein can be used to detect nucleic acids outside of spores (e.g., no lysis is required). 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 biological organisms. Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from biological samples can be used, including art-known DNA preparation methods 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, Wisconsin), and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, New Jersey), or combinations thereof. U.S. Patent No. 7,888,006 provides a DNA purification method, but does not disclose the compositions (e.g., lysis buffer, protectant) and methods provided herein.

[0075] In some embodiments, a cell lysis procedure is performed. Cell lysis may be performed before initiating the amplification reaction described herein (e.g., to release DNA and / or RNA from cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical methods (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or a combination thereof), physical methods (e.g., French press and sonication, etc.), or electrolytic lysis. For example, chemical methods generally involve disrupting cells using a lysing agent, extracting nucleic acids from the cells, followed by treatment with a chaotropic salt. In some embodiments, cell lysis involves the use of a detergent (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis involves the use of an ionic detergent (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods, such as freeze / thaw trituration and the use of a cell press, may also be useful. High-salt lysis procedures can also be used. For example, alkaline lysis procedures can be used. The latter procedure traditionally incorporates the use of a phenol-chloroform solution, although an alternative phenol-chloroform-free procedure involving three solutions can also be used. In the latter procedure, for example, one solution may contain 15 mM Tris (pH 8.0), 10 mM EDTA, and 100 μg / ml RNase A, a second solution may contain 0.2 N NaOH and 1% SDS, and a third solution may contain 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and components described herein.

[0076] Nucleic acids for performing the methods described herein can be provided without processing a sample containing the nucleic acid. For example, nucleic acids for performing the amplification methods described herein can be provided without prior nucleic acid purification. In some embodiments, target sequences are amplified directly from a sample (e.g., without any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids for performing the methods described herein are provided after processing a sample containing the nucleic acid. For example, nucleic acids can be extracted, isolated, purified, or partially purified from a sample. The term "isolated" generally refers to nucleic acids that have been removed from their original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed) and thus have been altered from their original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid" can refer to nucleic acids removed from a subject (e.g., a human subject). Isolated nucleic acids can be provided that have fewer non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. A composition containing isolated nucleic acids may contain no more than about 50%-99% of non-nucleic acid components. A composition comprising an isolated nucleic acid may be free of more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of non-nucleic acid components. The term "purified" generally refers to a nucleic acid that contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. A composition comprising a purified nucleic acid may be free of more than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of other non-nucleic acid components.

[0077] Nucleic acids can be provided for performing the methods described herein without modifying the nucleic acid, for example, by denaturing, digesting, nicking, unwinding, incorporating and / or ligating heterologous sequences, adding epigenetic modifications, adding labels (e.g., 32P, 33 P, 125 I, or 35 Examples of such labels include radioactive labels such as S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, and fluorescent dyes. Thus, in some embodiments, unmodified nucleic acids are amplified.

[0078] The methods disclosed herein for detecting target nucleic acid sequences (single-stranded or dsDNA and / or RNA) in a sample can detect target nucleic acid sequences (e.g., DNA or RNA) with high sensitivity. In some embodiments, the methods can be used to detect target DNA / RNA present in a sample containing multiple RNAs / DNAs (including a target RNA / DNA and multiple non-target RNAs / DNAs), where the target RNA / DNA is 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 It is present in one or more copies per non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meaning and shall refer to DNA, or RNA, or a combination of DNA and RNA.

[0079] The detection threshold of a method for detecting target RNA / DNA in a sample can be, for example, 10 nM or lower. The term "detection threshold" shall be given its ordinary meaning and shall describe the minimum amount of target RNA / DNA that must be present in a sample for detection to occur. As an illustrative example, if the detection threshold is 10 nM, a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the methods of the present disclosure provide a method for detecting target RNA / DNA at a concentration of 5 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower, 0.05 nM or lower, 0.01 nM or lower, 0.005 nM or lower, 0.001 nM or lower, 0.0005 nM or lower, 0.0001 nM or lower, 0.00005 nM or lower, 0.00001 nM or lower, or 0.00001 nM. or lower, 10 pM or lower, 1 pM or lower, 500 fM or lower, 250 fM or lower, 100 fM or lower, 50 fM or lower, 500 aM (attomolar) or lower, 250 aM or lower, 100 aM or lower, 50 aM or lower, 10 aM or lower, or 1 aM or lower. In some embodiments, the disclosed compositions or methods exhibit attamolar (aM), femtomolar (fM), picomolar (pM), and / or nanomolar (nM) detection sensitivity.

[0080] A sample may contain sample nucleic acids (e.g., multiple sample nucleic acids). The term "multiple" is used herein to mean two or more. Thus, in some embodiments, a sample contains 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 disclosed methods can be used as highly sensitive methods 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 contains 5, 10, 20, 25, 50, 100, 500, 10, 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 seeds or 10 7 The sample may contain DNA / RNA from 50 or more species, each differing in sequence from the others. In some embodiments, the sample contains DNA / RNA from cells (e.g., eukaryotic, mammalian, or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0081] As used herein, the term "sample" 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 a biological sample or an environmental sample. A sample may be derived from any source; for example, 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 derived from a patient (e.g., for diagnostic purposes). A sample may be derived from permeabilized cells, crosslinked cells, tissue sections, or combinations thereof. A sample may be derived from tissue prepared by crosslinking followed by delipidation and adjustment to a uniform refractive index. A sample may contain a target nucleic acid (e.g., target DNA / RNA) and multiple species of non-target DNA / RNA. In some embodiments, the target DNA / RNA is 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.

[0082] Patient-related samples 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, and samples that have been manipulated in some way after procurement (e.g., treatment with reagents); washed; or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA). Samples may include 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., RNA-containing cell lysates or other cell extracts). In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof.

[0083] 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 be infected or uninfected - and the sample may be any biological sample collected from an individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, stool 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.), and cultures thereof. The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens may be viruses, fungi, helminths, protozoans, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites, etc. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny headworms, and tapeworms (Cestodes). Protozoan infections include infections by 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, and Candida albicans.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza virus, dengue fever, West Nile virus, herpes virus, yellow fever virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, and papillomavirus. Pathogenic viruses include papovaviruses (e.g., HPV, polyomavirus); 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, and pityriasis rosea). 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, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pneumoniae, Cryptococcus neoformans, Histoplasma capsulatum, and Haemophilus influenzae.influenzae B, Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, human serum parvo-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 brucei rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria species (e.g., chicken cecal coccidia (tenella)), Onchocercavolvulus), Leishmania species (e.g., Leishmania tropica), Streptococcus pneumoniae, Pneumocystis carinii, Trichophyton rubrum, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora species, SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), herpes simplex, herpesvirus 6, herpesvirus 7, JC virus, and type A Influenza, influenza B, influenza C, rotavirus, human adenovirus, human enterovirus, hantavirus, Legionella dumophila, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsii, Ehrlichia species (e.g., Ehrlichia chaffeensis), Borrelia burgdorferi, Yersinia pestis, Chlamydia pneumoniae, Trichinella spiralis spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma species (e.g., Mycoplasma arthritidis), M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae.

[0084] amplification Methods for amplifying nucleic acids are provided herein. In some embodiments, nucleic acids are amplified using a suitable amplification process. Nucleic acid amplification typically involves enzymatic synthesis of nucleic acid amplicons (copies) containing sequences complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is performed in a single vessel, a single chamber, and / or a single volume (i.e., adjacent volumes). In some embodiments, the amplification method and the detection method (e.g., the detection methods described herein) are performed in a single vessel, a single chamber, and / or a single volume (i.e., adjacent volumes).

[0085] 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. "Amplifying" can also refer to a linear increase in the number of target nucleic acids, but is distinct from a single, 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 for some amplification but stops amplification before the exponential phase, typically producing approximately 500 copies of the desired nucleotide sequence. Preamplification can be used to limit inaccuracies associated with reactant depletion in a particular amplification reaction and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a single primer extension step can be performed prior to linear or exponential amplification.

[0086] A general description of the amplification process is provided herein. For example, when a primer (e.g., an oligonucleotide described herein) and a target nucleic acid are contacted, 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, a hybridized primer-target, or a primer-target duplex. The terms "near" or "adjacent" when referring to a nucleotide sequence of interest refer 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 refers to a range of about 1 nucleotide to about 50 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides) from the nucleotide or nucleotide sequence of interest. 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., 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.

[0087] Components of an amplification reaction (e.g., one or more amplification reagents) can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, and multiple primer sets for multiplex amplification), a nucleic acid target (e.g., a target nucleic acid derived from a sample), one or more polymerases, nucleotides (e.g., dNTPs), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, a monovalent ion, and a divalent ion). The amplification reaction may further include one or more of a reverse transcriptase, a reverse transcription primer, and one or more detection agents. Nucleic acid amplification can be performed in the presence of natural nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derivatized nucleotides. Natural nucleotides generally refer to adenylate, guanylate, cytidylate, thymidylate, or uridylate. Derivatized nucleotides generally are nucleotides other than natural nucleotides. Ribonucleoside triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, or U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be referred to 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 containing a detectable label (e.g., a fluorescent label or a colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, such as 32 P, 33 P, 125 I, or 35 The amplification 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, an antigen, a hapten, or a fluorescent dye. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., TriLink's CleanAmp™ dNTPs).

[0088] 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. In some embodiments, the non-enzymatic components 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. In some embodiments, the enzymatic components consist of a polymerase or a polymerase and a reverse transcriptase. Thus, such enzymatic components would 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, and recombinases.

[0089] In some embodiments, amplification conditions include enzymatic activity (e.g., enzymatic activity provided by a polymerase, or enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include enzymatic activity provided by enzymes other than the polymerase and / or reverse transcriptase, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. 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). Nucleic acid amplification may include non-thermal cycling PCR. In some embodiments, nucleic acid amplification includes an isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process performed at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature is maintained essentially constant during the amplification reaction. Isothermal amplification conditions generally do not include a thermal cycling (i.e., cycling between upper and lower temperature limits) component to the amplification process. When amplifying under isothermal conditions, the reaction can be maintained at an essentially constant temperature, meaning that the temperature does not need to be maintained at exactly one temperature. For example, isothermal amplification processes may experience small temperature fluctuations (e.g., ±1-5°C) due to environmental or equipment-based variables. Often, the entire reaction volume is maintained at an essentially constant temperature, and isothermal reactions, as used herein, generally do not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0090] The isothermal amplification reaction herein can be carried out at an essentially constant temperature. In some embodiments, the isothermal amplification reaction herein is carried out at a temperature of about 55°C to about 75°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C, or a temperature approximately these values, or a value or range between any two of these values. In some embodiments, a temperature element (e.g., a heat source) is maintained at an essentially constant temperature, for example, about 75°C or less, about 70°C or less, about 65°C or less, or about 60°C or less.

[0091] The amplification process herein can be carried out for a certain length of time, for example, until a detectable nucleic acid amplification product and / or quality control product is produced. The nucleic acid amplification product and / or quality control product can be detected by any suitable detection process and / or detection process described herein. The amplification process can be carried out within about 20 minutes or less, or within about 10 minutes or less. For example, the amplification process can be carried out within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or within any value or range between any two of these values. In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, nucleic acid targets 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 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 can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, and proteins (e.g., enzymatic agents).

[0092] In some embodiments, the methods disclosed herein do not involve heat denaturation (e.g., heating a solution containing nucleic acids to an elevated temperature, such as 75°C, 80°C, 90°C, or 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of nucleic acids. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions provided herein do not include a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and / or a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions and methods provided herein do not include intercalating agents, alkylating agents, and / or chemicals such as formamide, glycerol, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods of the present disclosure do not include contacting nucleic acids with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding). In some embodiments, the amplification step (e.g., step (c)) does 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). 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).

[0093] Nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-strand binding proteins), or any combination thereof. For example, nucleic acid targets can be amplified without exposure to helicases, including, but not limited to, DNA helicases and RNA helicases. Amplification conditions that do not include the use of helicases are helicase-free amplification conditions.

[0094] Nucleic acid targets can be amplified without exposure to recombinases, including, but not limited to, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX. In some embodiments, nucleic acid targets are amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY). Nucleic acid targets can be amplified without exposure to nucleic acid binding proteins (e.g., single-strand binding protein or single-stranded DNA binding protein (SSB)), such as T4 gp32. In some embodiments, nucleic acid targets are amplified without exposure to topoisomerases. Nucleic acid targets can be amplified with or without exposure to agents or conditions that destabilize nucleic acids. As used herein, the term "destabilization" shall be given its ordinary meaning and shall refer to the disruption of the overall organization and geometric orientation (e.g., double helix structure) of nucleic acid molecules by one or more of tilt, rotation, twist, slip, and flip effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, page 17). Destabilization generally does not refer to the melting or separation (e.g., denaturation) of nucleic acid strands. Nucleic acid destabilization can be achieved by exposure to agents, such as intercalating or alkylating agents and / or chemicals, such as formamide, urea, dimethyl sulfoxide (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. In some embodiments, the nucleic acid target is amplified without exposure to a ligase and / or an RNA replicase.

[0095] In some embodiments, nucleic acid targets can be amplified without cleavage or digestion. For example, nucleic acid targets can be amplified without prior exposure to one or more cleavage agents, resulting in an intact nucleic acid being amplified. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents during amplification. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents after amplification. Amplification conditions that do not include the use of a cleavage agent may be referred to herein as cleavage agent-free amplification conditions. The term "cleavage agent" generally refers to an agent, possibly a chemical or enzyme, that can cleave nucleic acids at one or more specific or non-specific sites. Specific cleavage agents often specifically cleave specific sites according to a specific nucleotide sequence. Cleavage agents can include endonucleases (e.g., restriction enzymes and nicking enzymes); 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.

[0096] Nucleic acid targets can be amplified without the use of restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without prior exposure to restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes during amplification. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNAse, RNAse (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. In some embodiments, nucleic acids are amplified without exonuclease treatment before, during, and / or after amplification. Amplification conditions that do not include the use of exonucleases are exonuclease-free amplification conditions. In some embodiments, the nucleic acid is amplified without DNAse and / or RNAse treatment. In some embodiments, the nucleic acid is amplified without RNAse H treatment.

[0097] Amplified nucleic acids may be referred to herein as nucleic acid amplification products or amplicons. In some embodiments, amplification products include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and the like, as well as combinations of the foregoing. Amplification products typically have a nucleotide sequence that is identical or substantially identical to the sequence of a sample nucleic acid (e.g., a target sequence) 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, with variations being the result of poor polymerase fidelity or other variables.

[0098] In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is contiguously complementary or substantially identical to a target sequence in the sample nucleic acid. Contiguous complementary generally refers to a nucleotide sequence in a first strand in which, for example, 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 contiguous. Stated differently, contiguous complementary generally refers to every contiguous base in a nucleotide sequence in a first strand being complementary to the corresponding contiguous base in a nucleotide sequence in a second strand. A contiguous complementary sequence may be about 5 to about 25 contiguous bases in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is contiguously complementary to or substantially identical to a target sequence in the sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not contain any additional sequence (e.g., at the 5' and / or 3' end or within the product) that is not contiguously complementary to or substantially identical to the target sequence, such as additional sequences incorporated into the amplification product by tail primers or ligation, and / or additional sequences that provide cleavage agent recognition sites (e.g., nicking enzyme recognition sites). Generally, unless the target sequence contains tandem repeats, the amplification product does not contain products in the form of tandem repeats.

[0099] The nucleic acid amplification product can comprise sequences complementary to or substantially identical to one or more primers used in the amplification reaction, hi some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is contiguous to or identical to a first primer sequence and a second nucleotide sequence that is contiguous to or identical to a second primer sequence.

[0100] Nucleic acid amplification products can include spacer sequences. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is contiguous with 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 to or substantially identical to one or more primers used in the amplification reaction. The spacer sequence flanked by sequences in the amplification product is generally located between a first sequence (complementary to or substantially identical to the first primer) and a second sequence (complementary to or substantially identical to the 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 primers. The spacer sequence may be or include approximately 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 of or consists essentially of a first nucleotide sequence that is contiguously complementary to or identical to the first primer sequence, a second nucleotide sequence that is contiguously complementary to 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 contiguously complementary to 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 mechanism incorporated into the amplification product. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguously complementary to 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 mechanism incorporated into the amplification product.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or another extra base (e.g., at the 5' and / or 3' end or within the product) introduced into the product due to, for example, errors or promiscuity in the amplification process.

[0101] Nucleic acid amplification products may be up to 50 bases in length, including 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number or range of bases between any two of these values. In some embodiments, nucleic acid amplification products of a given target sequence have the same or substantially the same length (e.g., within 1-10 bases). Thus, nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel) and generally do not produce multiple signals representing multiple lengths (e.g., a ladder or smear on an electrophoresis gel). In multiplex reactions, nucleic acid amplification products of different target sequences may have different lengths.

[0102] The methods and components described herein can be used for multiplex amplification, which generally refers to the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence). For example, multiplex amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample. For example, the amplifying step can include multiplex amplification of two or more target nucleic acid sequences, and the detecting step can include multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. The two or more target nucleic acid sequences can be specific to two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C). Multiplex amplification can also refer to the simultaneous or sequential amplification of one or more sequences present in multiple samples. For example, multiplex amplification can be used to amplify at least two amplifiable target sequences (e.g., the amplification reaction includes appropriate primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is configured to detect at least two target sequences, but only one of the target sequences is present in the sample being tested, so that both sequences are amplifiable, but only one sequence is amplified. In some embodiments, when two target sequences are present, the amplification reaction results in the amplification of both target sequences. A multiplex amplification reaction including appropriate primers and enzymes can result in the amplification of one, some, or all of the target sequences. In some embodiments, the amplification reaction is configured to detect two sequences using a pair of primers, one sequence being the target sequence and one sequence being a control sequence (e.g., a synthetic sequence that is amplifiable with the same primers as the target sequence but has spacer bases or a sequence different from the target). In some embodiments, the amplification reaction is configured to detect multiple sets of sequences using corresponding primer pairs, each set including a target sequence and a control sequence.

[0103] Primer Nucleic acid amplification is generally performed in the presence of one or more primers. A primer is generally characterized as an oligonucleotide comprising 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., target sequence). A primer can, for example, enable specific determination of the nucleotide sequence of a target nucleic acid or detection of the target nucleic acid or a characteristic 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 for 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 substantially less recognition, contact, or complex formation between either of those two molecules and other molecules. 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.

[0104] Primers can be designed and synthesized using any suitable process and may be of any length suitable for hybridizing to a target sequence and carrying out the amplification processes described herein. Primers are often designed according to the sequence of the target nucleic acid. In some embodiments, primers may be about 5 to about 30 bases in length, e.g., 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. Primers may be composed of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides), or mixtures thereof. 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 modifier), 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 (PS) 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, modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS linkages, 3' phosphate groups, and spacer bases (such as C3 spacers or other spacer bases). For example, a primer may contain 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 an RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in a primer to, for example, increase stability and binding affinity with the target sequence. In some embodiments, a primer can contain one or more phosphorothioate (PS) linkages (e.g., PS linkage modifications). PS linkages can substitute a sulfur atom for a non-bridging oxygen in the phosphate backbone of the primer. This modification typically renders the internucleotide linkage resistant to nuclease degradation. PS bond can be introduced between approximately the last 3-5 nucleotides at the 5' or 3' end of the primer to inhibit exonuclease degradation, for example. In some embodiments, PS bond inclusion throughout the primer can help reduce endonuclease attack. Primers may contain, for example, 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, primers contain 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 the 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.

[0105] A primer may comprise DNA bases, RNA bases, or both, and one or more of the DNA and RNA bases may be modified or unmodified. For example, a primer may be a mixture of DNA bases and RNA bases. A primer may comprise DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, a primer comprises unmodified DNA bases. In some embodiments, a primer comprises modified DNA bases. A primer may comprise RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, a primer comprises unmodified RNA bases. In some embodiments, a primer comprises modified RNA bases. In some embodiments, a primer does not comprise RNA bases. In some embodiments, a primer does not comprise DNA bases. In some embodiments, a primer does not comprise a cleavage agent recognition site (e.g., does not comprise a nicking enzyme recognition site). In some embodiments, a primer does not comprise a tail (e.g., does not comprise a tail comprising a nicking enzyme recognition site).

[0106] In some embodiments, all or a portion of the primer sequence may be complementary or substantially complementary to the target nucleic acid. Substantially complementary, in the context of sequences, generally refers to nucleotide sequences that will hybridize to each other. The stringency of hybridization conditions can be varied to allow for varying amounts of sequence mismatch. The target sequence and primer sequence may be at least 75% complementary to each other, including, for example, 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. A primer that is substantially complementary to a target nucleic acid sequence is typically also substantially identical to the complement of the target nucleic acid sequence (i.e., the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid may be at least 75% identical in sequence, for example, 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% identical to each other.

[0107] In some embodiments, the primers comprise a pair of primers. A pair of primers may comprise a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid). In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, 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, oligonucleotides other than primers, probes, etc.). In some embodiments, the primers consist of a pair of primers. 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 include additional primers, oligonucleotides, or probes for a detection process that is not considered part of the amplification. In some embodiments, primers are used in sets. An amplification primer set may comprise 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.

[0108] Nucleic acids (e.g., amplification products, sample nucleic acids, target nucleic acid sequences) described herein can comprise a first strand and a second strand that are complementary to each other. Amplification reaction components may comprise or consist of a first primer (first oligonucleotide) that is complementary to a target sequence in the first strand (e.g., sense strand, forward strand) of the sample nucleic acid, and a second primer (second oligonucleotide) that is complementary to a target sequence in the second strand (e.g., antisense strand, reverse strand) of the sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) comprises a first polynucleotide that is contiguously complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide that is contiguously complementary to the target sequence in the second strand of the sample nucleic acid. Contiguously complementary, with respect to primer-target, generally refers to a nucleotide sequence of a primer in which each base pairs sequentially with a corresponding, ordered base in the target sequence, with no gaps, additional sequence, or unpaired bases present within the sequence considered contiguously complementary. In some embodiments, the primer does not include any additional sequence (e.g., at the 5' and / or 3' end or within the primer) that is not contiguous with the target sequence, such as additional sequence present in a tail primer or loop primer, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site). In some embodiments, the amplification reaction components do not include primers that include additional sequence (i.e., sequence other than sequence that is contiguous with the target sequence), such as a tail primer, a loop primer, a step-loop structure, a primer that can form a hairpin structure, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site).

[0109] In some embodiments, a primer can contain modifications such as one or more inosines, abasic sites, LNAs, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifier that alters the binding properties of the primer. In some embodiments, a primer can include a detectable molecule or entity (e.g., a fluorophore, a radioisotope, a colorimetric reagent, a particle, an enzyme, etc.).

[0110] polymerase Amplification reaction components (e.g., one or more amplification reagents) can 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, into a nucleic acid target sequence (e.g., to which the primer anneals). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that can exhibit activity at high reaction temperatures (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C). Hyperthermophilic polymerases are sometimes referred to as hyperthermophilic polymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, the polymerase can incorporate from about 1 to about 50 nucleotides in a single synthesis, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range of nucleotides between any two of these values ​​in a single synthesis.

[0111] Amplification reaction components include 9°N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA polymerase; Therminator™ II DNA polymerase; Therminator™ III DNA polymerase; Therminator™ γ DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo); T4 DNA polymerase; T7 DNA polymerase; DeepVentR™ (exo) DNA polymerase; DeepVentR™ DNA polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start DNA polymerase; Phusion™ High-Fidelity The polymerase may include one or more DNA polymerases selected from 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; Taq DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase beta; and ThermoPhi DNA polymerase.

[0112] In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermostable at high temperatures). The half-life of the hyperthermophilic DNA polymerase may be about 5-10 hours at 95°C and about 1-3 hours at 100°C. For example, the amplification reaction components may include one or more hyperthermophilic DNA polymerases from Archaea (e.g., a hyperthermophilic DNA polymerase from Thermococcus or a hyperthermophilic DNA polymerase from Thermococcaceaen archaean). In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from the genus Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0113] In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase or a functional fragment thereof. A functional fragment generally retains one or more functions of a full-length polymerase, such as the ability to polymerize DNA (e.g., in an amplification reaction). In some cases, the functional fragment performs a function (e.g., polymerizing DNA in an amplification reaction) at a level that is at least about 50%, at least about 75%, at least about 90%, or at least about 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, such as those described herein. In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, an amplification reaction component (e.g., one or more amplification reagents) comprises a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase or a functional fragment thereof, hi some embodiments, an amplification reaction component comprises a polymerase comprising an amino acid sequence at least about 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment thereof.

[0114] The polymerase may have reverse transcriptase activity. In such embodiments, the amplification reaction can amplify an RNA target in a single step, for example, without the use of a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase activity include Bst (large fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, and Therminator™ II. The amplification reaction components can include one or more separate reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction can include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0115] 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. In some embodiments, the polymerase with no or low exonuclease activity comprises 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, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the 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 or corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:1 (e.g., D141A, E143A, E143D, and A485L).

[0116] Detection and Quantification The methods described herein can include detecting and / or quantifying the nucleic acid amplification product(s) and / or quality control product(s). The amplification product(s) can be detected and / or quantified, for example, by any suitable detection and / or quantification method described herein (e.g., signal-generating oligonucleotides).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' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating / binding dyes, absorbance methods (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension methods (e.g., iPLEX™), Affymetrix Molecular Inversion Probes (MIR), and the like. Probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and LNA probes, AlphaScreen, SNPstream, genetic bit The methods include, but are not limited to, GBA, multiplex minisequencing, SNaPshot, GOOD assay, microarray miniseq, arrayed primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed incorporation (TDI), 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, detecting the nucleic acid amplification products and / or quality control 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, detecting nucleic acid amplification products involves 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, either directly incorporated into the target sequence or incorporated into a probe containing 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 generating (or referencing) standard curves and / or look-up tables for quantification of nucleic acid amplification products and / or quality control products.

[0117] Detection of nucleic acid amplification products and / or quality control products may involve the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule whose detectable property is detectable under certain conditions, thereby enabling the molecule to function as a specific, useful signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or speed of passing through an aperture of a known size. A molecular beacon for detecting nucleic acid molecules may be, for example, a hairpin-shaped oligonucleotide containing a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin may contain a probe sequence 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 molecule may be covalently linked to 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 FRET. When the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product and / or a quality control product), hybridization can occur, converting the loop structure into a stable, more rigid conformation, causing the fluorophore molecule and the quencher molecule to separate, resulting in fluorescence. Because the probe is specific, the generation of fluorescence is generally due only 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 to 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 to 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). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with different colored fluorophores and different target sequences, allowing for the simultaneous detection of several products in the same reaction (eg, in a multiplex reaction).In the case of quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each cycle of amplification, and unhybridized molecular beacons are dark, so there is no need to isolate the probe-target hybrid to quantitatively determine the amount of amplification product. The signal obtained is proportional to the amount of amplification product. Detection using molecular beacons can be performed in real time or as an end-point detection method.

[0118] Detection of nucleic acid amplification products and / or quality control products may involve the use of lateral flow. The use of lateral flow typically involves the use of lateral flow devices, including, but not limited to, dipstick assays and thin-layer chromatography plates with various appropriate coatings. Various binding reagents for the sample, binding partners for the sample or conjugates containing binding partners, and signal generation systems are immobilized in the flow channels. Detection of nucleic acid amplification products and / or quality control products may involve the use of FRET, 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 of the donor molecule as it returns to its ground state can transfer the excitation energy to the acceptor molecule via long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored and is a function of the distance between the donor and acceptor, the overlap between the donor emission spectrum and 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, for quantifying 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. Probe-target hybridization changes the distance or orientation between the donor and acceptor, and a FRET change is observed.

[0119] Detection of nucleic acid amplification products and / or quality control products may involve the use of fluorescence polarization (FP). FP techniques are based on the principle that when a fluorescently labeled compound is excited with linearly polarized light, it will emit fluorescence with a degree of polarization inversely proportional to its rotation rate. Thus, when a molecule, such as a fluorescently labeled tracer-nucleic acid conjugate, is excited with linearly polarized light, the fluorophore is constrained from rotating between light absorption and emission, resulting in a highly polarized emission. When a free tracer compound (i.e., not bound to a nucleic acid) is excited with linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, resulting in a more random molecular orientation and depolarized emitted light. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction. Detection of nucleic acid amplification products and / or quality control products may involve the use of surface capture, achieved, for example, by immobilizing specific oligonucleotides on a surface to create a biosensor with both high sensitivity and selectivity. Examples of surfaces that can be used to attach probes include gold and carbon. Detection of nucleic acid amplification products and / or quality control products may involve the use of 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN). For example, TAQMAN probes are hydrolysis probes that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The principle of TAQMAN probes relies on 1) the 5'-3' exonuclease activity of Taq polymerase, which cleaves a dual-labeled probe upon hybridization to a complementary target sequence, and 2) fluorophore-based detection. The resulting fluorescent signal allows for quantitative measurement of the accumulation of amplification product during the exponential phase of amplification, and TAQMAN probes can significantly increase the specificity of detection.

[0120] Detecting nucleic acid amplification products and / or quality control products can include the use of intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes exhibit enhanced fluorescence upon binding to DNA or RNA). Dyes can include DNA or RNA intercalating fluorophores, including, but not limited to, SYTO® 82, acridine orange, ethidium bromide, Hoechst dye, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Detection of nucleic acid amplification products and / or quality control products can also include the use of absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260 nm) to concentration. Direct measurements of nucleic acids can be converted to concentrations using the Beer-Lambert law, which relates absorbance to concentration using the path length and extinction coefficient of the measurement. Detection of nucleic acid amplification products and / or quality control products can involve the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and quantity of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample is ionized, and the resulting ions can be separated according to their mass-to-charge ratio in an electric and / or magnetic field, and the mass-to-charge ratio of the ions is measured by a detector. Mass spectrometry methods include, for example, MALDI, MALDI-TOF, and electrospray. These methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can achieve high throughput due to fast signal acquisition from solid surfaces and automated analysis.

[0121] Detection of nucleic acid amplification products and / or quality control products may involve the use of nucleic acid sequencing. The entire or partial sequence of the amplification product may be determined, and the determined nucleotide sequence may be referred to as a read. For example, linear amplification products may be directly analyzed without further amplification (e.g., by using single-molecule sequencing). In some embodiments, linear amplification products are subjected to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single-molecule sequencing, multiplex sequencing, solid-phase single-nucleotide sequencing, and nanopore sequencing. Detecting nucleic acid amplification products and / or quality control products may involve the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm®).

[0122] Lysis buffer Soluble drugs As disclosed herein, the dissolving agent can include a detergent. The detergent can include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. Anionic surfactants have NH4 as a counterion. + , K. + , Na + , or Li + The cationic surfactant may contain I as a counter ion. - , Br - , or Cl - may also include:

[0123] The lysis agents provided herein may be capable of acting as denaturing agents. As used herein, "denaturing agent" or "denaturant" shall be given its ordinary meaning and include any compound or substance that will cause reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined by both the properties and concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturing agents include chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents can include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(dioleyoxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergents), 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). Organic, water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols, such as ethanol or isopropanol), or lower alkanediols (especially C2-C4 alkanediols, such as ethylene glycol) can be used as denaturing agents.The phospholipid may be a naturally occurring phospholipid such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or a synthetic phospholipid derivative or variant such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0124] Suitable surfactant levels may be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, it may be advantageous to use anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof). Anionic surfactants useful herein include the water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl radical, and the water-soluble salts of sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant.

[0125] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having a single long alkyl chain containing about 8 to 18 carbon atoms, such as lauryltrimethylammonium chloride, cetylpyridinium chloride, benzalkonium chloride, cetyltrimethylammonium bromide, di-isobutylphenoxyethyl-dimethylbenzylammonium chloride, alkyltrimethylammonium nitrite of coconut, cetylpyridinium fluoride, etc. Certain cationic surfactants can also act as antiseptics in the compositions disclosed herein.

[0126] Suitable nonionic surfactants that can be used in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds produced by the condensation of an organic hydrophobic compound, which can be aliphatic and / or aromatic, with an alkylene oxide group (hydrophilic nature).Examples of suitable nonionic surfactants include: poloxamers; sorbitan derivatives such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of aliphatic alcohols or alkylphenols; condensation products of ethylene oxide with the reaction product of propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides; and mixtures of such substances.These substances are useful for stabilizing foam without contributing to excessive viscosity increase of consumer product compositions.

[0127] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, where the aliphatic radical may be straight or branched chain, and one of the aliphatic substituents contains about 8 to 18 carbon atoms and one contains an anionic water-solubilizing group, such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of anionic short-chain surfactants include alkyl sulfates, alkyl sulfonates, alkylbenzene sulfonates, saturated or unsaturated fatty acids, and their salts. The polar head group-containing moiety in cationic surfactants can include, for example, quaternary ammonium, pyridinium, sulfonium, and / or phosphonium groups. For example, the polar head group can include trimethylammonium. Exemplary cationic short-chain surfactants include alkyltrimethylammonium halides, alkyltrimethylammonium tosylates, and N-alkylpyridinium halides.

[0128] reducing agent The lysis buffer and / or reagent composition (e.g., dried composition) can include one or more reducing agents. A "reducing agent" can be a compound or group of compounds. As used herein, a "reducing agent," also known as a "reductant," "reducing agent," or "reducing equivalent," can refer to an element or compound that donates electrons to another chemical species. In particular, reducing agents are generally compounds that cleave disulfide bonds by reduction, thereby overcoming tertiary protein folding and quaternary protein structure (multimeric subunits) stabilized by disulfide bonds. Examples of suitable reducing agents include, but are not limited to, 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED. In some embodiments of the methods, compositions, and kits provided herein, the lysis buffer and / or reagent composition (e.g., the dried composition) does not include one or more reducing agents.

[0129] Reagent Composition The reagent compositions (e.g., dry compositions) described herein can be provided in a "dry form," or 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, e.g., sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, e.g., arginine or histidine; lyotropic salts, e.g., magnesium sulfate; polyols, e.g., 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-dried" refer to a process in which the material to be dried is first frozen, and then the ice or frozen solvent is removed by sublimation under reduced pressure. "Lyophile" refers to a lyphophilized material.

[0130] The reagent composition (e.g., a dried composition) may be frozen, lyophilized, or spray-dried. The reagent composition may be heat-dried. The reagent composition may include one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). The sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymers may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The lyophilized reagent may include poly rA, EGTA, EDTA, Tween 80, and / or Tween 20.

[0131] Frozen or lyophilized or spray-dried or heat-dried compositions, or aqueous compositions for preparing frozen or lyophilized or spray-dried compositions, may be prepared using the following: (i) non-aqueous solvents, such as ethylene glycol, glycerol, dimethyl sulfoxide, and dimethylformamide; (ii) surfactants, such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic acid, also known as poloxamer; The composition may include one or more of the following: F127 (polyoxyethylene-polyoxypropylene copolymer), poloxamine, and sodium dodecyl sulfate; (iii) dissacharides, such as trehalose, sucrose, lactose, and maltose; (iv) polymers (which may have different MW), such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, ficoll, and albumin; and (v) amino acids, such as one or more of glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and gamma-aminobutyric acid.

[0132] The reagent composition (e.g., a dried composition) can include one or more protecting agents and one or more amplification reagents. The one or more protecting agents can include a cyclodextrin compound. Cyclodextrins (CDs) can be used for complexation with soluble agents (e.g., SDS). Cyclodextrins (CDs) can be cyclic oligosaccharides resembling truncated cones with a hydrophobic interior cavity and a hydrophilic exterior surface. The most commonly used natural cyclodextrins contain 6, 7, and 8 glucose units, designated α-, β-, and γ-CD. Natural CDs can have solubility. Chemically modified CDs, such as hydroxypropyl derivatives, improve solubility in aqueous media by up to 50%. CAVASOL® is a trade name for cyclodextrin derivatives from WACKER, encompassing a variety of α-, β-, and γ-CD derivatives. β-CD can form a strong inclusion complex (stronger than α-CD and β-CD) with sodium dodecyl sulfate (SDS) in a predominantly 1:1 stoichiometry. The binding constant of β-CD to SDS is 2100 M -1 ~2500M -1 The range can be as follows:

[0133] kit Disclosed herein is a kit for monitoring an amplification reaction. In some embodiments, the kit comprises a quality control template disclosed herein; a quality control primer disclosed herein; a signal-generating oligonucleotide disclosed herein; and / or an auxiliary quality control primer disclosed herein. The kit can comprise a reagent composition comprising one or more amplification reagents including one or more components (e.g., forward primer, reverse primer) for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the quality control template, signal-generating oligonucleotide, quality control primer, auxiliary quality control primer, and / or one or more components for amplification are in lyophilized or freeze-dried form and / or present in the reagent composition. The kit can include at least one component that provides real-time detection activity for nucleic acid amplification products and / or quality control products. The real-time detection activity can be provided by a molecular beacon. The reagent composition (e.g., a dried composition) can include a reverse transcriptase and / or a reverse transcription primer.

[0134] In some embodiments, the molar ratio of the one or more protectants to the one or more amplification reagents is about 10:1 to about 1:10 (e.g., about 2:1). In some embodiments, the one or more additives include Tween 20, Triton X-100, Tween 80, a non-ionic detergent (e.g., a non-ionic surfactant), or any combination thereof. In some embodiments, the one or more protectants include a cyclodextrin compound. In some embodiments, the one or more solubility reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) (e.g., about 0.2% (wt / vol)) of the processed sample. In some embodiments, the one or more solubility agents include a detergent. The detergent can include one or more of a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and an amphoteric surfactant. In some embodiments, it may be advantageous for one or more protecting agents to sequester one or more solubility agents, thereby preventing the one or more solubility agents from denaturing one or more amplification reagents.

[0135] The kit may include, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, as described herein. If one target is being amplified, a pair of primers (forward and reverse) may be included in the kit. If multiple target sequences are being amplified, multiple primer pairs may be included in the kit. The kit may include a control polynucleotide, and if multiple target sequences are being amplified, multiple control polynucleotides may be included in the kit. An enzyme with hyperthermophile polymerase activity can have an amino acid sequence that is at least about 90% or 95% identical to the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof. For example, an enzyme with hyperthermophile polymerase activity can include the amino acid sequence of SEQ ID NO: 1. The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may include (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length. The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoan.

[0136] The kit may also contain 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 a single container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a container separate 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 primers are present in a single container in lyophilized or heat-dried form.

[0137] The kits may include, for example, dNTPs or modified nucleotides used in the reactions, vessels, cuvettes, or other containers, or vials of water or buffer for rehydrating lyophilized or heat-dried components. The buffers used may be suitable for both polymerase activity and primer annealing activity, for example. The kits may also include instructions for performing one or more of the methods described herein and / or descriptions of one or more of the components described herein. The instructions and / or descriptions may be in printed form or may be included in a kit insert. The kits may also include a written description of an internet location providing such instructions or descriptions. The kits may also include reagents used in detection methods, such as reagents used in FRET, lateral flow devices, dipsticks, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. [Example]

[0138] 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.

[0139] Example 1 Hairpin Internal Standard (HPIC) Proof of Principle This example demonstrates monitoring of amplification reactions using the methods and compositions described herein. Figure 1 depicts a non-limiting, exemplary embodiment of the DNA hairpin internal standard assay reaction—the two-component MB-IC hairpin approach—disclosed herein and examined in this example. The IC primer extends on a molecular beacon (e.g., a hairpin-shaped probe), ultimately resulting in the generation of two hairpin products that have the same stem sequence and are complementary only in the loop (spacer) region. Real-time detection of the hairpin products can be achieved using the same molecular beacon. The HPIC assay components are one IC primer and a molecular beacon IC (Table 1). Figure 2 depicts the thermal setup for the hairpin IC (HPIC) isothermal amplification reaction described in this example. In the reaction described in this example, the molecular beacon IC (HPIC) was detected with the HEX fluorophore, and the target (Neisseria gonorrhoeae (Ng)) was detected with the ROX fluorophore. The IC template used is also shown in Table 1. In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 5' modification (e.g., 5HEX). In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 3' modification (e.g., 3IABkFQ).

[0140] [Table 1] The performance of a duplex system for hairpin IC (400 nM) and target Neisseria gonorrhoeae (Ng) detection was evaluated in clean versus 10% normal urine samples and using HPIC template dose settings. Figures 3A-3B show data for target (Ng) (ROX) detection and hairpin IC detection (HEX) in clean samples (Figure 3A) compared to samples containing 10% normal urine (Figure 3B). Figure 4A shows the Ng-only reaction, while Figures 4B-4D depict reactions with increasing levels of IC template (ICT): 0 ICT (Figure 4B), 50k ICT (Figure 4C), and 500k ICT (Figure 4D) in clean and 10% normal urine samples. These results demonstrate that HPIC (e.g., MB2-HEX) acts as both a beacon and a template for detection. The consequences of including additional IC template in the mixture were examined and it was found that increasing the IC template may make the IC too strong to obtain accurate detection and, without being bound by any particular theory, may compete with the target (e.g., Ng) signal.

[0141] Next, the performance of the duplex system for hairpin IC (400 nM) and target (Ng) detection was evaluated in samples with increasing levels of inhibitory urine: 10% (Figure 5A), 20% (Figure 5B), and 30% (Figure 5C). The MB hairpin IC was found to be detectably amplified in samples containing 10% or 20% inhibitory urine. Figure 6A depicts data for Ng detection in normal urine in the absence of an internal standard. The Ng ROX probe was used for target detection, and Syto 82 intercalating dye was used for both specific and nonspecific product detection. Figure 6B depicts data for Ng / hairpin IC detection in normal urine, where the hairpin IC probe (HPIC) is detected with the HEX fluorophore and the target (Ng) is detected with the ROX fluorophore. The MB hairpin IC used in this example also shows similar results across different lots. Figures 7A-7B depict data showing a comparison of target (Ng) (ROX) detection and hairpin IC detection (HEX) in samples containing 20% ​​inhibitory urine and hairpin IC from different lots: 9.14 (Figure 7A) and 10.12 (Figure 7B).

[0142] In summary, this example demonstrates that hairpin IC MBs can function as both IC beacons and templates without the need for the addition of additional IC templates. The addition of hairpin IC MBs did not negatively interfere with target (Ng) detection. Hairpin ICs can be fine-tuned to track target amplification for matrix inhibition. IC reactions performed well in samples containing interfering substances (e.g., in 10% normal urine, 10% inhibited urine, and 20% inhibited urine matrices). IC signals can increase as target (Ng) decreases. In some cases, there is a difference of approximately 1 minute between the Ng signal Td and the IC signal Td in 10% inhibited urine and approximately 2 minutes in 20% inhibited urine. In a sample with 10% inhibited urine and 10 cp / ul of target sequence, Td was approximately 4 minutes for the target, and the IC signal appeared at approximately 5 minutes. In samples with 20% inhibited urine and 10 cp / ul target, the Td was approximately 7 minutes for the target and the IC signal appeared at approximately 9 minutes.

[0143] Example 2 Hairpin internal standard (HPIC) molecular beacon modification This example demonstrates exemplary modifications of the signal-generating oligonucleotides (e.g., molecular beacons) described herein. To facilitate amplification and detection, the product hybridization melting temperature (Tm) can be designed to exceed or equal the product hairpin Tm. Signal-generating oligonucleotides (e.g., molecular beacons) modified with LNAs in the spacer region can be used for IC product detection. In this example, a hairpin-shaped internal standard target was amplified in an APA reaction for 10 minutes and simultaneously detected by a molecular beacon (Figure 9A) and an intercalating dye (Figure 9D). After the reaction, the temperature of the reaction was immediately ramped from the assay temperature to 90°C for melting curve analysis (Figures 9B and 9E) and melting derivative evaluation (Figures 9C and 9F). The sequences of the two molecular beacons (50 nM) and internal standard primer (500 nM) are shown in Table 2. The two molecular beacon designs (HpIC1b MB1 and HpIC1b MB2) contain the same fluorophore and quencher pair and have the same sequence. They differ only in the location of the LNA modification on the beacon. The resulting melting temperature difference for the two beacons MB1 (red curve, upper right) and MB2 (green curve, lower right) is 9°C. In some embodiments, the probes (e.g., molecular beacons) provided herein contain a 5' modification (e.g., 5HEX). In some embodiments, the probes (eg, molecular beacons) provided herein comprise a 3' modification (eg, 3IABkFQ).

[0144] [Table 2]

[0145] 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 can 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. With respect to the use of substantially any plural and / or singular terminology herein, those 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 in this specification and 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 stated otherwise.

[0146] 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.). Furthermore, those skilled in the art will understand that where a specific number is intended in an introduced claim recitation, such intention will be expressly recited in the claim; otherwise, no such intention 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 claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means limiting any particular claim containing such an introduced claim recitation to embodiments containing only one such recitation, even if the same claim also 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. Additionally, even if a particular number is explicitly recited in an introduced claim recitation, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a base 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 a person 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, a system having A only, B only, C only, 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 a person 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, a system having A only, B only, C only, 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 of those terms, or both terms. Additionally, when features or aspects of the disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is also thereby described in terms of every individual member or subgroup of members of the Markush group.

[0147] As one of ordinary skill in the art would understand, 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 that 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. Furthermore, as one of ordinary skill in the art would understand, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the recited numerical values ​​and that can be subsequently divided into subranges as discussed above. Finally, as one of ordinary skill in the art would understand, a range includes each individual member. Thus, for example, a group containing 1 to 3 items refers to groups containing 1, 2, or 3 items. Similarly, a group containing 1 to 5 items refers to groups containing 1, 2, 3, 4, or 5 items, etc.

[0148] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not limitation, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for monitoring an amplification reaction, comprising: 5' subdomain; the 3' subdomain; and a loop domain between the 5' subdomain and the 3' subdomain wherein intramolecular nucleotide base pairing between the 5′ subdomain and the 3′ subdomain is capable of forming a paired stem domain; and a quality control primer capable of hybridizing to at least a portion of the 3' subdomain of the quality control template; preparing a subjecting the quality control template and the quality control primer to an amplification reaction capable of producing a first quality control product; and detecting said first quality control product.

2. 2. The method of claim 1, wherein the amplification reaction is carried out in an amplification reaction mixture under amplification conditions, optionally wherein the amplification conditions are isothermal amplification conditions.

3. subjecting the quality control template and the quality control primer to an amplification reaction capable of producing a first quality control product, amplifying said quality control template using said quality control primers in said amplification reaction mixture under said amplification conditions, thereby producing said first quality control product.

3. The method of claim 1 or 2, comprising:

4. The method according to any one of claims 1 to 3, wherein the amplification reaction comprises a reverse transcription reaction.

5. providing an enzyme having polymerase activity, optionally wherein the enzyme having polymerase activity is an enzyme having hyperthermophilic polymerase activity, and further optionally wherein the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity; and / or Preparing reverse transcriptase The method of any one of claims 1 to 4, further comprising:

6. The amplification reaction contacting the quality control primer with the quality control template for hybridization; and extending the quality control primer hybridized to the quality control template with an enzyme having polymerase activity, thereby generating a first quality control product. The method according to any one of claims 1 to 5, comprising:

7. The amplification reaction contacting the quality control primer with the first quality control product for hybridization; and extending the quality control primer hybridized to the first quality control product with an enzyme having polymerase activity, thereby generating a second quality control product. The method according to any one of claims 1 to 6, comprising:

8. The amplification reaction contacting the quality control primer with the second quality control product for hybridization; and extending the quality control primer hybridized to the second quality control product with an enzyme having polymerase activity, thereby producing a first quality control product. The method according to any one of claims 1 to 7, comprising:

9. the first quality control product and the second quality control product comprise a 5' subdomain and the 3' subdomain capable of forming a paired stem domain; the first quality control product and the second quality control product have the same stem domain; and / or The method of any one of claims 1 to 8, wherein the first quality control product and the second quality control product comprise loop domains that are complementary to each other.

10. The method of any one of claims 1 to 9, wherein the amplification reaction comprises linear and / or exponential amplification of the first and second quality control products.

11. The method of any one of claims 1 to 10, wherein the 5' subdomain of the quality control template comprises at least a portion of the sequence of the quality control primer.

12. The method according to any one of claims 1 to 11, wherein the first quality control product and the second quality control product are both capable of forming a hairpin structure.

13. the quality control template comprises a 5' terminal domain 5' to the 5' subdomain; and / or The method of any one of claims 1 to 12, wherein the quality control template comprises a 3' terminal domain 3' of the 3' subdomain.

14. 14. The method of any one of claims 1 to 13, wherein the 5' terminal domain of the quality control template comprises at least a portion of the sequence of the quality control primer, and optionally the combined sequence of the 5' terminal domain and the 5' subdomain comprises the entire sequence of the quality control primer.

15. 15. The method of any one of claims 1 to 14, wherein detecting the first quality control product comprises detecting the first quality control product using a signal-generating oligonucleotide, optionally wherein the signal-generating oligonucleotide is capable of hybridizing to the first quality control product.

16. The method of any one of claims 1 to 15, wherein the detecting step comprises contacting the first quality control product with the signal-generating oligonucleotide for hybridization.

17. 17. The method of any one of claims 1 to 16, wherein the signal-generating oligonucleotide comprises a quencher, a label, or both, optionally wherein the label comprises a quenchable label, and further optionally wherein the quenchable label is a fluorophore.

18. The method of any one of claims 1 to 17, wherein the signal-generating oligonucleotide comprises a quencher, optionally the quencher being capable of quenching the label.

19. 19. The method of any one of claims 1 to 18, wherein the detecting step comprises contacting the first quality control product with the signal-generating oligonucleotide for hybridization.

20. the label is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the first quality control product; and / or After the signal-generating oligonucleotide hybridizes to the first quality control product, the label generates a signal; 20. The method of any one of claims 1 to 19, wherein optionally the signal is fluorescence.

21. 21. The method of any one of claims 1 to 20, wherein detecting the first quality control product comprises detecting a signal generated by the label of the signal-generating oligonucleotide, optionally wherein the label is a fluorophore and the signal is fluorescence.

22. 22. The method of any one of claims 1 to 21, wherein the detecting step comprises detecting the signal of the label before the amplification reaction, during the amplification reaction, after the amplification reaction, or any combination thereof.

23. providing a signal-generating oligonucleotide; subjecting said signal-generating oligonucleotide to said amplification reaction; and detecting said first quality control product using said signal-generating oligonucleotide. The method of any one of claims 1 to 22, further comprising:

24. The method of any one of claims 1 to 23, wherein the quality control template is a signal-generating oligonucleotide.

25. 25. The method of any one of claims 1 to 24, wherein the quality control template is (i) a template for the synthesis of the first quality control product, and (ii) a means for detecting the first quality control product.

26. 26. The method of any one of claims 1 to 25, wherein the signal-generating oligonucleotide is capable of (i) detecting the first quality control product, and (ii) serving as a template for quality control primer-driven synthesis of the first quality control product.

27. 27. The method of any one of claims 1 to 26, wherein (i) the quality control template, (ii) the quality control primer, and (iii) the signal-generating oligonucleotide are present in the same molecule.

28. The 5' terminal domain of the quality control template comprises: one or more RNA nucleotides; and / or the sequence of at least a portion of said quality control primer The method of any one of claims 1 to 27, comprising:

29. The quality control template does not contain a 3' terminal domain; and / or The method of any one of claims 1 to 28, wherein the 3' end of the quality control template is complementary to the 5' end of the 5' subdomain of the quality control template.

30. 30. The method of any one of claims 1 to 29, wherein a reverse transcriptase is capable of using the one or more RNA nucleotides of the 5'-terminal domain of the quality control template as a template to extend the 3'-end of the quality control template, thereby generating an extended quality control template.

31. The method of any one of claims 1 to 30, wherein the 3' end of the extended quality control template comprises a sequence complementary to at least a portion of the quality control primer.

32. 32. The method of any one of claims 1 to 31, wherein the amplification reaction comprises contacting a reverse transcriptase with the quality control template, thereby generating an extended quality control template, and optionally the extended quality control template comprises cDNA.

33. The amplification reaction contacting the quality control primer with the 3' end of the extended quality control template for hybridization; and extending the quality control primer hybridized to the 3' end of the extended quality control template with an enzyme having reverse transcriptase and / or polymerase activity, thereby generating a first quality control product. The method of any one of claims 1 to 32, comprising:

34. 34. The method of any one of claims 1 to 33, wherein the quality control template is a signal-generating oligonucleotide, the signal-generating oligonucleotide comprises a label, the loop domain comprises one or more RNA nucleotides, optionally the label comprises a quenchable label, and further optionally the quenchable label is a fluorophore.

35. The signal-generating oligonucleotide comprises a quencher and optionally: the label is in the 3'-terminal domain and the quencher is in the 5'-terminal domain; and / or The method of any one of claims 1 to 34, wherein the label is in the 5'-terminal domain and the quencher is in the 3'-terminal domain.

36. The amplification reaction contacting the quality control primer with the quality control template for hybridization; and 36. The method of any one of claims 1 to 35, comprising extending the quality control primer hybridized to the quality control template with a reverse transcriptase, thereby generating a first quality control product, optionally wherein the reverse transcriptase comprises RNase H activity.

37. 37. The method of any one of claims 1 to 36, wherein the reverse transcriptase cleaves the quality control template at the one or more RNA nucleotides during production of the first quality control product, thereby generating a first cleavage product and a second cleavage product that comprise a label.

38. 38. The method of any one of claims 1 to 37, wherein detecting the first quality control product comprises detecting a signal generated by the first cleavage product comprising a label, optionally wherein the label is a fluorophore and the signal is fluorescence.

39. Providing a supplemental quality control primer; and subjecting said supplemental quality control primers to said amplification reaction. The method of any one of claims 1 to 38, further comprising:

40. the signal-generating oligonucleotide is from about 10 nucleotides to about 100 nucleotides in length; the quality control template is from about 10 nucleotides to about 100 nucleotides in length; the quality control primer and / or the auxiliary quality control primer are from about 5 nucleotides to about 25 nucleotides in length; and / or 40. The method of any one of claims 1 to 39, wherein the 5' subdomain, the 3' subdomain, the loop domain, the 5' terminal domain and / or the 3' terminal domain are from about 1 nucleotide to about 25 nucleotides in length.

41. 41. The method of any one of claims 1 to 40, wherein the signal-generating oligonucleotide, the quality control template and / or the quality control primer comprise one or more phosphorothioate linkages and / or one or more locked nucleic acids.

42. 42. The method of any one of claims 1 to 41, wherein the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide.

43. 43. The method of any one of claims 1 to 42, wherein the signal-generating oligonucleotide comprises one or more locked nucleic acids (LNAs), optionally the one or more LNAs are within the loop domain and / or stem domain, and further optionally the one or more LNAs enhance detectability of the first quality control product.

44. 44. The method of any one of claims 1 to 43, wherein the signal-generating oligonucleotide is configured such that the melting temperature (Tm) of a first quality control product / signal-generating oligonucleotide duplex is equal to or exceeds the melting temperature (Tm) of the paired stem domain of the signal-generating oligonucleotide, and optionally comprises one or more LNAs in the loop domain and / or stem domain.

45. 45. The method of any one of claims 1 to 44, wherein the signal-generating oligonucleotide does not contain a dye capable of quenching the label.

46. 46. ​​The method of any one of claims 1 to 45, wherein the signal-generating oligonucleotide does not comprise a moiety capable of quenching the label other than a nucleotide of the signal-generating oligonucleotide.

47. the 5' terminal domain of the quality control template and / or the signal-generating oligonucleotide comprises the label; the 5' terminal domain and / or the 5' subdomain of the quality control template and / or the signal-generating oligonucleotide comprises one or more cytosine bases; the 3' terminal domain and / or the 3' subdomain of the quality control template and / or the signal-generating oligonucleotide comprises one or more guanine and / or adenine bases; 47. The method of any one of claims 1 to 46, wherein optionally, the one or more guanine and / or adenine bases are capable of quenching the label after the quality control template and / or the signal-generating oligonucleotide form a hairpin structure.

48. the 3' terminal domain of the quality control template and / or the signal-generating oligonucleotide comprises the label; the 3' terminal domain and / or the 3' subdomain of the quality control template and / or the signal-generating oligonucleotide comprises one or more cytosine bases; the 5' terminal domain and / or the 5' subdomain of the quality control template and / or the signal-generating oligonucleotide comprises one or more guanine and / or adenine bases; 48. The method of any one of claims 1 to 47, wherein optionally, the one or more guanine and / or adenine bases are capable of quenching the label after the quality control template and / or the signal-generating oligonucleotide form a hairpin structure.

49. 49. The method of any one of claims 1 to 48, wherein detecting the first quality control product comprises detecting a decrease in the amount of signal generated by a label of the quality control primer, optionally wherein the label is a fluorophore and the signal is fluorescence.

50. 50. The method of any one of claims 1 to 49, wherein the production of the first quality control product and the second quality control product correlates with the decay of the detected signal.

51. 51. The method of any one of claims 1 to 50, wherein the 5' end of the quality control primer comprises a label, and the quality control primer comprises one or more pyrimidine bases adjacent to the label, and optionally one or more cytosine and / or thymine bases adjacent to the label.

52. 52. The method of any one of claims 1 to 51, wherein the 3' terminal domain and / or the 3' subdomain of the quality control template, first quality control product and / or second quality control product comprises one or more guanine and / or adenine bases.

53. after the quality control primer binds to the quality control template and / or second quality control product and is extended by an enzyme having polymerase activity to generate a first quality control product, the one or more guanine and / or adenine bases present in the 3′-terminal domain and / or the 3′ subdomain of the first quality control product are capable of quenching the label after the first quality control product forms a hairpin structure; and / or 53. The method of any one of claims 1 to 52, wherein after the quality control primer binds to the first quality control product and is extended by an enzyme having polymerase activity to generate a second quality control product, the one or more guanine bases and / or adenine bases present in the 3'-terminal domain and / or the 3'-subdomain of the second quality control product are capable of quenching the label after the second quality control product forms a hairpin structure.

54. 54. The method of any one of claims 1 to 53, wherein detecting the first quality control product comprises contacting the first quality control product with a fluorescent dye.

55. 55. The method of any one of claims 1 to 54, wherein the step of providing the quality control primer, the quality control template, and / or the signal-generating oligonucleotide comprises the step of providing a reagent composition comprising the quality control primer, the quality control template, and / or the signal-generating oligonucleotide.

56. 56. The method of any one of claims 1 to 55, wherein subjecting the quality control primer, the quality control template, and / or the signal-generating oligonucleotide to an amplification reaction comprises contacting the reagent composition with a processed sample to produce the amplification reaction mixture.

57. 57. The method of any one of claims 1 to 56, further comprising detecting a target nucleic acid sequence in the sample.

58. subjecting the target nucleic acid sequence to an amplification reaction capable of producing a nucleic acid amplification product; and 58. The method of any one of claims 1 to 57, comprising detecting the nucleic acid amplification product using a target signal-generating oligonucleotide, wherein the target signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product.

59. subjecting the target nucleic acid sequence to an amplification reaction capable of producing a nucleic acid amplification product, 59. The method of any one of claims 1 to 58, comprising amplifying the target nucleic acid sequence in the amplification reaction mixture under said amplification conditions, thereby producing a nucleic acid amplification product.

60. contacting a sample containing biological entities with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence; and contacting the reagent composition with the processed sample to produce the amplification reaction mixture, the reagent composition comprising one or more amplification reagents; 60. The method of any one of claims 1 to 59, comprising:

61. The one or more amplification reagents are reverse transcriptase; An enzyme having hyperthermophilic polymerase activity, optionally having reverse transcriptase activity. Forward primer; reverse primer; a reverse transcription primer; and / or dNTP 61. The method of any one of claims 1 to 60, comprising:

62. The method of any one of claims 1 to 61, wherein the sample nucleic acid comprises a nucleic acid comprising the target nucleic acid sequence.

63. The step of amplifying the target nucleic acid sequence comprises: The method includes a step of amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplifying step comprises amplifying a nucleic acid comprising the target nucleic acid sequence by: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) Enzymes with hyperthermophilic polymerase activity 63. The method of any one of claims 1 to 62, comprising contacting said nucleic acid amplification product with

64. The nucleic acid is double-stranded DNA; and / or 64. The method of any one of claims 1 to 63, wherein the nucleic acid is a product of a reverse transcription reaction, optionally wherein the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid, and further optionally, wherein the amplifying step comprises generating the nucleic acid by a reverse transcription reaction.

65. The amplification reaction is carried out for a period of about 5 minutes to about 60 minutes; amplifying the quality control template includes producing the first quality control product and / or the second quality control product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes; amplifying the target nucleic acid sequence comprises producing the nucleic acid amplification product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes; and / or 65. The method of any one of claims 1 to 64, wherein the detecting step is carried out in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition is contacted with the treated sample.

66. the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA; and / or 66. The method of any one of claims 1 to 65, wherein the pH of the lysis buffer is from about 1.0 to about 10.0, and optionally the pH of the lysis buffer is about 2.

2.

67. 67. The method of any one of claims 1 to 66, wherein the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid, optionally wherein the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof.

68. The reagent composition may be lyophilized, heat-dried, and / or contain one or more additives, the one or more additives being: Tween 20, Triton X-100 and / or tween 80; amino acid; sugars or sugar alcohols, optionally including sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or Optionally, a polymer including polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. and optionally, contacting the reagent composition with the processed sample comprises dissolving the reagent composition in the processed sample.

69. The one or more solubility reagents are Constituting from about 0.001% (wt / vol) to about 1.0% (wt / vol) of the processed sample, optionally about 0.2% (wt / vol) of the processed sample; and / or 69. The method of any one of claims 1 to 68, comprising a detergent, optionally wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant.

70. carried out in a single reaction vessel; does not involve the use of any enzymes other than the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity; does not involve the use of any enzyme other than the enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid and / or quality control template during the amplification step; and / or 70. The method of any one of claims 1 to 69, which does not include contacting the nucleic acid and / or quality control template with a signal-stranded DNA binding protein.

71. the target nucleic acid sequence comprises a length of about 20 nucleotides or less to about 90 nucleotides or less, and optionally, the target nucleic acid sequence comprises a length of about 30 nucleotides; the forward primer, the reverse primer and / or the reverse transcription primer are about 8 to 16 bases in length; the nucleic acid amplification product is about 20 to 40 bases in length; and / or 71. The method of any one of claims 1 to 70, wherein the spacer sequence comprises a portion of the target nucleic acid sequence, and optionally the spacer sequence is 1 to 10 bases in length.

72. 72. The method of any one of claims 1 to 71, wherein the isothermal amplification conditions comprise a constant temperature of about 30°C to about 72°C, further optionally about 55°C to about 75°C, optionally about 56°C to about 67°C.

73. The amplifying step includes: is carried out for a period of about 5 minutes to about 60 minutes, optionally wherein the amplifying step is for a period of about 15 minutes; and / or 73. The method of any one of claims 1 to 72, carried out under helicase-free, signal strand binding protein-free, cleavage agent-free and recombinase-free isothermal amplification conditions.

74. 74. The method of any one of claims 1 to 73, wherein the enzyme with 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; optionally, the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:1; further optionally, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:1; and optionally, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity.

75. The method of any one of claims 1 to 74, wherein the sample ribonucleic acid is contacted with the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity simultaneously; optionally, the sample ribonucleic acid is contacted with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, and the forward and reverse primers simultaneously; and further optionally, the sample ribonucleic acid is contacted with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer simultaneously.

76. The biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle; The biological entity comprises a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof; and / or 76. The method of any one of claims 1 to 75, wherein the target nucleic acid sequence is a viral, bacterial, fungal or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a virus, bacterium, fungus or protozoan.

77. the sample is a biological sample or an environmental sample; the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof; and / or 77. The method of any one of claims 1 to 76, wherein the biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, faeces, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

78. 78. The method of any one of claims 1 to 77, wherein the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally the two or more target nucleic acid sequences being specific to two or more different organisms, and further optionally the two or more different organisms comprising one or more of SARS-CoV-2, influenza A, influenza B and / or influenza C.

79. 79. The method of any one of claims 1 to 78, wherein the amplifying step comprises one or more of 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), optionally wherein the amplifying step does not comprise loop-mediated isothermal amplification (LAMP).

80. (ix) purifying the nucleic acid amplification product; (xi) removing the one or more lytic agents from the processed sample or the amplification reaction mixture; (xii) thermally and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the processed sample or the amplification reaction mixture.

81. The method, the reagent composition and / or the amplification reaction mixture comprises: a template capable of generating said first quality control product other than said quality control template; a probe capable of detecting said first quality control product other than said signal-generating oligonucleotide; a double-stranded template capable of generating said first quality control product; a linear template capable of generating said first quality control product; and / or a primer capable of hybridizing to the quality control template, the first quality control product, and / or the second quality control product other than the quality control primer The method of any one of claims 1 to 80, wherein the method does not include

82. 82. The method of any one of claims 1 to 81, comprising determining the presence, absence and / or amount of said first quality control product.

83. The presence, absence and / or amount of the signal the presence, absence, and / or amount of said first quality control product; the presence, absence, and / or amount of one or more interfering components in the amplification reaction mixture; (i) the integrity of the one or more amplification reagents in the amplification reaction mixture; (ii) a failure of the apparatus in which the amplification reaction is carried out; and / or (iii) sample-related inhibition of the amplification reaction, optionally including matrix-related inhibition; and / or The degree to which amplification of the target nucleic acid sequence is inhibited in the amplification reaction The method according to any one of claims 1 to 82, wherein

84. The method, the reagent composition and / or the amplification reaction mixture may be used to generate at least about 50,000 copies to about 10 12 84. The method of any one of claims 1 to 83, comprising about 20 copies of said quality control template, and optionally a comparable method of monitoring the amplification reaction uses about 20 copies to about 100 copies of an internal standard template.

85. 85. The method of any one of claims 1 to 84, wherein the method, the reagent composition and / or the amplification reaction mixture comprises at least about 1.1 times more copies of the quality control template and / or quality control primer compared to a comparable method of monitoring an amplification reaction that does not comprise the quality control template and / or the quality control primer, and optionally the comparable method comprises an internal standard template that is not capable of forming a hairpin structure.

86. 86. The method of any one of claims 1 to 85, wherein a comparable method of monitoring an amplification reaction that does not include the quality control template and / or the quality control primers inhibits the amplification of the target nucleic acid sequence and / or detection of nucleic acid amplification products by at least about 1.1 times more than the method of any one of claims 1 to 85, and optionally, the comparable method includes an internal control template that is not capable of forming a hairpin structure.

87. 87. The method of any one of claims 1 to 86, wherein the quality control template and / or the quality control primer is not capable of hybridizing to the target nucleic acid sequence.

88. 88. The method of any one of claims 1 to 87, wherein the presence of the quality control template and / or the quality control primers does not inhibit the amplification of the target nucleic acid sequence and / or detection of the nucleic acid amplification product.

89. 89. The method of any one of claims 1 to 88, wherein the presence of the quality control template and / or the quality control primer in the amplification reaction mixture improves the amplification of the target nucleic acid sequence and / or the detection of the nucleic acid amplification product by at least about 1.1 fold compared to a comparable method in which the quality control template and / or the quality control primer are absent from the amplification reaction mixture, and optionally the comparable method includes an internal control template that is not capable of forming a hairpin structure.

90. 90. The method of any one of claims 1 to 89, wherein the number of false priming events and / or generation of primer-dimers is reduced by at least about 1.1 fold compared to a comparable method of monitoring an amplification reaction that does not include said quality control template and / or said quality control primers, and optionally said comparable method includes an internal control template that is not capable of forming a hairpin structure.

91. 1. A kit for monitoring an amplification reaction, comprising: A quality control mold according to any one of claims 1 to 90; A quality control primer according to any one of claims 1 to 90; A signal-generating oligonucleotide according to any one of claims 1 to 90; and / or A supplemental quality control primer according to any one of claims 1 to 90. Kit including:

92. a lysis buffer comprising one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, said sample nucleic acids being suspected of containing a target nucleic acid sequence, optionally wherein said one or more lysis agents comprise a detergent, said detergent comprising one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant and an amphoteric surfactant; and / or A reagent composition comprising one or more amplification reagents containing one or more components for amplifying said target nucleic acid sequence under isothermal amplification conditions.

92. The kit of any one of claims 91, comprising:

93. The one or more components for amplification include: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, optionally having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof, optionally having an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO:1, and further optionally being a polymerase comprising the amino acid sequence of SEQ ID NO:

1.

93. The kit of claim 91 or 92, comprising:

94. 94. The kit of any one of claims 91 to 93, wherein the quality control template, the signal-generating oligonucleotide, the quality control primer, the auxiliary quality control primer and / or the one or more components for amplification are in lyophilized or freeze-dried form and / or are present in the reagent composition.

95. A quality control mold according to any one of claims 1 to 90; A quality control primer according to any one of claims 1 to 90; A signal-generating oligonucleotide according to any one of claims 1 to 90; A supplemental quality control primer according to any one of claims 1 to 90; a target nucleic acid sequence; and / or one or more additional primers and / or one or more probes specific to the target nucleic acid sequence; A reaction mixture comprising:

96. The one or more additional primers and / or one or more probes specific to the target nucleic acid sequence are a forward primer and / or a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or a target signal-generating oligonucleotide capable of hybridizing to a nucleic acid amplification product produced by amplifying said target nucleic acid sequence; 96. The reaction mixture of claim 95, comprising:

97. 97. The reaction mixture of claim 95 or 96, comprising one or more of an enzyme having polymerase activity, dNTPs, and a buffer, optionally wherein the enzyme is an enzyme having hyperthermophilic polymerase activity.

98. 98. The reaction mixture of any one of claims 95 to 97, wherein the enzyme with 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 with hyperthermophilic polymerase activity comprises the amino acid sequence of SEQ ID NO:1.

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