Hairpin internal control for isothermal nucleic acid amplification

Abstract

The disclosure herein includes methods, compositions, and kits for monitoring amplification reactions. Some embodiments include a quality control template comprising a 5'subdomain, a 3 'subdomain, and a ring domain located between the 5' subdomain and the 3 'subdomain. And paired stem domains can be formed through intramolecular nucleotide base pairing between the 5'subdomain and the 3 'subdomain. The quality control primer may be capable of hybridizing with at least a portion of the 3'subdomain, An amplification reaction of the quality control template and the quality control primer may generate a first quality control product, The method may include detecting the first quality control product.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 374,835, filed September 7, 2022, pursuant to 35 U.S. SC §119(e), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] References to sequence lists

[0004] This application is filed together with an electronic sequence list. The sequence list is provided as a file entitled 68EB-317350-WO, created on September 6, 2023, and is 23,044 bytes in size. The information in the electronic sequence list is incorporated herein by reference in its entirety.

[0005] background

[0006] field

[0007] This disclosure generally relates to methods and compositions for amplifying (e.g., isothermal amplification) nucleic acids.

[0008] Description of the prior art

[0009] Nucleic acid-based diagnostics can be used for the rapid detection of infections, diseases, and / or genetic variations. For example, the identification of bacterial or viral nucleic acids in a sample can be used to diagnose a specific type of infection. Other examples include the identification of single nucleotide polymorphisms for disease management or forensic science, and the identification of genetic variations in food products indicating genetic modifications. Typically, nucleic acid-based diagnostic assays require the amplification of a specific portion of the nucleic acid 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 steps of denaturation (e.g., separation of strands in a double-stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short strands of complementary DNA sequences), and extension of the primers along the complementary target by a polymerase. Such thermal cycling can be a time-consuming process that typically requires specialized facilities. Therefore, there is a need for faster nucleic acid amplification methods that do not require thermal cycling. Such methods can be used, for example, in field testing and point-of-care diagnostics. In particular, there is a need for faster compositions and methods for nucleic acid detection in which internal controls (“IC”) do not interfere with target amplification efficiency. Two main IC strategies are employed in nucleic acid detection assays: competitive IC methods and non-competitive IC methods. The difference lies in whether the ICs share a common set of primers for both IC and target amplification. With a competitive IC strategy, there is always some competition between the target and IC, where target and IC fragments with the same primers on their flanks are amplified simultaneously. Competition for IC amplification can reduce target amplification efficiency and thus lead to a lower detection limit. Therefore, competitive IC methods require more IC optimization to achieve a sensitive detection limit. In non-competitive methods, the target and IC each use different primer sets for amplification. While the kinetics of each reaction are not affected by primer competition, IC amplification must be limited by controlled concentrations of IC-specific primers and / or IC templates to limit competition for primers and DNA polymerase between the target and IC reactions. Therefore, 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 that offer reduced assay complexity and reduced undesirable interactions between IC and target amplification.

[0010] Overview

[0011] This document discloses methods for monitoring amplification reactions. In some embodiments, the method includes providing a quality control template comprising: a 5' subdomain; a 3' subdomain; and a loop domain located between the 5' and 3' subdomains, wherein intramolecular nucleotide base pairing between the 5' and 3' subdomains is capable of forming a paired stem domain. In some embodiments, the method includes providing quality control primers capable of hybridizing with at least a portion of the 3' subdomain. In some embodiments, the method includes performing an amplification reaction on the quality control template and the quality control primers to generate 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, performing an amplification reaction on the quality control template and the quality control primers to generate a first quality control product includes amplifying the quality control template with the quality control primers in an amplification reaction mixture under amplification conditions to generate the first quality control product. In some embodiments, the amplification reaction includes a reverse transcription reaction.

[0012] In some embodiments, the method further includes: providing an enzyme with polymerase activity (e.g., an enzyme with hyperthermophilic biopolymerase activity); and / or providing reverse transcriptase. In some embodiments, the enzyme with hyperthermophilic biopolymerase activity has reverse transcriptase activity. In some embodiments, the amplification reaction includes: contacting a quality control primer with a quality control template for hybridization, and extending the quality control primer hybridized with the quality control template with an enzyme with polymerase activity, thereby generating a first quality control product. In some embodiments, the amplification reaction includes: contacting a quality control primer with the first quality control product for hybridization, and extending the quality control primer hybridized with the first quality control product with an enzyme with polymerase activity, thereby generating a second quality control product. In some embodiments, the amplification reaction includes: contacting a quality control primer with the second quality control product for hybridization, and extending the quality control primer hybridized with the second quality control product with an enzyme with polymerase activity, thereby generating a first quality control product. In some embodiments, the first and second quality control products comprise a 5' subdomain and a 3' subdomain capable of forming a paired stem domain. In some embodiments, the first and second quality control products have the same stem domain. In some embodiments, the first and second quality control products comprise complementary loop domains. In some embodiments, the amplification reaction includes linear amplification and / or exponential amplification of the first and second quality control products. 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 and second quality control products are capable of forming hairpin structures. In some embodiments, the quality control template comprises a 5' terminal domain located at the 5' of the 5' subdomain, and / or the quality control template comprises a 3' terminal domain located at the 3' of the 3' subdomain. In 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 the quality control primer.

[0013] Detection of the first quality control product may include detecting the first quality control product using a signal-generating oligonucleotide. In some embodiments, the signal-generating oligonucleotide is capable of hybridizing with the first quality control product. In some embodiments, detection includes contacting the first quality control product with the signal-generating oligonucleotide for hybridization. In some embodiments, the signal-generating oligonucleotide comprises a quencher, a label, or both. 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 quencher is capable of quenching the label. In some embodiments, detection 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 when the signal-generating oligonucleotide hybridizes with the first quality control product. In some embodiments, the label generates a signal when the signal-generating oligonucleotide hybridizes with the first quality control product. In some embodiments, the signal is fluorescence. In some embodiments, detection of the first quality control product includes detecting the signal generated by the label of the signal-generating oligonucleotide. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, detection includes detecting the signal of the label before, during, after, or any combination thereof.

[0014] In some embodiments, the method further includes: providing a signal-generating oligonucleotide; performing an amplification reaction on the signal-generating oligonucleotide; and detecting a first quality control product using the signal-generating oligonucleotide. In some embodiments, the quality control template is the signal-generating oligonucleotide. In some embodiments, the quality control template is (i) a template for the synthesis of the first quality control product, and (ii) 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.

[0015] In some embodiments, the 5' end 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 contain a 3' end 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, reverse transcriptase is capable of using one or more RNA nucleotides from the 5' end 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 reverse transcriptase with the quality control template to generate an extended quality control template. In some embodiments, the extended quality control template comprises cDNA. In some embodiments, the amplification reaction comprises: 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 reverse transcriptase and / or an enzyme with polymerase activity, thereby generating a first quality control product.

[0016] In some embodiments, the quality control template is a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide contains a label, and wherein the loop domain contains one or more RNA nucleotides. In some embodiments, the label contains a quenchable label (e.g., a fluorophore). In some embodiments, the signal-generating oligonucleotide contains a quencher. In some embodiments, the label is located in a 3' terminal domain and the quencher is located in a 5' terminal domain, and / or the label is located in a 5' terminal domain and the quencher is located in a 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 reverse transcriptase, thereby generating a first quality control product. In some embodiments, the reverse transcriptase contains RNase H activity. In some embodiments, the reverse transcriptase cleaves the quality control template at one or more RNA nucleotides during the generation of the first quality control product, thereby generating a first cleavage product and a second cleavage product containing the label. In some embodiments, detecting the first quality control product includes detecting a signal generated by the first cleavage product containing the label. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some implementations, the method further includes: providing supplemental quality control primers; and performing an amplification reaction on the supplemental quality control primers.

[0017] 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 supplemental quality control primer are 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 contain one or more phosphate-thioester bonds 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.

[0018] The signal-generating oligonucleotide may contain one or more LNAs. In some embodiments, one or more LNAs are located within a loop domain (e.g., one or more LNAs enhance the detectability of the first quality control product) and / or a stem loop. In some embodiments, the signal-generating oligonucleotide is configured such that the melting temperature (Tm) of the first quality control product / signal-generating oligonucleotide duplex is equal to or greater than the melting temperature (Tm) of the paired stem domain of the signal-generating oligonucleotide (e.g., via one or more LNAs located within the loop domain).

[0019] In some embodiments, the signal-generating oligonucleotide does not contain a dye capable of quenching the label. In some embodiments, the signal-generating oligonucleotide does not contain a quenchable portion other than the nucleotide of the signal-generating oligonucleotide. In some embodiments, the 5' terminal domain of the quality control template and / or the signal-generating oligonucleotide contains a label. In some embodiments, the 5' terminal domain and / or 5' subdomain of the quality control template and / or the signal-generating oligonucleotide contains one or more cytosine bases. In some embodiments, the 3' terminal domain and / or 3' subdomain of the quality control template and / or the signal-generating oligonucleotide contains one or more guanine bases and / or adenine bases. In some embodiments, one or more guanine bases and / or adenine bases are capable of quenching the label when 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 contains a label. In some embodiments, the 3' terminal domain and / or 3' subdomain of the quality control template and / or signal-generating oligonucleotide contains 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 contains one or more guanine bases and / or adenine bases. In some embodiments, one or more guanine bases and / or adenine bases are capable of quenching the label when the quality control template and / or signal-generating oligonucleotide forms a hairpin structure.

[0020] In some embodiments, detecting the first quality control product includes 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 fluorescence. In some embodiments, the generation of the first and second quality control products is correlated with the detected decrease in signal. In some embodiments, the 5' end of the quality control primer contains a label. In some embodiments, the quality control primer contains one or more pyrimidine bases adjacent to the label. In some embodiments, the quality control primer contains 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 contains one or more guanine bases and / or adenine bases. In some embodiments, when 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, one or more guanine bases and / or adenine bases present in the 3' terminal domain and / or 3' subdomain of the first quality control product are quenched when the first quality control product forms a hairpin structure. In some embodiments, when the quality control primer binds to the first quality control product and is extended by an enzyme with polymerase activity to generate the second quality control product, one or more guanine bases and / or adenine bases present in the 3' terminal domain and / or 3' subdomain of the second quality control product are quenched when the second quality control product forms a hairpin structure. In some embodiments, detecting the first quality control product includes contacting the first quality control product with a fluorescent dye.

[0021] In some embodiments, providing quality control primers, quality control templates, and / or signal-generating oligonucleotides includes providing a reagent composition comprising quality control primers, quality control templates, and / or signal-generating oligonucleotides. In some embodiments, performing an amplification reaction with the quality control primers, quality control templates, and / or signal-generating oligonucleotides includes contacting the reagent composition with a treated sample to generate an amplification reaction mixture. In some embodiments, the method further includes detecting a target nucleic acid sequence in the sample. In some embodiments, the method includes performing an amplification reaction on the target nucleic acid sequence capable of generating nucleic acid amplification products. In some embodiments, the method includes detecting nucleic acid amplification products with target signal-generating oligonucleotides, wherein the target signal-generating oligonucleotides are capable of hybridizing with the nucleic acid amplification products. In some embodiments, performing an amplification reaction on the target nucleic acid sequence capable of generating nucleic acid amplification products includes amplifying the target nucleic acid sequence in an amplification reaction mixture under amplification conditions to generate nucleic acid amplification products.

[0022] The method may include: contacting a sample containing a biological entity with a lysis buffer to generate a treated sample, wherein the lysis buffer contains one or more lysis agents capable of lysing the biological entity to release sample nucleic acids contained therein, and wherein the sample nucleic acids are suspected of containing a target nucleic acid sequence. The method may also include: contacting a reagent composition with the treated sample to generate an amplification reaction mixture, wherein the reagent composition contains one or more amplification reagents. In some embodiments, one or more amplification reagents contain: reverse transcriptase; an enzyme having hyperthermophilic polymerase activity (e.g., an enzyme having both 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 containing a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence comprises: amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplification comprises contacting the nucleic acid containing the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with the sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic biopolymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the nucleic acid is: double-stranded DNA; and / or a product of a reverse transcription reaction. In some embodiments, the nucleic acid is a product of a reverse transcription reaction generated from sample ribonucleic acid (e.g., amplification comprises generating nucleic acid via a reverse transcription reaction).

[0023] In some embodiments, the amplification reaction proceeds over a period of approximately 5 minutes to approximately 60 minutes. In some embodiments, the amplification quality control template includes the generation of a first quality control product and / or a second quality control product at a detectable level within approximately 20 minutes, approximately 15 minutes, or approximately 10 minutes. In some embodiments, the amplification of the target nucleic acid sequence includes the generation of a nucleic acid amplification product at a detectable level within approximately 20 minutes, approximately 15 minutes, or approximately 10 minutes. In some embodiments, the detection is performed within less than approximately 20 minutes, less than approximately 15 minutes, less than approximately 10 minutes, less than approximately 5 minutes, or less than approximately 2 minutes from the time the reagent composition comes into contact 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 approximately 1.0 to approximately 10.0. In some embodiments, the pH of the lysis buffer is approximately 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.

[0024] In some embodiments, the reagent composition is lyophilized, heat-dried, and / or contains one or more additives, wherein the one or more additives include: Tween 20, Triton X-100, and / or Tween 80; amino acids; sugars or sugar alcohols (e.g., sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof); and / or polymers (e.g., polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, peptides, collagen peptides, or any combination thereof). In some embodiments, contacting the reagent composition with the treated sample includes dissolving the reagent composition in the treated sample. In some embodiments, one or more lysis reagents comprise: about 0.001% (w / v) to about 1.0% (w / v) of the treated sample (e.g., about 0.2% (w / v)); and / or a detergent (e.g., a cationic surfactant, anionic surfactant, nonionic surfactant, amphoteric surfactant). In some embodiments, the method is: carried out in a single reaction vessel; does not include the use of any enzymes other than reverse transcriptase and enzymes with hyperthermophilic biopolymerase activity; does not include the use of any enzymes other than enzymes with hyperthermophilic biopolymerase activity; does not include thermal denaturation and / or enzymatic denaturation of nucleic acids and / or quality control templates during the amplification step; and / or does not include contacting nucleic acids and / or quality control templates with single-stranded DNA-binding proteins.

[0025] In some embodiments, the target nucleic acid sequence comprises a length of no more than about 20 nucleotides and no more than about 90 nucleotides. 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 long. In some embodiments, the nucleic acid amplification product is about 20 to 40 bases long. In some embodiments, the spacer region sequence comprises a portion of the target nucleic acid sequence. In some embodiments, the spacer region sequence is 1 to 10 bases long. In some embodiments, the isothermal amplification conditions include 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 amplification is performed for a period of about 5 minutes to about 60 minutes (e.g., about 15 minutes); and / or under isothermal amplification conditions free of helicase, single-strand binding proteins, cleavage agent-free, and recombinase.

[0026] In some embodiments, the enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof. In some embodiments, the enzyme having 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 having hyperthermophilic polymerase activity has low exonuclease activity or no exonuclease activity. In some embodiments, the sample ribonucleic acid is simultaneously contacted with reverse transcriptase and the enzyme having hyperthermophilic polymerase activity. In some embodiments, the sample ribonucleic acid is simultaneously contacted with reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, and forward and reverse primers. In some embodiments, the sample ribonucleic acid is simultaneously contacted with reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, forward primers, reverse primers, and reverse transcription primers.

[0027] In some embodiments, the biological entity includes one or more of prokaryotic cells, eukaryotic cells, viral particles, exosomes, protoplasts, and microvesicles. In some embodiments, the biological entity includes viruses, bacteria, fungi, protozoa, portions thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, bacteria, fungi, or protozoa. In some embodiments, the sample nucleic acid is derived from a virus, bacteria, fungi, or protozoa. In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the environmental sample is, or is obtained from, the following: food samples, beverage samples, paper surfaces, fabric surfaces, metal surfaces, wood surfaces, plastic surfaces, soil samples, freshwater samples, wastewater samples, saline samples, samples exposed to atmospheric air or other gases, their cultures, or any combination thereof. In some embodiments, the biological sample is, or is obtained from, the following: tissue samples, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, swabs from skin or mucous membrane surfaces, their cultures, or any combination thereof.

[0028] In some embodiments, the amplification includes multiplex amplification of two or more target nucleic acid sequences, and the detection step includes multiplex detection of two or more nucleic acid amplification products derived from said 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 virus, influenza B virus, and / or influenza C virus). In some embodiments, amplification includes one or more of the following: archaea polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand substitution amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple substitution amplification (MDA), branching amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustaining sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple substitution amplification (IMDA). In some embodiments, amplification does not include loop-mediated isothermal amplification (LAMP). In some embodiments, the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) addition of a ribonuclease inhibitor to the treated sample; (vii) addition of a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more lysis agents from the treated sample or the amplification reaction mixture; (xii) thermal denaturation and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0029] In some embodiments, the method, reagent composition, and / or amplification reaction mixture do not contain: a template other than a quality control template capable of generating a first quality control product; a probe other than a signal-generating oligonucleotide capable of detecting the first quality control product; a double-stranded template capable of generating the first quality control product; a linear template capable of generating the first quality control product; and / or primers other than quality control primers capable of hybridizing with the quality control template, the first quality control product, and / or the second quality control product.

[0030] 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 malfunction of the instrument in which the amplification reaction is performed; and / or (iii) inhibition of the sample source for the amplification reaction. In some embodiments, inhibition of the sample source includes inhibition of the matrix source. In some embodiments, the presence, absence, and / or amount of a signal indicates the degree to which the amplification of the target nucleic acid sequence is inhibited in the amplification reaction.

[0031] In some embodiments, the method, reagent composition, and / or amplification reaction mixture contains at least about 50,000 copies to about 500,000 copies of a quality control template. In some embodiments, a comparable method for monitoring the amplification reaction employs an internal control template of about 20 copies to about 100 copies. In some embodiments, the method, reagent composition, and / or amplification reaction mixture contains at least about 1.1 times more copies of a quality control template and / or quality control primers than a comparable method for monitoring an amplification reaction that does not contain a quality control template and / or quality control primers. In some embodiments, the comparable method includes an internal control template that cannot form a hairpin structure. In some embodiments, a comparable method for monitoring an amplification reaction that does not contain a quality control template and / or quality control primers inhibits the amplification of the target nucleic acid sequence and / or the detection of nucleic acid amplification products at least about 1.1 times more than the methods disclosed herein. In some embodiments, the quality control template and / or quality control primers are unable to hybridize with the target nucleic acid sequence. In some embodiments, the presence of the quality control template and / or quality control primers does not inhibit the amplification of the target nucleic acid sequence and / or the detection of nucleic acid amplification products. In some embodiments, the presence of a quality control template and / or quality control primers in the amplification reaction mixture improves the amplification of the target nucleic acid sequence and / or the detection of nucleic acid amplification products by at least about 1.1 times compared to comparable methods in which the amplification reaction mixture does not contain a quality control template and / or quality control primers. In some embodiments, the number of erroneous priming events and / or primer dimer generation is reduced by at least about 1.1 times compared to comparable methods for monitoring amplification reactions that do not contain a quality control template and / or quality control primers.

[0032] This document discloses kits for monitoring amplification reactions. In some embodiments, the kit includes: the quality control template disclosed herein; the quality control primers disclosed herein; the signal-generating oligonucleotides disclosed herein; and / or supplementary quality control primers disclosed herein.

[0033] The kit may contain: a lysis buffer comprising one or more lysis agents capable of lysing a biological entity to release sample nucleic acids contained therein, wherein the sample nucleic acids are suspected of containing a target nucleic acid sequence. In some embodiments, the one or more lysis agents comprise a detergent, and the detergent comprises one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. The kit may contain: a reagent composition comprising one or more amplification reagents comprising 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 comprise: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing with a sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with a sequence of the second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic biopolymerase activity capable of generating nucleic acid amplification products. In some embodiments, the enzyme having hyperthermophilic biopolymerase 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 having hyperthermophilic biopolymerase 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 having hyperthermophilic biopolymerase activity is a polymerase containing the amino acid sequence of SEQ ID NO:1. In some embodiments, the quality control template, signal generation oligonucleotide, quality control primer, supplemental 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.

[0034] The contents disclosed herein include reaction mixtures. In some embodiments, the reaction mixture comprises: the quality control template disclosed herein; the quality control primers disclosed herein; the signal-generating oligonucleotides disclosed herein; supplementary quality control primers 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.

[0035] In some embodiments, one or more additional primers and / or one or more probes specific to the target nucleic acid sequence comprise: a forward primer and / or a reverse primer, wherein the forward primer is capable of hybridizing with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with the sequence of the second strand of the target nucleic acid sequence; and / or a target signal capable of hybridizing with a nucleic acid amplification product generated by amplifying the target nucleic acid sequence to generate an oligonucleotide. The reaction mixture may comprise: an enzyme having polymerase activity, dNTPs, and a buffer, or one or more thereof. In some embodiments, the enzyme is an enzyme having hyperthermophilic biopolymerase activity. In some embodiments, the enzyme having hyperthermophilic biopolymerase 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. Brief description of the attached diagram

[0037] Figure 1 Non-limiting exemplary embodiments of the DNA hairpin internal control assay disclosed herein are described. IC primer, internal control primer; HpIC1 MB2, hairpin internal control molecular beacon; HpIC1 P1, hairpin internal control product 1; HpIC1 P2, hairpin internal control product 2.

[0038] Figure 2 A non-limiting exemplary embodiment of the thermal setup for hairpin IC (HPIC) isothermal amplification reaction described herein is depicted.

[0039] Figures 3A-3B The sample shown is compared with one containing 10% normal urine. Figure 3B Compared to clean samples, Figure 3A Data related to the detection of target Neisseria gonorrhoeae (Ng) (ROX) and hairpin IC detection (HEX) in [the study]. NTC, no target control; cp, copy. Molecular beacon IC (HPIC) was detected via HEX fluorophore, and the target (Ng) was detected via ROX fluorophore.

[0040] Figures 4A-4D Data related to the detection of target gonococcus (Ng)(ROX) and hairpin IC detection (HEX) are presented. Figure 4A The reaction of Ng alone was shown, and Figures 4B-4D The response with increasing levels of IC template (ICT) was described: 0ICT( Figure 4B ), 50k ICT Figure 4C ) and 500k ICT ( Figure 4D ). Figure 4DThe star-shaped indicator indicates that air bubbles are present in the pipette tip for this sample. NTC, no target control; cp, copy. Molecular beacon IC (HPIC) was detected via HEX fluorophore, and the target (Ng) was detected via ROX fluorophore.

[0041] Figures 5A-5C Data related to the detection of target gonococci (Ng)(ROX) and hairpin IC detection (HEX) in samples with increased levels of inhibitory urine are shown: 10% ( Figure 5A ), 20% Figure 5B ) and 30% Figure 5C NTC, no target control; cp, copy. Molecular beacon IC (HPIC) was detected via HEX fluorophore, and the target (Ng) was detected via ROX fluorophore.

[0042] Figure 6A Data related to the detection of Ng in normal urine in the absence of an internal control were presented. An Ng ROX probe was used for target detection, and a Syto 82 intercalation dye was used for amplification product detection. Figure 6B Data related to the detection of Ng / hairpin IC in normal urine are presented, wherein the hairpin IC probe (HPIC) is detected by HEX fluorophore and the target (Ng) is detected by ROX fluorophore.

[0043] Figures 7A-7B Data comparing the detection of target gonococci (Ng) (ROX) and the hairpin IC detection (HEX) in reactions using hairpin IC from different batches are presented: 9.14 ( Figure 7A ) and 10.12 Figure 7B NTC, no target control; cp, copy. Molecular beacon IC (HPIC) was detected via HEX fluorophore, and the target (Ng) was detected via ROX fluorophore.

[0044] Figures 8A-8B A non-limiting exemplary schematic diagram of the isothermal amplification reaction provided herein is shown.

[0045] Figures 9A-9F The description depicts the interaction with molecular beacons HpIC1b MB1 and HpIC1b MB2 ( Figures 9A-9C ) and intercalating dyes ( Figures 9D-9F Data related to the detection of the amplified internal control. The hairpin-shaped internal control target was amplified in the APA reaction for 10 minutes, while simultaneously detected by the probe ( Figure 9A ) and intercalating dyes ( Figure 9DThe detection was performed. A hairpin-shaped internal control was labeled with HEX at the 5' end and with IBFQ at the 3' end. In some such embodiments, it serves as both an internal control target and a detection probe. After the reaction, the reaction temperature was immediately increased from the assay temperature to 90°C for melt-curve analysis. Figure 9B and Figure 9E ) and chain-resolver derivative evaluation ( Figure 9C and Figure 9F ). Detailed Implementation

[0046] The following detailed description incorporates reference to the accompanying drawings, which form part of this document. In the drawings, like reference numerals generally identify like components unless the context otherwise indicates. The illustrative embodiments described in the detailed description, drawings, and claims are not intended 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 aspects of this disclosure as generally described herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form part of this disclosure.

[0047] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned in this article concerning relevant technologies are incorporated herein by reference in their entirety.

[0048] Unless otherwise defined, the 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 pertains. See, for example, 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 the purposes of this disclosure, the following terms are defined below.

[0049] This document discloses methods for monitoring amplification reactions. In some embodiments, the method includes providing a quality control template comprising: a 5' subdomain; a 3' subdomain; and a loop domain located between the 5' and 3' subdomains, wherein intramolecular nucleotide base pairing between the 5' and 3' subdomains is capable of forming a paired stem domain. In some embodiments, the method includes providing quality control primers capable of hybridizing with at least a portion of the 3' subdomain. In some embodiments, the method includes performing an amplification reaction on the quality control template and the quality control primers to generate 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, performing an amplification reaction on the quality control template and the quality control primers to generate a first quality control product includes amplifying the quality control template with the quality control primers in an amplification reaction mixture under amplification conditions to generate the first quality control product. In some embodiments, the amplification reaction includes a reverse transcription reaction.

[0050] This document discloses kits for monitoring amplification reactions. In some embodiments, the kit includes: the quality control template disclosed herein; the quality control primers disclosed herein; the signal-generating oligonucleotides disclosed herein; and / or supplementary quality control primers disclosed herein.

[0051] The contents disclosed herein include reaction mixtures. In some embodiments, the reaction mixture comprises: the quality control template disclosed herein; the quality control primers disclosed herein; the signal-generating oligonucleotides disclosed herein; supplementary quality control primers 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.

[0052] Hair clip interior comparison

[0053] The disclosures provided herein include methods, compositions, kits, and reaction mixtures that, in some embodiments, utilize archaea polymerase amplification (“APA”) to isothermally amplify regions of interest within a target nucleotide template for real-time analyte detection, while simultaneously monitoring or evaluating the amplification reaction (e.g., internal control (“IC”) assays). The methods, compositions, reaction mixtures, and kits provided herein overcome the challenges posed by competition for target amplification and address the aforementioned needs in the art by leveraging the stability of hairpin structures to reduce nonspecific interactions with primary target amplification. Advantages of the hairpin IC systems, methods, compositions, reaction mixtures, and kits provided herein may include reduced assay complexity and reduced undesirable interactions with target amplification. Surprisingly, in some embodiments, when double-stranded with a hairpin IC, target amplification exhibits improved low-copy detection compared to target-only assays.

[0054] This document discloses methods, compositions, reaction mixtures, and kits for internal control assays that can be double-stranded with a specific target assay and can report the integrity of core reagents, instrument malfunctions, and / or sample inhibition during the reaction in the absence of a specific signal from target amplification. In some embodiments, the internal control assay is designed to utilize APA to simultaneously amplify the DNA target and the internal control template for real-time detection under isothermal conditions.

[0055] Disclosed internal control methods can achieve simultaneous amplification of a specific target and an internal control (IC) by utilizing the structural stability of stem-loop hairpins. Unlike currently available IC methods that use linear DNA as an IC template, some embodiments of the methods and compositions provided herein use hairpin-shaped templates (e.g., quality control templates). Due to the complementary nature of the hairpin stem, in some embodiments, amplification of the hairpin IC target requires only a single primer complementary to the 3' end of the stem. The IC primer (e.g., a quality control primer) can be extended onto the hairpin template to generate a first round of hairpin products (e.g., a first quality control product) complementary to the IC template. Subsequent repeated extensions of the IC template driven by a single IC primer can generate two hairpin products having a stem structure and a complementary loop sequence. The generated IC products can be detected by signal-generating oligonucleotides (e.g., probes, molecular beacons). In some embodiments, the signal-generating oligonucleotides (e.g., molecular beacons) are modified with LNA to enhance the detectability of the hairpin products. In some embodiments, the hairpin template can be labeled with a signal-generating portion and function as both a template and a detection probe. In some implementations, an internal DNA control is provided, which includes IC primers and hairpin probes that also serve as IC templates.

[0056] The IC method described herein can be extended to RNA IC assays comprising IC primers, signal-generating oligonucleotides (e.g., molecular beacons), and / or hairpin-shaped IC templates. The IC template may contain an RNA segment at the 5' end and a hairpin-shaped DNA segment with RT primer sequences at the 3' end in the stem region. In the presence of reverse transcriptase, the RT primer in the hairpin's stem region can be extended over the RNA segment of the IC template, generating cDNA that is also complementary to the IC primers. Subsequent amplification driven by a single IC primer can generate a hairpin-shaped product complementary in the loop region, which can be detected by the signal-generating oligonucleotide (e.g., molecular beacon). In some embodiments, the signal-generating oligonucleotide (e.g., molecular beacon) is modified with LNA.

[0057] The methods, compositions, reaction mixtures, and kits disclosed herein advantageously employ a hairpin-based IC approach, which allows for 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 hindering target amplification, for example, at 50,000 to 500,000 copies, compared to 20-100 copies of the IC target commonly used in other IC systems to reduce competition with target amplification. In some embodiments provided herein, where the hairpin IC probe is also used as a quality control template, the copy number can be even higher (e.g., in the concentration range of 25 nmol / L to 50 nmol / L, or >10). 12 (One copy). In some implementations, only a single primer and a hairpin oligonucleotide partially labeled with a signal generation component are required, which functions as both a template and a detection probe. High concentrations of hairpin oligonucleotides can be used without affecting target amplification. Not bound by any particular theory, fewer primer-dimers and mis-priming can occur due to the use of a single primer and hairpin-shaped template and product.

[0058] In some embodiments, the internal control assay comprises a single primer for detecting the amplified hairpin product, a hairpin-shaped internal control template, and / or a signal-generating oligonucleotide (e.g., a molecular beacon). In some embodiments, the internal control assay comprises a single primer and a signal-generating oligonucleotide (e.g., a molecular beacon) that functions as both a template and a detector.

[0059] In some embodiments, the signal-generating oligonucleotide (e.g., a molecular beacon) contains a fluorophore at its 5' end and a quencher at its 3' end. In some embodiments, the signal-generating oligonucleotide (e.g., a molecular beacon) can be replaced by a hairpin-shaped DNA probe without a quencher to detect hybridization with hairpin amplification products. In the absence of IC amplification, the fluorescence of a hairpin probe with a fluorophore attached at its 5' end can be quenched by guanine and / or adenine bases in the complementary stem. Not bound by any particular theory, conformational rearrangement can occur during hybridization, leading to an increase in fluorescence intensity.

[0060] The fundamental technical principles underlying the design of the hairpin-based internal control presented herein can include the nearest neighbor (NN) theory for predicting DNA thermodynamics using energy values ​​and the secondary structure kinetics of DNA hairpins. Unbound by any particular theory, the mechanism of hairpin amplification and its advantages as an internal control can be related to the thermodynamics of the hairpin stem-loop structure.

[0061] Further embodiments of the methods and compositions provided herein include probe-free forms based on hairpin-based internal controls. In this method, a single IC primer labeled with a fluorophore at its 5' end and a hairpin template can be the only two components necessary 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 can be copied exponentially to produce a hairpin product, wherein the 5' fluorophore is quenched due to the proximity of the guanine base via photoinduced electron transfer. In some embodiments, a single IC primer labeled with a fluorophore at its 5' end can be the sole component necessary 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 copied exponentially to produce a palindromic hairpin product, wherein the 5' fluorophore is quenched due to the proximity of the guanine base via photoinduced electron transfer. In this single-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.

[0062] Three-component IC assay

[0063] In some embodiments, RNA hairpin IC assays include IC primers, signal-generating oligonucleotides (e.g., molecular beacons), and / or hairpin-shaped IC templates. The IC template may contain an RNA segment at its 5' end and a hairpin-shaped DNA segment with RT primer sequences at its 3' end in the stem region. In the presence of reverse transcriptase, the RT primer in the hairpin's stem region can extend over the RNA segment of the IC template, generating cDNA that is also complementary to the IC primer. Not bound by any particular theory, subsequent amplification can be entirely driven by a single IC primer, forming a pan-handle-shaped product. For ease of amplification and detection, the product hybridization melting temperature (Tm) can be designed to be greater than or equal to the product hairpin Tm. In some embodiments, signal-generating oligonucleotides (e.g., molecular beacons) modified with locked nucleic acids (LNAs) in the spacer region can be used for IC product detection. In some embodiments, fluorescent dyes can be used for the detection of amplified IC products.

[0064] DNA hairpin IC assays may comprise a hairpin-shaped IC template, a single IC primer, and / or a beacon. In some embodiments, the IC primer may be extended onto the IC template to generate two complementary "pot-and-handle" shaped products in the loop (spacer) region. Not bound by any particular theory, subsequent repeated extensions of the IC primer onto the pot-and-handle products can generate exponential amplification. For ease of amplification and detection, the product hybridization Tm may be designed to be greater than or equal to the product hairpin Tm. In some embodiments, signal-generating oligonucleotides modified with LNA (e.g., molecular beacons) may be used for IC product detection. In some embodiments, fluorescent dyes may be used for the detection of amplified IC products.

[0065] In some embodiments, the disclosed hairpin IC method can provide reduced primer-related interactions with the target being measured. In some embodiments, only three IC amplification components are required: primers for DNA or RNA IC assays, a template, and a probe (e.g., a molecular beacon). In the RNA IC assays provided herein, RT primers can be embedded in a chimeric IC template. In some embodiments, a combination of low IC primer concentrations and the formation of a "pot-handle" structure can further reduce non-specific interactions with the target being measured.

[0066] Two-component IC assay

[0067] In some implementations, the hairpin IC method comprises a single IC primer and a signal-generating oligonucleotide (e.g., a molecular beacon) for both IC amplification and detection. Figure 1The sequence of the signal-generating oligonucleotide (e.g., a molecular beacon) may include part or all of the IC primer, the spacer region of the IC template, and 4-5 nucleotides adjacent to the IC spacer region and complementary to the 3' end of the IC primer. The IC primer can hybridize with the beacon and generate a first-round extension product whose 3' end is complementary to the IC primer. Subsequent extensions of the IC primer can generate a "pot-handle" shaped internal control product. Not bound by any particular theory, subsequent repeated extensions of the IC primer on the pot-handle product can be driven entirely by a single IC primer. The generated IC product can be detected by using an IC beacon modified with, for example, LNA to enhance the detectability of the pot-handle product.

[0068] The hairpin-based two-component IC method described herein can be extended to RNA IC assays. In this case, a signal-generating oligonucleotide (e.g., a molecular beacon) can contain some RNA bases in a loop region. RT primers can extend along the RNA bases in the beacon to generate cDNA. During cDNA synthesis, the RNA bases in the loop region can be degraded by the RNase H activity of reverse transcriptase, resulting in the release of a fluorescent signal. Depending on the beacon design, in some embodiments, IC primers can be used to improve exponential amplification.

[0069] Advantageously, compared to alternative methods for assay monitoring, this method can have minimal primer-related interactions with the target being measured. In some embodiments, the DNA IC assay has only two IC components: primers for IC amplification and detection, both serving as internal controls, and a hairpin probe (e.g., a beacon). For the two-component RNA IC assay provided herein, the RT primers and the signal-generating oligonucleotide containing the RNA segment in the loop region (e.g., a molecular beacon) can be the minimal components, with optional IC primers used in some embodiments.

[0070] Methods for monitoring amplification reactions

[0071] This document discloses methods for monitoring amplification reactions. In some embodiments, the method includes providing a quality control template comprising: a 5' subdomain; a 3' subdomain; and a loop domain located between the 5' and 3' subdomains, wherein intramolecular nucleotide base pairing between the 5' and 3' subdomains is capable of forming a paired stem domain. In some embodiments, the method includes providing quality control primers capable of hybridizing with at least a portion of the 3' subdomain. In some embodiments, the method includes performing an amplification reaction on the quality control template and the quality control primers to generate 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, performing an amplification reaction on the quality control template and the quality control primers to generate a first quality control product includes amplifying the quality control template with the quality control primers in an amplification reaction mixture under amplification conditions to generate the first quality control product. The amplification reaction may include a reverse transcription reaction.

[0072] The method may include: providing an enzyme with polymerase activity (e.g., an enzyme with hyperthermophilic biopolymerase activity). The method may include: providing a reverse transcriptase. In some embodiments, the enzyme with hyperthermophilic biopolymerase activity has reverse transcriptase activity. The amplification reaction may include: contacting a quality control primer with a quality control template for hybridization, and extending the quality control primer hybridized with the quality control template using an enzyme with polymerase activity, thereby generating a first quality control product. The amplification reaction may include: contacting a quality control primer with the first quality control product for hybridization, and extending the quality control primer hybridized with the first quality control product using an enzyme with polymerase activity, thereby generating a second quality control product. The amplification reaction may include: contacting a quality control primer with the second quality control product for hybridization, and extending the quality control primer hybridized with the second quality control product using an enzyme with polymerase activity, thereby generating a first quality control product. The first and second quality control products may contain a 5' subdomain and a 3' subdomain capable of forming a paired stem domain. In some embodiments, the first and second quality control products have the same stem domain. The first and second quality control products may contain complementary loop domains. The amplification reaction may include linear and / or exponential amplification of the first and second quality control products. The 5' subdomain may contain at least a portion of the sequence of the quality control primer. Both the first and second quality control products may be able to form hairpin structures. As used herein, the term "hairpin structure" should be given its common meaning and should also refer to a double-helix region formed by base pairing between adjacent, reversed, complementary sequences in a single strand of RNA or DNA. The quality control template may contain a 5' terminal domain located at the 5' end of the 5' subdomain, and / or the quality control template may contain a 3' terminal domain located at the 3' end of the 3' subdomain. The 5' terminal domain of the quality control template may contain at least a portion of the sequence of the quality control primer. The combined sequence of the 5' terminal domain and the 5' subdomain may contain the entire sequence of the quality control primer.

[0073] Detection of the first quality control product may include detection of the first quality control product using a signal-generating oligonucleotide. The signal-generating oligonucleotide may be capable of hybridizing with the first quality control product. Detection may include contacting the first quality control product with the signal-generating oligonucleotide for hybridization. The signal-generating oligonucleotide may contain a quencher, a label, or both. The label may contain a quenchable label (e.g., a fluorophore). A quenchable label may be, for example, a fluorophore. As used herein, the term "fluorophore" should be given its general meaning and also refers to any reporter group whose presence can be detected by its luminescent properties. Non-limiting examples of fluorophores include: Cy2 TM (506), YO-PRO TM-1(509), YOYO TM -1 (509), Calcein (517), FITC (518), FluorX TM (519) Alexa TM (520), Rhodamine 110 (520), Oregon Green TM 500(522), Oregon Green TM 488(524), RiboGreen TM (525), Rhodamine Green TM (527), Rhodamine 123 (529), Magnesium Green TM (531) Calcium Green TM (533), TO-PRO TM -1(533), TOTO1(533), JOE(548), BODIPY530 / 550(550), Dil(565), BODIPY TMR(568), BODIPY558 / 568(568), BODIPY564 / 570(570), Cy3 TM (570), Alexa TM 546(570), TRITC(572), Magnesium Orange TM (575), Phycoerythrin R&B (575), Rhodamine phalloidin (575), Calcium Orange TM (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red TM (590), Cy3.5 TM (596),ROX(608),CalciumCrimson TM (615), Alexa TM 594 (615), Texas Red (615), Nile Red (628), YO-PRO TM -3(631), YOYO TM -3(631), R-phycocyanin(642), C-phycocyanin(648), TO-PRO TM -3(660), TOTO3(660), DiDDilC(5)(665), Cy5 TM(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 numbers in parentheses are the maximum emission wavelengths of the responding fluorophores in nanometers). In some embodiments, the fluorophore is Cy5. TM In some implementations, the fluorophore is hexachlorofluorescein (HEX).

[0074] Signal-generating oligonucleotides can contain quenchers. These quenchers can quench the label. Quenching can be mediated by fluorescence resonance energy transfer (FRET). FRET is based on the classical dipole-dipole interaction 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 reach up to [missing value]. The distance between the donor and acceptor spectral overlap and the relative orientation of the donor and acceptor transition dipole moments also play a role. Quenching of a fluorophore can also occur due to the formation of a non-fluorescent complex or a non-fluorescent molecule between two fluorophores. This mechanism is referred to 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, it is considered that a quencher component is not required to observe detectable changes in fluorescence, and that proximal-base quenching effects are sufficient to produce detectable fluorescence shifts to allow for the evaluation, monitoring, observation, and / or tracking of nucleic acid amplification reactions. 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.

[0075] Detection may include contacting a first quality control product with a signal-generating oligonucleotide for hybridization. The label may be capable of generating a signal upon hybridization of the signal-generating oligonucleotide with the first quality control product. In some embodiments, the label generates a signal upon hybridization of the signal-generating oligonucleotide with the first quality control product. The signal may be fluorescence. Detection of the first quality control product may include detecting the signal generated by the label of the signal-generating oligonucleotide. The label may be a fluorophore, and the signal may be fluorescence. Detection may include detecting the label's signal before, during, or after the amplification reaction, or any combination thereof.

[0076] The method may include: providing a signal-generating oligonucleotide; performing an amplification reaction on the signal-generating oligonucleotide; and detecting a first quality control product using the signal-generating oligonucleotide. The quality control template may be the signal-generating oligonucleotide. The quality control template may be (i) a template for the synthesis of the first quality control product, and (ii) a tool for detecting the first quality control product. The signal-generating oligonucleotide may 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.

[0077] In some embodiments, the 5' end 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 contain a 3' end 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, reverse transcriptase may be able to use one or more RNA nucleotides of the 5' end 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 contain a sequence complementary to at least a portion of the quality control primer. The amplification reaction may include contacting reverse transcriptase with the quality control template to generate the extended quality control template. The extended quality control template may contain cDNA. In some embodiments, the amplification reaction includes: contacting the quality control primer with the 3' end of the extended quality control template for hybridization, and extending the quality control primer hybridizing with the 3' end of the extended quality control template with reverse transcriptase and / or an enzyme with polymerase activity, thereby generating a first quality control product.

[0078] The quality control template may be a signal-generating oligonucleotide. The signal-generating oligonucleotide may contain a label. The loop domain may contain one or more RNA nucleotides. The label may contain a quenchable label (e.g., a fluorophore). The signal-generating oligonucleotide may contain a quencher. The label may be located in the 3' end domain, and the quencher may be located in the 5' end domain, and / or the label may be located in the 5' end domain, and the quencher may be located in the 3' end domain. The amplification reaction may 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 with reverse transcriptase, thereby generating a first quality control product. The reverse transcriptase may contain RNase H activity. In some embodiments, the reverse transcriptase cleaves the quality control template at one or more RNA nucleotides during the generation of the first quality control product, thereby generating a first cleavage product and a second cleavage product containing the label. Detection of the first quality control product may include detecting a signal generated by the first cleavage product containing the label. Detection of the first quality control product may include detecting a signal containing the label. The first cleavage product may contain the label. The amount of the detected signal may 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 fluorescence. The method may include: providing supplemental quality control primers; and performing an amplification reaction on the supplemental quality control primers.

[0079] The signal-generating oligonucleotide can be from approximately 10 nucleotides to approximately 100 nucleotides in length. The quality control template can be from approximately 10 nucleotides to approximately 100 nucleotides in length. The quality control primer and / or supplemental quality control primer can be from approximately 5 nucleotides to approximately 25 nucleotides in length. The 5' subdomain, 3' subdomain, loop domain, 5' terminal domain, and / or 3' terminal domain can be from approximately 1 nucleotide to approximately 25 nucleotides in length. The signal-generating oligonucleotide, quality control template, and / or quality control primer may contain one or more phosphate thioester bonds 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.

[0080] The signal-generating oligonucleotide may contain one or more LNAs. One or more LNAs may be located within a loop domain (e.g., one or more LNAs enhance the detectability of the first quality control product). The signal-generating oligonucleotide may be configured such that the melting temperature (Tm) of the first quality control product / signal-generating oligonucleotide duplex is equal to or greater than the melting temperature (Tm) of the paired stem domain of the signal-generating oligonucleotide (e.g., configured via one or more modified and / or modified bases, such as LNAs located in the loop domain). The LNA may be located in the stem domain. In some embodiments, the LNA is also placed in the stem region to enhance hairpin stability and improve detectability. Additionally, and without being bound by any particular theory, in some embodiments, when using archaea polymerase, the 3' terminal base is LNA-modified (or otherwise modified) to block the 3' to 5' exonuclease activity of the polymerase. Stem LNAs can most effectively configure the probe's Tm above the assay temperature and thereby (i) stabilize the hairpin and reduce the baseline signal and / or (ii) enhance the Tm of the quality control product / signal-generating oligonucleotide duplex, thus 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 located at the 3' end of the stem, the LNA enhances the initiation of the IC primer and increases the Tm of the quality control product / signal-generating oligonucleotide duplex. In some embodiments, when located at the 5' stem, the LNA can slow down or inhibit IC product generation, although they can enhance the Tm of the quality control product / signal-generating oligonucleotide duplex. Modifications and modified bases can include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modifications (e.g., Acrydite). TM Adenosine, azide (NHS ester), digoxin (NHS ester), cholesterol-TEG, I-Linker TM Amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link) TMAmino modifiers), alkynes (e.g., 5'-hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dibiotin, PC-biotin, desulfurized biotin-TEG), thiol modification (e.g., thiol modifier C3 SS, dithiol, thiol modifier C6 SS)); 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, reverse dT, reverse dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, Super Super Locked nucleic acids (LNAs), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA non-locked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), iso-dC, iso-dG, fluorine C, fluorine U, fluorine A, fluorine G); thiophosphate bond modifications (e.g., thiophosphorylated DNA bases, thiophosphorylated RNA bases, thiophosphorylated 2'-O-methyl bases, thiophosphorylated LNA bases); and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, thiophosphate bonds, 3' phosphate groups, spacer bases (e.g., C3 spacer bases or other spacer bases). One or more modified nucleotides may include spacers, base-free sites, unmethylated RNA bases, 2'-O-methylated nucleotides, and any combination thereof.

[0081] In some embodiments, the signal-generating oligonucleotide does not contain a dye capable of quenching the label. In some embodiments, the signal-generating oligonucleotide does not contain any quenchable portion other than the nucleotide of the signal-generating oligonucleotide. The 5' terminal domain of the quality control template and / or the signal-generating oligonucleotide may contain a label. The 5' terminal domain and / or 5' subdomain of the quality control template and / or the signal-generating oligonucleotide may contain one or more cytosine bases. The 3' terminal domain and / or 3' subdomain of the quality control template and / or the signal-generating oligonucleotide may contain one or more guanine bases and / or adenine bases. One or more guanine bases and / or adenine bases may be capable of quenching the label when 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 may contain a label. The 3' terminal domain and / or 3' subdomain of the quality control template and / or the signal-generating oligonucleotide may contain one or more cytosine bases. The 5' terminal domain and / or 5' subdomain of the quality control template and / or signal-generated oligonucleotide may contain one or more guanine and / or adenine bases. One or more guanine and / or adenine bases may be able to quench the marker when the quality control template and / or signal-generated oligonucleotide forms a hairpin structure.

[0082] Detection of the first quality control product may include detecting a decrease in the amount of signal generated by the label of the quality control primer. The label may be a fluorophore, and the signal may be fluorescence. The generation of the first and second quality control products may be correlated with the detected decrease in signal. The 5' end of the quality control primer may contain a label, and the quality control primer may contain 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 may contain one or more guanine and / or adenine bases. When 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, one or more guanine and / or adenine bases present in the 3' terminal domain and / or 3' subdomain of the first quality control product may be able to quench the label when the first quality control product forms a hairpin structure. When a quality control primer binds to a first quality control product and is extended by an enzyme with 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 can quench the label when the second quality control product forms a hairpin structure. Detection of the first quality control product may include contacting the first quality control product with a fluorescent dye.

[0083] Providing quality control primers, quality control templates, and / or signal-generating oligonucleotides may include providing a reagent composition comprising quality control primers, quality control templates, and / or signal-generating oligonucleotides. Amplification of the quality control primers, quality control templates, and / or signal-generating oligonucleotides may include contacting the reagent composition with a treated sample to generate an amplification reaction mixture. This method may include detecting a target nucleic acid sequence in a sample. This method may include: performing an amplification reaction on the target nucleic acid sequence capable of generating nucleic acid amplification products. This method may include: detecting nucleic acid amplification products with target signal-generating oligonucleotides, wherein the target signal-generating oligonucleotides are capable of hybridizing with nucleic acid amplification products. Performing an amplification reaction on the target nucleic acid sequence capable of generating nucleic acid amplification products may include amplifying the target nucleic acid sequence in an amplification reaction mixture under amplification conditions to generate nucleic acid amplification products. This method may include: contacting a sample containing a biological entity with a lysis buffer to generate a treated sample, wherein the lysis buffer contains one or more lysis agents capable of lysing the biological entity to release the sample nucleic acids contained therein, and wherein the sample nucleic acids are suspected of containing a target nucleic acid sequence. This method may include: contacting a reagent composition with a treated sample to generate an amplification reaction mixture, wherein the reagent composition contains one or more amplification reagents.

[0084] One or more amplification reagents may comprise: reverse transcriptase; an enzyme with hyperthermophilic polymerase activity (e.g., an enzyme with both hyperthermophilic polymerase and reverse transcriptase activity); a forward primer; a reverse primer; a reverse transcription primer; and / or dNTPs. The sample nucleic acid may contain a nucleic acid containing a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence comprises: amplifying a target nucleic acid sequence comprising a first and a second strand complementary to each other under isothermal amplification conditions, wherein the amplification comprises contacting the nucleic acid containing the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with the sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme with 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 may be a product of a reverse transcription reaction generated from sample ribonucleic acid (e.g., amplification comprises generating nucleic acid via a reverse transcription reaction). The amplification reaction may last for a period of approximately 5 minutes to approximately 60 minutes. In some implementations, the amplification quality control template may 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.

[0085] In some embodiments, the method is: carried out in a single reaction vessel; does not include the use of any enzymes other than reverse transcriptase and enzymes with hyperthermophilic biopolymerase activity; does not include the use of any enzymes other than enzymes with hyperthermophilic biopolymerase activity; does not include thermal denaturation and / or enzymatic denaturation of nucleic acids and / or quality control templates during the amplification step; and / or does not include contacting nucleic acids and / or quality control templates with single-stranded DNA-binding proteins.

[0086] In some embodiments, the method, reagent composition, and / or amplification reaction mixture do not contain: a template other than a quality control template capable of generating a first quality control product; a probe other than a signal-generating oligonucleotide capable of detecting the first quality control product; a double-stranded template capable of generating the first quality control product; a linear template capable of generating the first quality control product; and / or primers other than quality control primers capable of hybridizing with the quality control template, the first quality control product, and / or the second quality control product.

[0087] This method may include determining the presence, absence, and / or amount of a first quality control product. The presence, absence, and / or amount of a signal may indicate the presence, absence, and / or amount of the first quality control product. The presence, absence, and / or amount of a signal may 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 may indicate: (i) the integrity of one or more amplification reagents in the amplification reaction mixture; (ii) a malfunction of the instrument in which the amplification reaction is performed; and / or (iii) inhibition of the sample source of the amplification reaction (e.g., inhibition of the matrix source). The presence, absence, and / or amount of a signal may indicate the degree to which the amplification of the target nucleic acid sequence is inhibited in the amplification reaction.

[0088] The method, reagent composition, and / or amplification reaction mixture may contain at least about 50,000 copies to about 500,000 copies of a quality control template (e.g., in a 3-component assay). The method, reagent composition, and / or amplification reaction mixture may contain an IC template / probe at a concentration of about 25 nM to 50 nM, which may be about 10 12A copy of the IC template (e.g., in a two-component assay). Comparable methods for monitoring amplification reactions may employ an internal control template of approximately 20 to approximately 100 copies. In some embodiments, the method, reagent composition, and / or amplification reaction mixture contain at least approximately 1.1 times (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or any two of these values) more copies of the quality control template and / or quality control primers compared to comparable methods monitoring amplification reactions that do not contain a quality control template and / or quality control primers. In some embodiments, the comparable method includes an internal control template that is unable to form a hairpin structure. In some embodiments, comparable methods for monitoring amplification reactions that do not contain quality control templates and / or quality control primers inhibit the amplification of the target nucleic acid sequence and / or the detection of nucleic acid amplification products by at least about 1.1 times more than the methods disclosed herein (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or any two of these values, by a number or range). In some embodiments, the quality control templates and / or quality control primers are unable to hybridize with the target nucleic acid sequence. In some embodiments, the presence of the quality control templates and / or quality control primers does not inhibit the amplification of the target nucleic acid sequence and / or the detection of nucleic acid amplification products. The presence of a quality control template and / or quality control primers in the amplification reaction mixture can improve the amplification of the target nucleic acid sequence and / or the 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 any value or range between any two of these values) compared to comparable methods that do not contain a quality control template and / or quality control primers. The number of erroneous priming events and / or primer-dimer generation can 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 any value or range between any two of these values) compared to comparable methods that do not contain a quality control template and / or quality control primers.

[0089] The disclosure herein includes a reaction mixture. In some embodiments, the reaction mixture comprises: the quality control template disclosed herein; the quality control primers disclosed herein; the signal-generating oligonucleotides disclosed herein; supplementary quality control primers 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 may contain one or more of an enzyme with polymerase activity, dNTPs, and buffers.

[0090] Amplifying the target nucleic acid sequence may include generating nucleic acid amplification products and / or quality control products at detectable levels within approximately 20 minutes, 15 minutes, or 10 minutes. Detection may be performed within less than approximately 20 minutes, 15 minutes, 10 minutes, or 5 minutes from the time the reagent composition comes into contact with the treated sample.

[0091] The lysis buffer may contain one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. The pH of the lysis buffer may be from about 1.0 to about 10.0 (e.g., about 2.2). Sample nucleic acids may include sample ribonucleic acid and / or sample deoxyribonucleic acid. Sample nucleic acids may contain cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or combinations thereof. In some embodiments, one or more amplification reagents include: reverse transcriptase; an enzyme with hyperthermophilic biopolymerase activity; and / or dNTPs. In some embodiments, the enzyme with hyperthermophilic biopolymerase activity has reverse transcriptase activity; a forward primer; a reverse primer; and a reverse transcription primer. The reagent composition may be lyophilized, heat-dried, and / or contain one or more additives. In some embodiments, one or more additives include: Tween 20, Triton X-100, and / or Tween 80; amino acids; sugars or sugar alcohols; and / or polymers. Sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof. Polymers may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, Ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. Contacting the reagent composition with the treated sample may include dissolving the reagent composition in the treated sample. In some embodiments, one or more lytic agents comprise: about 0.001% (w / v) to about 1.0% (w / v) of the treated sample (e.g., about 0.2% (w / v) of the treated sample); and / or a detergent (e.g., one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants).

[0092] In some embodiments, the method is: carried out in a single reaction vessel; does not include the use of any enzymes other than reverse transcriptase and enzymes with hyperthermophilic biopolymerase activity; does not include the use of any enzymes other than enzymes with hyperthermophilic biopolymerase activity; does not include thermal denaturation and / or enzymatic denaturation of nucleic acids during the amplification step; and / or does not include contacting nucleic acids with single-stranded DNA-binding proteins.

[0093] The target nucleic acid sequence may be no longer than about 20 nucleotides to no longer than about 90 nucleotides (e.g., about 30 nucleotides). The forward primer, reverse primer, and / or reverse transcription primer may be about 8 to 16 bases long. The nucleic acid amplification product may be about 20 to 40 bases long. The spacer region sequence may contain a portion of the target nucleic acid sequence. The spacer region sequence may be 1 to 10 bases long. Isothermal amplification conditions may include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, or optionally about 56°C to about 67°C. Amplification may be performed for a period of about 5 minutes to about 60 minutes (e.g., about 15 minutes). Amplification may be performed under isothermal amplification conditions free of helicase, single-strand binding proteins, cleavage agents, and recombinase. Amplification can be performed using methods 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, immune amplification, nucleic acid sequence-based amplification (NASBA), autonomous sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). PCR can be real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0094] An 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. An 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. An enzyme with hyperthermophilic polymerase activity can be a polymerase containing the amino acid sequence of SEQ ID NO:1. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low exonuclease activity or no exonuclease activity. Sample ribonucleic acid can be simultaneously contacted with reverse transcriptase and an enzyme with hyperthermophilic polymerase activity. Sample ribonucleic acid can be simultaneously contacted with reverse transcriptase, an enzyme with hyperthermophilic polymerase activity, and forward and reverse primers. Sample ribonucleic acid can be simultaneously contacted with reverse transcriptase, an enzyme with hyperthermophilic polymerase activity, forward primers, reverse primers, and reverse transcription primers.

[0095] In some embodiments, quality control primers are provided. The quality control primers may contain at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any number or range between these values) identical to SEQ ID NO:3 or SEQ ID NO:9. The quality control primers may contain sequences having one, two, three, four, or more mismatched or generic nucleotides relative to SEQ ID NO:3 or SEQ ID NO:9. In some embodiments, a quality control template (e.g., a hairpin internal control (HPIC) molecular beacon) is provided. The quality control template may contain at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any number or range between these values) identical to SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:11. The quality control template may contain sequences having one, two, three, four, or more mismatched or generic nucleotides relative to SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:11. The quality control primers and quality control template may contain one or more modifications (e.g., phosphorylated DNA bases, LNA). The quality control template may contain 5' modifications (e.g., 5HEX) and / or 3' modifications (e.g., 3IABkFQ). The IC assay components provided herein can be used in multiple assays (e.g., in conjunction with assays for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae).

[0096] In some implementations, amplification includes and / or excludes 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 implementations, amplification does not include LAMP.

[0097] In some embodiments, the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) addition of a ribonuclease inhibitor to the treated sample; (vii) addition of a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more lysis agents from the treated sample or the amplification reaction mixture; (xii) thermal denaturation and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0098] The term "isothermal amplification reaction" should be given its usual meaning and should also include reactions in which the temperature does not change significantly during the reaction. In some embodiments, the temperature deviation of the isothermal amplification reaction during the main enzymatic reaction step in which amplification occurs does not exceed 10°C, for example, not more than 5°C or not more than 2°C. Different enzymes can be used for amplification depending on the method of nucleic acid isothermal amplification. Isothermal amplification compositions and methods are described in PCT application published as WO2017176404, the contents of which are incorporated herein by reference in their entirety.

[0099] In some embodiments, the methods and components described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer resists precipitation formation for a period of time under storage conditions (e.g., a storage-stable lysis buffer). Compositions, kits, and methods for resisting precipitation with lysis buffers are described in International Application No. PCT / US23 / 61980, entitled “NON-OPAQUELYTIC BUFFER COMPOSITION FORMULATIONS,” filed February 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0100] Apart from acidic and / or low pH conditions, some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote chain dissociation and / or promote de-strutation). Compositions, kits, and methods for nucleic acid detection in which nucleic acid chains dissociate under low pH conditions (e.g., via contact with acidic lysis buffer) to facilitate subsequent rapid amplification and detection are described in International Application No. PCT / US23 / 61978 entitled “METHOD FOR SEPARATING GENOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS”, filed February 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0101] In some embodiments, the methods and compositions described herein may comprise lysis buffers and / or reagent compositions. Lysis buffers comprising lysis agents and reducing agents, and reagent compositions comprising amplifying agents and one or more protective agents capable of isolating lysis agents (e.g., cyclodextrin compounds), are described in International Application No. PCT / US22 / 21015 entitled “ISOTHERMAL AMPLIFICATION OF PATHOGENS”, filed March 18, 2022, the contents of which are incorporated herein by reference in their entirety.

[0102] In some embodiments, the methods and compositions described herein may comprise signal-generating oligonucleotides containing one or more polymerase terminators (e.g., protected signal-generating oligonucleotides). Compositions, kits, and methods for nucleic acid detection, wherein the protected signal-generating oligonucleotides are capable of reducing nonspecific product formation and / or fewer false positives, are described in U.S. Provisional Patent Application No. 63 / 374,772, entitled “MODIFIED MOLECULAR BEACONS FOR IMPROVED DETECTION SPECIFICITY,” filed September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0103] Some embodiments of the methods and compositions described herein may include probes that denature at temperatures different from the optimal APA reaction temperature, enabling multiplexing of the target and / or internal control. Compositions, kits, and methods for multiplexed nucleic acid detection are described in U.S. Provisional Patent Application No. 63 / 374,831, entitled “ARCHEAL POLYMERASE AMPLIFICATION,” filed September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0104] In some embodiments, some embodiments of the methods and compositions described herein may 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 entitled “METHODS AND COMPOSITIONS FOR PATHOGEN DETECTION”, filed September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0105] Nucleic acid, subjects, samples and nucleic acid processing

[0106] This document provides methods and compositions for amplifying nucleic acids. The terms “nucleic acid” and “nucleic acid molecule” are used interchangeably herein. These terms refer to nucleic acids having any composition, such as DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), etc.), RNA (e.g., messenger RNA (mRNA), short repressive 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, etc.), RNA / DNA hybrids, and polyamide nucleic acids. Nucleic acids (PNAs), all of which can be in single-stranded or double-stranded form, and unless otherwise limited, may include known analogs of natural nucleotides capable of functioning in a manner similar to naturally occurring nucleotides. Nucleic acids can be or may be derived from plasmids, bacteriophages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or other nucleic acids capable of replicating or being replicated in vitro or in the host cell, cell nucleus, mitochondria, or cytoplasm of the cell. Unless specifically defined, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, interspecific homologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly indicated sequences. In particular, degenerate codon substitutions can be achieved by mixing at the third position of one or more selected (or all) codons. Sequences substituted with bases and / or deoxyinosine residues are used. The term nucleic acid is used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene. As equivalents, the term may also include derivatives, variants, and analogs of RNA or DNA synthesized from nucleotide analogs, single-stranded (“sense” or “antisense”, “positive” or “negative” strand, “positive” or “negative” reading frame, “positive” or “negative” reading frame, “positive” or “negative” strand), and double-stranded polynucleotides. It typically includes regions before and after the coding regions involved in the transcription / translation of the gene product and the regulation of transcription / translation (leader and trailer), as well as intermediate sequences (introns) between the various 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)). For RNA, the thymine base is replaced by uracil. The length or size of nucleic acids can be expressed by the number of bases.

[0107] 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, target polynucleotide, and / or 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. When the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may contain a single-stranded target sequence. When the target nucleic acid is double-stranded, it typically comprises a first strand and a second strand. The first and second strands may be referred to as the forward and reverse strands, and are typically complementary to each other. When the target nucleic acid is single-stranded, a complementary strand may be generated, for example, by polymerization and / or reverse transcription, making the target nucleic acid double-stranded and having a first / forward strand and a second / reverse strand.

[0108] The target sequence can refer to the sense or antisense strand of a nucleic acid sequence, or it can refer to a sequence present on the original target nucleic acid, an amplified copy, or an amplification product. The target sequence can be a subsequence within a larger polynucleotide. For example, the target sequence can be a short sequence (e.g., 20 to 50 bases) within a nucleic acid fragment, chromosome, or plasmid that is targeted for amplification. In some embodiments, the target sequence can refer to a sequence in the target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used for amplifying the nucleic acid. Therefore, the target sequence can refer to the entire sequence targeted for amplification, or it can refer to a subsequence of the target nucleic acid bound to an oligonucleotide. The amplification product can be a larger molecule containing the target sequence and at least one other sequence or other nucleotide. The amplification product can be approximately the same length as the target sequence, for example, exactly the same length as the target sequence. The amplification product can contain or consist of the target sequence.

[0109] The length of the target sequence and / or the concentration (percentage) of guanine cytosine (GC) can depend in part on the temperature at which the amplification reaction is run, and this temperature can depend in part on the stability of the polymerase used in the reaction. Sample assays can be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, when the polymerase is stable at up to 60°C to 65°C, the target sequence length can be, for example, from 19 to 50 nucleotides, or the length can be, for example, from about 40 to 50, 20 to 45, 20 to 40, or 20 to 30 nucleotides. The GC concentration under these conditions can be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0110] 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 from any genome, such as the genomes of animals, plants, insects, viruses, and bacteria (e.g., genomes present in spores). In some embodiments, genomic target nucleic acids are located at a specific genomic locus or more than one genomic locus. Genomic loci may include any one or combination of open reading frame DNA, non-transcribed DNA, intron sequences, exon sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequences considered to be associated with a given genomic locus.

[0111] The target sequence may contain one or more repeating elements (e.g., multiple repeats, inverted repeats, palindromes, tandem repeats, microsatellites, microsatellites, etc.). In some embodiments, the target sequence exists as a repeating element (e.g., multiple repeats, inverted repeats, palindromes, tandem repeats, microsatellite repeats, microsatellite repeats, etc.) within the sample nucleic acid (e.g., within nucleic acid fragments, chromosomes, genomes, plasmids). For example, the target sequence may appear multiple times as a repeating element, and one, some, or all occurrences of the target sequence within the repeating element can be amplified using the methods described herein (e.g., using a single pair of primers). In some embodiments, the target sequence exists as a repeat and / or intraspecific homolog within the sample nucleic acid (e.g., within nucleic acid fragments, chromosomes, genomes, plasmids).

[0112] Target nucleic acids may include microRNAs. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21 to 23 nucleotides long) and single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNAs and are partially complementary to messenger RNAs. Target nucleic acids may include microRNA precursors, such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop structures that have been further processed into miRNAs. Target nucleic acids may include short interfering RNAs (siRNAs), which are short (e.g., about 20 to 25 nucleotides long) and at least partially double-stranded RNA molecules involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0113] The nucleic acids used in the methods described herein can be obtained from any suitable biological sample or specimen, such as those 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 protozoa. Any human or non-human animal can be selected, including but not limited to mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovids (e.g., cattle), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), swine (e.g., pigs), camels (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), bears (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 (e.g., embryo, fetus, infant, child, adult).

[0114] A sample or test sample can be any sample isolated from or obtained from a subject or a portion thereof. Non-limiting examples of samples include fluids or tissues from a subject, including but not limited to blood or blood products (e.g., serum, plasma, etc.), cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, cerebrospinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneum, catheter, ear, arthroscopy), serum, plasma, urine, aspirate, biopsy samples, paracentesis samples, cells (e.g., blood cells) or portions thereof (e.g., mitochondria, nuclei, extracts, etc.), female reproductive tract washes, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, mammary gland fluid, hard tissues (e.g., liver, spleen, kidney, lung, or ovary), etc., or combinations thereof. The term blood includes whole blood, blood products, or any fraction of blood, such as serum, plasma, erythrocyte sedimentation rate (ESR), or similar substances as conventionally defined. Plasma refers to the whole blood fraction obtained by centrifuging blood treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after a blood sample has clotted. Fluid or tissue samples are typically collected according to standard protocols usually followed in hospitals or clinics. For blood, an appropriate amount of peripheral blood is usually collected (e.g., between 3 and 40 ml) and can be stored before or after preparation according to standard procedures.

[0115] Samples may include those containing spores, viruses, cells, nucleic acids from prokaryotes or eukaryotes, and / or any free nucleic acids. For example, the methods described herein can be used to detect nucleic acids outside spores (e.g., without lysis). Samples can be isolated from any material suspected of containing the target sequence, such as from the subject described above. In some embodiments, the target sequence is present in air, plants, soil, or other materials suspected of containing biological organisms.

[0116] Nucleic acids can be derived from one or more sources (e.g., isolated, extracted, purified) by methods known in the art. Any suitable method can be used to isolate, extract, and / or purify nucleic acids from biological samples, including DNA preparation methods in the art, and a variety of commercially available reagents or kits, such as Qiagen's QIAamp Cyclic Nucleic Acid Kit, QiaAmp DNA Microkit, or QiaAmp DNA Blood Microkit (Qiagen, Hilden, Germany), GenomicPrep... TM Blood DNA Isolation Kit (Promega, Madison, Wis.), GFX TM Genomic blood DNA purification kits (Amersham, Piscataway, NJ) or combinations thereof. U.S. Patent No. 7,888,006 discloses a DNA purification method but does not disclose the compositions (e.g., lysis buffers, protectants) and methods described herein.

[0117] In some embodiments, a cell lysis procedure is performed. Cell lysis can be performed before initiating the amplification reaction described herein (e.g., to release DNA and / or RNA from the cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or combinations thereof), physical (e.g., French press, sonication, etc.), or electrolytic lysis methods. For example, chemical methods typically use lysing agents to disrupt cells and extract nucleic acids from cells, followed by treatment with a dissociative salt. In some embodiments, cell lysis includes the use of detergents (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis includes the use of ionic detergents (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods such as freezing / thawing followed by grinding, using cell crushing, etc., can also be useful. High-salt lysis procedures can also be used. For example, an alkaline lysis procedure can be used. The latter procedure traditionally involves the use of a phenol-chloroform solution, and alternative phenol-chloroform-free procedures involving three solutions can be used. In the latter procedure, one solution may contain 15 mM Tris, pH 8.0; 10 mM EDTA and 100 μg / ml RNase A; the second solution may contain 0.2 N NaOH and 1% SDS; and the third solution may, for example, 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.

[0118] Nucleic acids can be provided for performing the methods described herein without processing the sample containing nucleic acids. For example, nucleic acids can be provided for performing the amplification methods described herein without prior nucleic acid purification. In some embodiments, the target sequence is amplified directly from the sample (e.g., without performing any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids are provided for performing the methods described herein after processing the sample containing nucleic acids. For example, nucleic acids can be extracted, isolated, purified, or partially purified from the sample. The term "isolated" generally refers to nucleic acids taken from their original environment (e.g., the natural environment if it is naturally present, or the host cell if it is exogenously expressed) and thus altered from their original environment by human intervention (e.g., "by human hands"). The term "isolated nucleic acid" can refer to nucleic acids taken from a subject (e.g., a human subject). Isolated nucleic acids can provide a smaller amount of non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than those present in the source sample. Compositions containing isolated nucleic acids may contain no more than 50% to more than 99% of non-nucleic acid components. Compositions containing isolated nucleic acids may be free of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of non-nucleic acid components. The term "purified" generally refers to the fact that the provided nucleic acids contain less of the non-nucleic acid components (e.g., proteins, lipids, carbohydrates) present prior to the purification process of the nucleic acids. Compositions containing purified nucleic acids may be free of about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of other non-nucleic acid components.

[0119] Methods for using nucleic acids as described herein can be provided without modifying the nucleic acids. Modifications may include, for example, denaturation, digestion, nicking, unwinding, incorporation and / or ligation of heterologous sequences, addition of epigenetic modifications, and addition of markers (e.g., radioactive markers such as...). 32 P, 33 P, 125 I or 35 S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, fluorescent dyes, etc. Therefore, in some embodiments, unmodified nucleic acids are amplified.

[0120] The method disclosed herein for detecting target nucleic acid sequences (single-stranded or double-stranded DNA and / or RNA) in a sample can detect target nucleic acid sequences (e.g., DNA or RNA) with high sensitivity. In some embodiments, the method can be used to detect target DNA / RNA present in a sample containing more than one type of RNA / DNA (including target RNA / DNA and more than one type of non-target RNA / DNA), wherein the target RNA / DNA is present in quantities of 10, 20, 25, 50, 100, 500, 10 3 5×10 3 10 4 5×10 4 10 5 5×10 5 10 6 One or 10 7 One or more copies of a non-target DNA / RNA are present. As used herein, the terms “RNA / DNA” and “RNAs / DNAs” should be given their ordinary meaning and should also refer to DNA, or RNA, or a combination of DNA and RNA.

[0121] For methods of detecting target RNA / DNA in a sample, the detection threshold can be, for example, 10 nM or less. The term "detection threshold" should be given its general meaning and should also be described as the minimum amount of target RNA / DNA that must be present in the sample for detection to be performed. As an illustrative example, when 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 implementations, the disclosed method has detection thresholds of 5 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, 0.005 nM or less, 0.001 nM or less, 0.0005 nM or less, 0.0001 nM or less, 0.00005 nM or less, 0.00001 nM or less, 10 pM or less, 1 pM or less, 500 fM or less, 250 fM or less, 100 fM or less, 50 fM or less, 500 aM (attomolar, micromolars per liter) or less, 250 aM or less, 100 aM or less, 50 aM or less, 10 aM or less, or 1 aM or less. In some embodiments, the disclosed compositions or methods exhibit detection sensitivities of atomolar / liter (aM), femtomolar / liter (fM), picomolar / liter (pM), and / or nanomolar / liter (nM).

[0122] The sample may contain sample nucleic acids (e.g., more than one type of sample nucleic acid). The term "more than one" is used herein to mean two or more. Thus, in some embodiments, the sample contains two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more) sample nucleic acids (e.g., DNA / RNA). The disclosed methods can be used as a highly sensitive way to detect the presence of target nucleic acids in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, the sample contains 5, 10, 20, 25, 50, 100, 500, 1 ... 3 Seed, 5×10 3 10 species 4 Seed, 5×10 4 10 species 5 Seed, 5×10 5 10 species 6 species or 10 7 One, 50 or more DNA / RNA species. In some embodiments, the sample contains DNA / RNA from cells (e.g., eukaryotic cells, mammalian cells or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0123] The term "sample" as used herein shall be given its general meaning and shall include any sample containing RNA and / or DNA (e.g., for determining the presence of target DNA and / or target RNA 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 cell lysate, DNA / RNA-rich cell lysate, or DNA / RNA isolated and / or purified from cell lysate. A sample may be from a patient (e.g., for diagnostic purposes). A sample may be from permeabilized cells, cross-linked cells, tissue sections, or combinations thereof. A sample may be from tissue prepared by cross-linking followed by delipidization and conditioning to form a uniform refractive index. A sample may contain a target nucleic acid (e.g., target DNA / RNA) and more than one non-target DNA / RNA. In some embodiments, target DNA / RNA is expressed in units of 10, 20, 25, 50, 100, 500, 10 3 5×10 3 10 4 5×10 4 10 5 5×105 10 6 One or 10 7 One copy of a non-target DNA / RNA exists in the sample.

[0124] Patient samples include blood and other liquid samples of biological origin, solid tissue samples such as biopsy samples or tissue cultures or cells derived therefrom and their progeny, and samples that have been manipulated (e.g., by treatment with reagents), washed, or enriched with certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA) after they are obtained. Samples may include, or include, biological samples, including but not limited to, clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), and also include tissue obtained by surgical resection, tissue obtained by biopsy, cells in cultures, cell supernatants, cell lysates, tissue samples, organs, bone marrow, etc. Biological samples may include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), such as RNA-containing samples obtained from such cells (e.g., RNA-containing cell lysates or other cell extracts). In some implementations, environmental samples are, or are obtained from, the following: food samples, beverage samples, paper surfaces, fabric surfaces, metal surfaces, wood surfaces, plastic surfaces, soil samples, freshwater samples, wastewater samples, saline samples, samples exposed to atmospheric air or other gases, their cultures, or any combination thereof.

[0125] The sample may originate from (or be suspected of being) diseased cells, fluids, tissues, or organs; or from normal (non-diseased) cells, fluids, tissues, or organs. In some embodiments, the sample originates from (or is suspected of being) pathogen-infected cells, tissues, or organs. For example, the sample may originate from an individual who may or may not be infected, and the sample may be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, fecal sample, cerebrospinal fluid, fine needle aspirate, swab samples (e.g., oral swabs, cervical swabs, nasal swabs), interstitial fluid, synovial fluid, nasal discharge, tears, erythrocyte sedimentation rate (ESR), mucosal samples, epithelial cell samples (e.g., epithelial cell scrapings), etc.), and their cultures. The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens can be viruses, fungi, worms, protozoa, malaria parasites, Plasmodium, Toxoplasma, Schistosoma, etc. "Hernias" include roundworms, heartworms, and phytophagous nematodes (Nematoda), trematodes (Trematoda), acanthocephalans, and tapeworms (Cestoda). Protozoan infections include infections with Giardia spp. and Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, giardiasis, Chaga's 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*.Pathogenic viruses include, for example, immunodeficiency viruses (e.g., HIV); influenza viruses; dengue fever; West Nile virus; herpesvirus; yellow fever virus; hepatitis C virus; hepatitis A virus; hepatitis B virus; human papillomavirus, etc. Pathogenic viruses can include DNA viruses, such as: papillomaviruses (e.g., HPV, polyomaviruses); hepatotropic DNA viruses; herpesviruses (e.g., HSV (e.g., HSV I, HSV II), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasis rosea, Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., thymviruses, avian adenoviruses, ichtadenoviruses, mammalian adenoviruses, siadenoviruses); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, sheep pox virus, pseudococcosis, bovine papular stomatitis virus; tanapoxetine virus, yaba monkeytumor virus). Virus); Molluscum contagiosum virus (MCV); Parvovirus (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, brucellosis virus, human parvovirus 4G1); Geminiviridae; Nanoviridae; Phycodnaviridae; etc. Non-limiting examples of pathogens include Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Haemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, and 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, blue tongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus Virus), simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma krusei, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria sp. (e.g., Eimeria tenella), Onchocercavolvulus, Leishmania sp. (e.g., Leishmania tropicala), Streptococcus pneumoniae pneumonia, Pneumocystis carinii, Trichophyton rubrumrubrum), Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., SARS-CoV-2, Human Immunodeficiency Virus Type 1 (HIV-1), Human T-Cell Lymphotrophic Virus Type 1 (HTLV-1), Herpes Simplex Virus, Herpesvirus 6, Herpesvirus 7, JC Virus, Influenza A Virus, Influenza B Virus, Influenza C Virus, Rotavirus, Human Adenovirus, Human Enterovirus Enteroviruses, Hantavirus, Legionella dumoffii, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsii, Ehrlichia sp. (e.g., Ehrlichiachaffeensis), Borrelia burgdorferi, Yersiniapestis, Chlamydia pneumoniae, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti), Mycoplasma(e.g., Mycoplasma arthritidis), Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini, Acholeplasma laidlawii, Mycoplasma salivarium, and Mycoplasma pneumoniae.

[0126] Amplification

[0127] This document provides methods for amplifying nucleic acids. In some embodiments, suitable amplification methods are used to amplify nucleic acids. Nucleic acid amplification typically involves the enzymatic synthesis of nucleic acid amplicon (copy), which contains a sequence complementary to the nucleotide sequence to be amplified. In some embodiments, the amplification method is performed in a single container, a single chamber, and / or a single volume (i.e., a continuous volume). In some embodiments, the amplification method and the detection method (e.g., the detection method described herein) are performed in a single container, a single chamber, and / or a single volume (i.e., a continuous volume).

[0128] The terms “amplify,” “amplification,” “amplification reaction,” or “amplifying” refer to any in vitro process used to double the number of copies of a target nucleic acid. Amplification sometimes refers to an “exponential” increase in the number of target nucleic acids. “Amplification” can also refer to a linear increase in the number of target nucleic acids, but this is distinct from a single, single primer extension step. In some implementations, a limited amplification reaction, also known as pre-amplification, can be performed. Pre-amplification is a method in which a limited amount of amplification occurs due to a small number of cycles, such as 10 cycles. Pre-amplification allows for some amplification but stops amplification before the exponential phase and typically yields approximately 500 copies of the desired nucleotide sequence. The use of pre-amplification can limit inaccuracies associated with depleted reactants in certain amplification reactions and can also reduce amplification bias due to the nucleotide sequence or abundance of the target. In some implementations, a single primer extension can be performed as a prelude to linear or exponential amplification.

[0129] This document presents a general description of the amplification process. Primers (e.g., oligonucleotides described herein) contact the target nucleic acid, and, for example, complementary sequences anneal or hybridize with each other. Primers may anneal with the target nucleic acid at or near the sequence of interest (e.g., adjacent, contiguous, etc.). Primers annealed to the target may be referred to as primer-target hybrids, hybridized primer-targets, or primer-target duplexes. When referring to the nucleotide sequence of interest, the term "near" or "contiguous" refers to the distance (e.g., number of bases) or region between the terminator terminator end and one or more nucleotides (e.g., nucleotide sequences) of the target. Typically, "contiguous" is 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, forward and reverse primers, a first oligonucleotide and a second oligonucleotide) anneal within about 1 to 20 nucleotides from the nucleotide or nucleotide sequence of interest and produce an amplification product. In some embodiments, primers are annealed within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended along the target (i.e., the template strand) by a polymerase to produce a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplification product is produced. 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.

[0130] The components of an amplification reaction (e.g., one or more amplification reagents) may include, for example, one or more primers (e.g., single primers, primer pairs, primer sets, oligonucleotides, multiple primer sets for multiplex amplification, etc.), a nucleic acid target (e.g., target nucleic acid from a sample), one or more polymerases, nucleotides (e.g., dNTPs, etc.), and a suitable buffer (e.g., a buffer containing detergents, reducing agents, monovalent ions, and divalent ions). The amplification reaction may also include one or more of the following: reverse transcriptase, reverse transcription primers, and one or more detection reagents.

[0131] Nucleic acid amplification can be performed in the presence of native nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derived nucleotides. Native nucleotides typically refer to adenosine, guanylic acid, cytidine, thymidine, or uridine. Derived nucleotides are typically nucleotides distinct from native nucleotides. Ribonucleotide triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, or U. Deoxyribonucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be represented herein as A, G, C, T, or U, without specific mention of DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs may be used, such as analogs containing a detectable label (e.g., fluorescent or colorimetric label). For example, nucleic acid amplification can be performed in the presence of labeled dNTPs, such as radiolabeled ones, such as... 32 P, 33 P, 125 I or 35 S; enzyme labeling, such as alkaline phosphatase; fluorescent labeling, such as fluorescein isothiocyanate (FITC); or other labeling, such as biotin, avidin, digoxigenin, antigen, hapten, or fluorescent dye. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, for example, thermally activated dNTPs (e.g., CleanAmp from TriLink). TM dNTP).

[0132] One or more amplification reagents may include non-enzymatic components and enzyme components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions. In some embodiments, the non-enzymatic component does 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 endonucleases, nickases, recombinases, etc. In some embodiments, the enzyme component consists of a polymerase or a combination of a polymerase and a reverse transcriptase. Therefore, such an enzyme component excludes other proteins (e.g., nucleic acid-binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, etc.

[0133] In some embodiments, amplification conditions include enzymatic activity (e.g., enzymatic activity provided by polymerase or enzymatic activity provided by polymerase and reverse transcriptase). In some embodiments, enzymatic activity does not include enzymatic activity provided by enzymes other than polymerase and / or reverse transcriptase, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, etc. Polymerase activity and reverse transcriptase activity may be provided by a single enzyme or a single enzyme type (e.g., polymerase and reverse transcriptase), or by a single enzyme or enzyme type (e.g., polymerase).

[0134] Nucleic acid amplification can include non-thermal cycling types of PCR. In some embodiments, nucleic acid amplification includes isothermal amplification processes, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is typically 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 remains substantially constant during the amplification reaction process. Isothermal amplification conditions typically do not include the thermal cycling component (i.e., cycling between higher and lower temperatures) of the amplification process. When amplified under isothermal conditions, the reaction can be maintained at a substantially constant temperature, meaning that the temperature can not be precisely maintained at one temperature. For example, during isothermal amplification, small temperature fluctuations (e.g., ±1°C to 5°C) may occur due to, for example, environmental or device-based variables. Typically, the entire reaction volume is maintained at a substantially constant temperature, and isothermal reactions as described herein typically do not include amplification conditions that depend on temperature gradients generated within the reaction vessel and / or temperature cycling based on convection.

[0135] The isothermal amplification reaction described herein can be carried out at a substantially constant temperature. In some embodiments, the isothermal amplification reaction described herein is carried out at a temperature of about 55°C to about 75°C, for example at or around the following temperatures: 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or about 75°C, or a number or range between any two of these values. In some embodiments, the temperature element (e.g., a heat source) is maintained at a substantially constant temperature, for example at or below about 75°C, at or below about 70°C, at or below about 65°C, or at or below about 60°C.

[0136] The amplification process described herein can be performed for a certain duration, such as until detectable nucleic acid amplification products and / or quality control products are generated. The nucleic acid amplification products and / or quality control products can be detected by any suitable detection process and / or the detection process described herein. The amplification process can be performed for a duration of approximately 20 minutes or less, or approximately 10 minutes or less. For example, the amplification process can be performed for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or a number or range between any two of these values.

[0137] In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that denature nucleic acids. In some embodiments, the nucleic acid target can be amplified during the amplification step (and / or other steps) without exposure to agents or conditions that promote strand separation. In some embodiments, the nucleic acid target can be amplified during the amplification step (and / or other steps) without exposure to agents or conditions that promote denaturation. Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote denaturation may include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, proteins (e.g., enzymes), etc.

[0138] In some embodiments, the methods disclosed herein do not include thermal denaturation of nucleic acids (e.g., heating a solution containing nucleic acids to high temperatures, such as above 75°C, 80°C, 90°C, or 95°C or higher) or protein-based (e.g., enzymatic) denaturation. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, RNA replicases, and nucleic acid-binding proteins (e.g., single-stranded binding proteins). In some embodiments, the compositions provided herein do not contain helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, RNA replicases, and / or nucleic acid-binding proteins (e.g., single-stranded binding proteins). In some embodiments, the compositions and methods provided herein do not contain 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 disclosed methods 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 chain separation and / or promote melting). In some embodiments, the amplification step (e.g., step (c)) does not include agents and / or conditions other than polymerases (e.g., hyperthermophilic biopolymerases) that denature nucleic acids (e.g., promote chain separation and / or promote melting). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions other than polymerases (e.g., hyperthermophilic biopolymerases) and / or low pH conditions (e.g., contact with one or more acids) that denature nucleic acids (e.g., promote chain separation and / or promote melting).

[0139] Nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such agents or conditions include helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, 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 excluding the use of helicases are helicase-free amplification conditions.

[0140] The nucleic acid target 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, the nucleic acid target is amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY). The nucleic acid target can be amplified without exposure to nucleic acid binding proteins (e.g., single-strand binding proteins or single-strand DNA binding proteins (SSBs)), such as T4gp32. In some embodiments, the nucleic acid target is amplified without exposure to topoisomerases. The nucleic acid target can be amplified with or without exposure to agents or conditions that destabilize nucleic acids. As used herein, the term “destabilization” should be given its general meaning and should also refer to the disruption of the overall organization and geometric orientation of a nucleic acid molecule (e.g., a double helix structure) by one or more of the following effects: tilting, rolling, twisting, sliding, and flipping (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 unwinding or separation of nucleic acid chains (e.g., denaturation). Nucleic acid destabilization can be achieved, for example, by exposure to agents such as intercalating agents 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 involve 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 ligases and / or RNA replicases.

[0141] In some embodiments, the nucleic acid target can be amplified without cleavage or digestion. For example, the nucleic acid target can be amplified without prior exposure to one or more cleavage agents, and the intact nucleic acid is amplified. In some embodiments, the nucleic acid target is amplified without exposure to one or more cleavage agents during amplification. In some embodiments, the nucleic acid target is amplified without exposure to one or more cleavage agents after amplification. Amplification conditions excluding the use of cleavage agents may be referred to herein as cleavage-free amplification conditions. The term "cleavage agent" generally refers to an agent, sometimes a chemical substance or enzyme, that can cleave nucleic acids at one or more specific or non-specific sites. Specific cleavage agents typically cleave based on a specific nucleotide sequence at a specific site. Cleavage agents may include endonucleases (e.g., restriction endonucleases, cleavage enzymes, etc.); exonucleases (DNases, RNases (e.g., RNase H), 5' to 3' exonucleases (e.g., exonucleases II), 3' to 5' exonucleases (e.g., exonucleases I), and multi(A)-specific 3' to 5' exonucleases); and chemical cleavage agents.

[0142] Nucleic acid targets can be amplified without the use of restriction endonucleases and / or nickases. In some embodiments, the nucleic acid is amplified without prior exposure to restriction endonucleases and / or nickases. In some embodiments, the nucleic acid is amplified without exposure to restriction endonucleases and / or nickases during amplification. In some embodiments, the nucleic acid is amplified without exposure to restriction endonucleases and / or nickases after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNases, RNases (e.g., RNase H), 5' to 3' exonucleases (e.g., exonuclease II), 3' to 5' exonucleases (e.g., exonuclease I), and multi(A)-specific 3' to 5' exonucleases. In some embodiments, the nucleic acid is amplified without exonuclease treatment before, during, and / or after amplification. Amplification conditions excluding the use of exonucleases are exonuclease-free amplification conditions. In some embodiments, the nucleic acid is amplified without DNase treatment and / or RNase treatment. In some implementations, nucleic acids are amplified without treatment with RNase H.

[0143] The amplified nucleic acid may be referred to herein as a nucleic acid amplification product or amplicon. In some embodiments, the amplification product comprises naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and combinations thereof. The amplification product typically has a nucleotide sequence that is identical or substantially identical to the sequence in the sample nucleic acid (e.g., the target sequence) or its complement. The “substantially identical” nucleotide sequence in the amplification product will typically have a high sequence identity with the amplified nucleotide sequence or its complement (e.g., approximately 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), and variation is sometimes the result of polymerase infidelity or other variables.

[0144] In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid. Sequential complementarity typically refers to a nucleotide sequence in the first strand, where each ordered base (e.g., read from 5' to 3') pairs with a corresponding ordered base in the second strand, and there are no gaps, additional sequences, or unpaired bases in the sequence considered sequentially complementary. In other words, sequential complementarity typically means that all consecutive bases in the nucleotide sequence in the first strand are complementary to the corresponding consecutive bases in the nucleotide sequence in the second strand. Sequentially complementary sequences are sometimes about 5 to about 25 consecutive bases in length, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 consecutive bases, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is sequentially complementary to or substantially identical to the target sequence in the sample nucleic acid. Therefore, in some embodiments, the nucleic acid amplification product does not contain any additional sequence that is discontinuously complementary to or substantially identical to the target sequence (e.g., at the 5' and / or 3' ends, or within the product), such as additional sequences incorporated into the amplification product via tailing primers or ligation, and / or additional sequences that provide cleavage recognition sites (e.g., cleavage enzyme recognition sites). Typically, the amplification product does not contain a product in the form of a tandem repeat sequence unless the target sequence contains a tandem repeat sequence.

[0145] The nucleic acid amplification product may contain sequences complementary to or substantially identical to one or more primers used in the amplification reaction. In some embodiments, the nucleic acid amplification product contains a first nucleotide sequence that is sequentially complementary to or identical to the first primer sequence, and a second nucleotide sequence that is sequentially complementary to or identical to the second primer sequence.

[0146] Nucleic acid amplification products may contain spacer sequences. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is sequentially complementary to or substantially identical to a portion of the target sequence in the sample nucleic acid, and 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 flanking the sequence in the amplification product is typically located between the first sequence (complementary to or substantially identical to the first primer) and the second sequence (complementary to or substantially identical to the second primer). Therefore, the amplification product typically comprises the first sequence, followed by the spacer sequence, followed by the second sequence. The spacer sequence is typically not complementary to or substantially identical to the sequence in the primers. The spacer sequence may be or may include the following: 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 comprises, or substantially comprises, a first nucleotide sequence sequentially complementary to or identical to the first primer sequence, a second nucleotide sequence sequentially complementary to or identical to the second primer sequence, and a spacer region sequence. 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 sequentially complementary to or identical to the first and second primer sequences and is not part of the spacer region sequence, for example, an additional sequence incorporated into the amplification product by tailing or circularizing primers, ligation, or other mechanisms. In some embodiments, the nucleic acid amplification product generally does not contain any additional sequence (e.g., at the 5' and / or 3' end; or within the product) that is not sequentially complementary to or identical to the first and second primer sequences and is not part of the spacer region sequence, for example, an additional sequence incorporated into the amplification product by tailing or circularizing primers, ligation, or other mechanisms. However, in such implementations, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or one or more additional bases (e.g., at the 5' end and / or 3' end; or within the product) introduced into the product, for example, through errors or contamination during the amplification process.

[0147] The length of nucleic acid amplification products can be up to 50 bases, including 10, 15, 20, 25, 30, 35, 40, 45, 50 bases, or any number or range between any two of these values. In some embodiments, nucleic acid amplification products for a given target sequence have the same length or substantially the same length (e.g., within 1 to 10 bases). Therefore, nucleic acid amplification products for a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel) and typically do not produce multiple signals indicating multiple lengths (e.g., ladders or tails on an electrophoresis gel). For multiplex reactions, nucleic acid amplification products for different target sequences can have different lengths.

[0148] 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 amplification step may include multiplex amplification of two or more target nucleic acid sequences, and the detection step may include multiplex detection of two or more nucleic acid amplification products derived from said two or more target nucleic acid sequences. The two or more target nucleic acid sequences may be specific to two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A virus, influenza B virus, and / or influenza C virus). Multiplex amplification can also refer to the simultaneous or stepwise amplification of one or more sequences present in multiple samples. For example, multiplex amplification can be used to amplify at least two target sequences that can be amplified (e.g., the amplification reaction includes suitable primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is prepared to detect at least two target sequences, but only one target sequence is present in the sample being tested, such that although both sequences can be amplified, 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. Multiplex amplification reactions can result in the amplification of one, some, or all of the target sequences, comprising appropriate primers and enzymes. In some embodiments, the amplification reaction is prepared to detect two sequences using a primer pair, one of which is the target sequence and the other is a control sequence (e.g., a synthetic sequence capable of being amplified by primers identical to the target sequence but having a different spacer region base or sequence from the target). In some embodiments, the amplification reaction is prepared to detect multiple sets of sequences with corresponding primer pairs, each set including a target sequence and a control sequence.

[0149] Primers

[0150] Nucleic acid amplification is typically performed in the presence of one or more primers. Primers are generally characterized as oligonucleotides, which consist of nucleotide sequences capable of hybridizing or annealing to the target nucleic acid at or near (e.g., adjacent) a specific region of interest (i.e., the target sequence). For example, primers can allow for the specific determination of the target nucleic acid nucleotide sequence or the detection of the target nucleic acid or its characteristics (e.g., the presence or absence of the sequence). Primers can be naturally occurring or synthetic. The term specificity, or particularity, generally refers to the binding or hybridization of one molecule with another molecule, such as primers for target polynucleotides. That is, specificity or particularity refers to the recognition, contact, and formation of a stable complex between two molecules, compared to the recognition, contact, or complex formation of either of those molecules with any other molecule. The term annealing or hybridization generally refers to the formation of a stable complex between two molecules. When referring to primers, the terms primer, oligomer, or oligonucleotide are used interchangeably herein.

[0151] Primers can be designed and synthesized using suitable methods and can have any length suitable for hybridization with the target sequence and for the amplification process described herein. Primers are typically designed based on the sequence in the target nucleic acid. In some embodiments, primer lengths can be from about 5 to about 30 bases, for example, lengths of 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. Primers can contain naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides) or mixtures thereof. Modifications and modified bases can include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemicals or adapter modifications (e.g., Acrydite). TM Adenosine, azide (NHS ester), digoxin (NHS ester), cholesterol-based TEG, I-Linker TM Amino-modified compounds (e.g., amino-modified C6, amino-modified C12, amino-modified C6 dT, Uni-Link™ amino-modified compounds), alkynes (e.g., 5'-hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, double biotin, PC biotin, desulfurized biotin-TEG), thiol modification (e.g., thiol-modified C3 SS, dithiol, thiol-modified C6 SS)); fluorophores (e.g., Freedom). TM Dyes, Alexa Dyes, LI-COR ATTO™ dyes, Rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas -X(NHS ester), 640 (NHS ester), Dy 750 (NHS ester), Iowa Dark quencher modification (e.g., Iowa) FQ, Iowa RQ), dark quencher modification (e.g., Black Hole) -1. Black Hole -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-bromodU, deoxyuridine, reverse dT, reverse dideoxy-T, dideoxy-C, 5-methyldC, deoxyinosine, Super Super Locked nucleic acids (LNAs), 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, fluorine C, fluorine U, fluorine A, fluorine G); phosphate thioester (PS) bond modifications (e.g., phosphorylated DNA bases, phosphorylated RNA bases, phosphorylated 2'-O-methyl bases, phosphorylated LNA bases); and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS linkages, 3' phosphate groups, and spacer bases (such as C3 spacer bases or other spacer bases). For example, primers 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 RNAs. Primers containing 2'-O-methyl RNA bases can be synthesized directly. For example, this modification can increase the Tm of the RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. The 2'-O-methyl RNA base can be included in the primer, for example, to increase stability and binding affinity to the target sequence. In some embodiments, the primer may contain one or more phosphate thioester (PS) links (e.g., PS bond modification). The PS bond replaces a non-bridging oxygen atom in the primer's phosphate backbone with a sulfur atom. This modification generally makes the internucleotide linkage resistant to nuclease degradation. For example, a PS bond can be introduced between approximately the last 3 to 5 nucleotides at the 5' or 3' end of the primer to inhibit exonuclease degradation. In some embodiments, PS bonds included throughout the primer can help reduce endonuclease attack. For example, the primer may contain a 3' phosphate group. In some cases, 3' phosphorylation can inhibit degradation by certain 3' exonucleases and can be used to block elongation by DNA polymerases. In some embodiments, the primer contains one or more spacer bases (e.g., one or more C3 spacers). The C3 spacer phosphoramide may be incorporated into the primer interior or 5' end. For example, multiple C3 spacers may be added to either end of the primer to introduce long hydrophilic spacer arms for attaching fluorophores or other pendant groups.

[0152] Primers may contain DNA bases, RNA bases, or both, with one or more DNA and RNA bases being modified or unmodified. For example, primers may be a mixture of DNA and RNA bases. Primers may consist of DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, primers consist of unmodified DNA bases. In some embodiments, primers consist of modified DNA bases. Primers may consist of RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, primers consist of unmodified RNA bases. In some embodiments, primers consist of modified RNA bases. In some embodiments, primers do not contain RNA bases. In some embodiments, primers do not contain DNA bases. In some embodiments, primers do not contain cleavage recognition sites (e.g., do not contain cleavage enzyme recognition sites). In some embodiments, primers do not contain tails (e.g., do not contain tails containing cleavage enzyme recognition sites).

[0153] In some implementations, the primer sequences may be wholly or partially complementary to or substantially complementary to the target nucleic acid. Substantially complementary sequences generally refer to the nucleotide sequences that will hybridize to each other. The stringency of the hybridization conditions can be varied to tolerate different numbers of sequence mismatches. The target sequence and primer sequences may, for example, be at least 75% complementary to each other, including 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. Primers substantially complementary to the target nucleic acid sequence are also generally substantially identical to the complementary sequence of the target nucleic acid (i.e., the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid can 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.

[0154] In some embodiments, primers comprise a pair of primers. A pair of primers may include a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of the target nucleic acid). In some embodiments, primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Therefore, in some embodiments, a pair of primers is used for amplifying the target sequence, and additional primers or oligonucleotides are not included in the amplification of the target sequence (e.g., the amplification reaction component does not contain additional primer pairs for a given target sequence, does not contain nested primers, does not contain bumper primers, does not contain oligonucleotides other than primers, does not contain probes, etc.). In some embodiments, 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, primers consist of a pair of primers; however, 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 groups. An amplification primer set may include a pair of forward and reverse primers for a given target sequence. For multiplex amplification, the primers amplifying the first target sequence are considered the primer set, and the primers amplifying the second target sequence are considered a different primer set.

[0155] The nucleic acids described herein (e.g., amplification products, sample nucleic acids, target nucleic acid sequences) may comprise a first strand and a second strand complementary to each other. Amplification reaction components may comprise, or consist of, a first primer (first oligonucleotide) complementary to the target sequence in the first strand (e.g., sense strand, forward strand) of the sample nucleic acid and a second primer (second oligonucleotide) complementary to the 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 sequentially complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide sequentially complementary to the target sequence in the second strand of the sample nucleic acid. Sequential complementarity between primer and target is typically a nucleotide sequence in the primer in which each base is sequentially paired with a corresponding ordered base in the target sequence, and gaps, additional sequences, or unpaired bases in the sequence are not considered sequentially complementary. In some embodiments, the primers do not contain any additional sequences that are discontinuously complementary to the target sequence (e.g., at the 5' and / or 3' ends, or within the primer), such as additional sequences present in tailed or circularized primers, and / or additional sequences that provide cleavage recognition sites (e.g., cleavage enzyme recognition sites). In some embodiments, the amplification reaction components do not contain primers containing additional sequences (i.e., sequences other than those continuously complementary to the target sequence), such as tailed primers, circularized primers, primers capable of forming stem-loop structures, hairpin structures, and / or additional sequences that provide cleavage recognition sites (e.g., cleavage enzyme recognition sites).

[0156] In some embodiments, primers may contain modifications such as one or more inosines, a base-free site, LNA, minor groove binding agents, double-strand stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifiers that alter primer binding properties. In some embodiments, primers may contain detectable molecules or entities (e.g., fluorophores, radioisotopes, colorimetric agents, particles, enzymes, etc.).

[0157] polymerase

[0158] Amplification reaction components (e.g., one or more amplification reagents) may contain one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides into a nucleic acid target sequence (e.g., a primer annealed thereto) to extend the 3' hydroxyl terminus of a primer molecule (e.g., the amplification primers described herein). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that are active at elevated reaction temperatures (e.g., above 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C). Hyperthermophilic polymerases may or may not have strand substitution capability. In some embodiments, the polymerase may incorporate about 1 to about 50 nucleotides in a single synthesis, for example, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in a single synthesis, or any number or range between any two of these values.

[0159] The amplification reaction components may contain one or more DNA polymerases selected from the following: 9°N DNA polymerase; 9°Nm TM DNA polymerase; Therminator TM DNA polymerase; Therminator TM II DNA polymerase; Therminator TM III DNA polymerase; Therminator TM 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' exonuclease); T4 DNA polymerase; T7 DNA polymerase; Deep VentR TM (Exo-nucleotide) DNA polymerase; Deep VentR TM DNA polymerase; DyNAzyme TM EXT DNA; DyNAzyme TMII. Hot-start DNA polymerase; Phusion TM High-fidelity DNA polymerase; DNA polymerase; (Exonuclease) DNA polymerase; RepliPHI TM Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm) TM DNA polymerase); MasterAmp TM AmpliTherm TM DNA polymerase; Taq DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase β; and ThermoPhi DNA polymerase.

[0160] In some embodiments, the amplification reaction component comprises one or more hyperthermophilic DNA polymerases (e.g., thermostable hyperthermophilic DNA polymerases). The hyperthermophilic DNA polymerase may have a half-life of about 5 to 10 hours at 95°C and a half-life of about 1 to 3 hours at 100°C. For example, the amplification reaction component may comprise one or more hyperthermophilic DNA polymerases from archaea (e.g., hyperthermophilic DNA polymerases from the genus *Thermococcus*, or hyperthermophilic DNA polymerases from the archaea of ​​the family *Thermococcaceae*). In some embodiments, the amplification reaction component comprises one or more hyperthermophilic DNA polymerases from the genus *Pyrococcus*, family *Methanococcaceae*, genus *Methanococcus*, or genus *Thermus*. In some implementations, the amplification reaction components contain one or more hyperthermophilic DNA polymerases derived from *Thermus thermophiles*.

[0161] In some embodiments, the amplification reaction component comprises a thermophilic biological DNA polymerase or a functional fragment thereof. The functional fragment typically retains one or more functions of the full-length polymerase, such as the ability to polymerize DNA (e.g., in the amplification reaction). In some cases, the functional fragment performs its function at a level of at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the full-length polymerase's functional level (e.g., in the amplification reaction). The level of polymerase activity can be assessed, for example, using detectable nucleic acid amplification methods, such as those described herein. In some embodiments, the amplification reaction component comprises a thermophilic biological DNA polymerase containing the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or the functional fragment of SEQ ID NO:1 or SEQ ID NO:2.

[0162] In some embodiments, the amplification reaction component (e.g., one or more amplification reagents) comprises a polymerase containing an amino acid sequence that is at least about 90% identical to that of a hyperthermophilic biopolymerase or a functional fragment thereof. In some embodiments, the amplification reaction component comprises a polymerase containing an amino acid sequence that is 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.

[0163] Polymerases can possess reverse transcription capabilities. In such implementations, the amplification reaction can amplify the RNA target in a single step, for example, without using a separate reverse transcriptase. Non-restrictive examples of polymerases with reverse transcriptase capabilities include Bst (large fragment), 9°N DNA polymerase, and 9°Nm... TM DNA polymerase, Therminator TM Therminator TM II. The amplification reaction components may contain one or more individual reverse transcriptases. In some embodiments, the amplification reaction contains more than one polymerase. For example, the amplification reaction may contain a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0164] In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases without exonuclease activity or with low exonuclease activity are used during amplification. In some embodiments, polymerases without exonuclease activity or with low exonuclease activity contain one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, a polymerase with a modification that reduces exonuclease activity may have 10% or less exonuclease activity compared to an unmodified polymerase, such as less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase does not have 5' to 3' exonuclease activity or has low 5' to 3' exonuclease activity, and / or does not have 3' to 5' exonuclease activity or has low 3' to 5' exonuclease activity. In some embodiments, the polymerase lacks single-strand-dependent exonuclease activity or has low single-strand-dependent exonuclease activity, and / or lacks double-strand-dependent exonuclease activity or has low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that reduce or eliminate the exonuclease activity of the polymerase include substitutions of one or more amino acids at positions 141 and / or 143 and / or 458 of SEQ ID NO:1 (e.g., D141A, E143A, E143D, and A485L) or at positions corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:1.

[0165] Detection and quantification

[0166] The methods described herein may include the detection and / or quantification of nucleic acid amplification products and / or quality control products. For example, any suitable detection and / or quantification method described herein (e.g., signal-generating oligonucleotides) may be used to detect and / or quantify amplification products. Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, endpoint), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface trapping, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalation / binding dyes, absorbance methods (e.g., colorimetry, turbidimetry), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension methods (e.g., iPLEX). TMThe following technologies from Affymetrix are used: Molecular Inverted Probe (MIP) technology, Restriction Fragment Length Polymorphism (RFLP) analysis, Allele-Specific Oligonucleotide (ASO) analysis, Methylation-Specific PCR (MSPCR), Pyrosequencing analysis, Acycloprime analysis, Reverse Dot Blotting, GeneChip microarrays, Dynamic Allele-Specific Hybridization (DASH), Peptide Nucleic Acid (PNA) and LNA probes, AlphaScreen, SNPstream, Genetic Bit Analysis (GBA), Multiplex Microsequencing, SNaPshot, GOOD assay, Microarray Microsequencing, Array Primer Extension (APEX), Microarray Primer Extension, Tag Arrays, Coded Microspheres, Template-Directed Incorporation (TDI), Colorimetric Oligonucleotide Ligation Assay (OLA), Sequence-Encoded OLA, Microarray Ligation, Ligase Chain Reaction, Padlock Probes, and Invader Assay. 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, microarrays, and combinations thereof. In some embodiments, detection of nucleic acid amplification products and / or quality control products includes using real-time detection methods (i.e., detecting and / or continuously monitoring products during the amplification process). In some embodiments, detection of nucleic acid amplification products includes using endpoint detection methods (i.e., detecting products after the amplification process is completed or stopped). Nucleic acid detection methods may also employ labeled nucleotides used in probes directly incorporated into the target sequence or containing a target complementary sequence. Such labeling may be radioactive and / or fluorescent in nature and may be distinguishable in any manner discussed herein. In some embodiments, quantification of nucleic acid amplification products may be achieved using one or more detection methods described below. In some embodiments, detection methods may be used in conjunction with the measurement of signal intensity and / or the generation (or reference) of standard curves and / or lookup tables for quantifying nucleic acid amplification products and / or quality control products.

[0167] Detection of nucleic acid amplification products and / or quality control products may include the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule whose detectable properties are detectable under certain conditions, thereby enabling the molecule to function as a specific and informative signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or rate of passage through an opening of known size. A molecular beacon used to detect nucleic acid molecules may be, for example, a hairpin oligonucleotide containing a fluorophore at one end and a quenching dye at the other. The loop of the hairpin may contain a probe sequence complementary to the target sequence, while the stem is formed by annealing a complementary arm sequence located on either side of the probe sequence. The fluorophore and quenching molecule may be covalently linked at opposite ends of each arm. Fluorescent and quenching molecules approach each other under conditions that prevent hybridization of the oligonucleotide to its complementary target, or when the molecular beacon is free in solution, thereby preventing FRET. When a molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product and / or quality control product), hybridization can occur, and the loop structure transforms into a stable, more rigid conformation, leading to the separation of the fluorophore and quencher molecule, thus producing fluorescence. Due to the specificity of the probe, fluorescence generation is usually entirely due to the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes with a sequence in the amplification product that is identical or complementary to the sequence in the target nucleic acid. In other cases, the molecular beacon probe sequence hybridizes with a sequence in the amplification product that is not identical or complementary to the sequence in the target nucleic acid (e.g., hybridization with a sequence added to the amplification product via tailing primers or 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, enabling the simultaneous detection of several products in the same reaction (e.g., in multiplex reactions). For quantitative amplification (QA), molecular beacons specifically bind to the amplified target after each amplification cycle. Because unhybridized molecular beacons are dark, it is unnecessary to separate the probe-target hybrid to quantitatively determine the amount of amplified product. The generated signal is proportional to the amount of amplified product. Detection using molecular beacons can be performed in real time and can also be used as an endpoint detection method.

[0168] Detection of nucleic acid amplification products and / or quality control products may include the use of lateral flow. The use of lateral flow typically includes the use of lateral flow devices, including but not limited to test strip assays and thin-layer chromatography plates with various suitable coatings. Immobilized along the flow path are various binding reagents, binding couplers, or conjugates relating to binding couplers used in sample and signal generation systems.

[0169] Detection of nucleic acid amplification products and / or quality control products can include the use of FRET, an energy transfer mechanism between two chromophores of a donor and acceptor molecule. In short, the donor fluorophore molecule is excited at a specific excitation wavelength. When the donor molecule returns to its ground state, its subsequent emission transfers the excitation energy to the acceptor molecule via long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored and varies with the distance between the donor and acceptor, the overlap of the donor and acceptor emission spectra, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be used for quantitative molecular dynamics, for example, in DNA-DNA interactions described by 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. Probe-target hybridization causes changes in the distance or orientation of the donor and acceptor, and FRET changes are observed.

[0170] Detection of nucleic acid amplification products and / or quality control products may include the use of fluorescence polarization (FP). FP technology is based on the principle that when excited by linearly polarized light, a fluorescently labeled compound emits fluorescence, the degree of polarization being inversely proportional to its rotation rate. Therefore, when a fluorescently labeled molecule, such as a tracer-nucleic acid conjugate, is excited by linearly polarized light, the emitted light remains highly polarized because the fluorophore is restricted from rotating between the time of light absorption and emission. When a free tracer compound (i.e., not bound to a nucleic acid) is excited by linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, and the molecule is more randomly oriented; therefore, the emitted light is depolarized. Thus, fluorescence polarization provides a quantitative method for measuring the amount of tracer-nucleic acid conjugate produced in the amplification reaction.

[0171] Detection of nucleic acid amplification products and / or quality control products can include the use of surface capture, such as by immobilizing specific oligonucleotides onto a surface, resulting in a biosensor with both high sensitivity and selectivity. Examples of surfaces that can be used for probe attachment include gold and carbon. Detection of nucleic acid amplification products and / or quality control products can also include the use of 5' to 3' exonucleases to hydrolyze probes (e.g., TAQMAN). For example, TAQMAN probes are hydrolyzing probes that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The TAQMAN probe principle relies on 1) the 5' to 3' exonuclease activity of Taq polymerase to cleave the double-labeled probe during hybridization with a complementary target sequence and 2) fluorophore-based detection. The resulting fluorescent signal allows for the quantitative measurement of amplification product accumulation during the exponential phase of amplification, and TAQMAN probes can significantly increase the specificity of detection.

[0172] Detection of nucleic acid amplification products and / or quality control products may include the use of intercalation and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalation dyes exhibit enhanced fluorescence when bound to DNA or RNA). Dyes may include DNA or RNA intercalation fluorophores, including but not limited to… 82. Acridine Orange, Ethidium Bromide, Hoechst Dyes Propranolol iodide, I (Asymmetric cyanine dye) II, TOTO (thiazole orange dimer), and YOYO (oxazolium yellow dimer). Detection of nucleic acid amplification products and / or quality control products may include the use of absorbance methods (e.g., colorimetry, turbidimetry). 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 concentration using Beer-Lambert's law, which correlates absorbance with concentration using the measured path length and extinction coefficient. Detection of nucleic acid amplification products and / or quality control products may include 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 can be ionized, and the resulting ions are separated in an electric and / or magnetic field according to their mass-to-charge ratio, and a detector measures the mass-to-charge ratio of the ions. Mass spectrometry methods include, for example, MALDI, MALDI-TOF, and electrospray ionization. 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 high-speed signal acquisition and automated analysis away from solid surfaces.

[0173] Detection of nucleic acid amplification products and / or quality control products may include the use of nucleic acid sequencing. The entire sequence or a portion of the amplification product can be determined, and the determined nucleotide sequence may be referred to as a read. For example, linear amplification products can be analyzed directly without further amplification (e.g., by using single-molecule sequencing methods). In some embodiments, the linear amplification products are further amplified and then analyzed (e.g., using ligation sequencing or pyrosequencing methods). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, ligation sequencing, pyrosequencing, synthetic sequencing, single-molecule sequencing, multiplex sequencing, solid-phase single-nucleotide sequencing, and nanopore sequencing. Detection of nucleic acid amplification products and / or quality control products may include the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., company).

[0174] Lysis buffer

[0175] pyrolysis agent

[0176] As disclosed herein, the pyrolysis agent may include a detergent. The detergent may include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. Anionic surfactants may include NH4. + K + Na + Or Li + As a counterion, cationic surfactants may contain I... - ,Br - or Cl - As a counter ion.

[0177] The cleavage agents described herein can also act as denaturants. The terms "denaturing agent" or "denaturant" as used herein should be given their general meaning and include any compound or material that causes reversible protein unfolding. The strength of a denaturing agent or denaturant will be determined by both the properties and concentration of the specific agent or denaturant. Suitable denaturing agents or denaturants include cleaving agents, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations of two or more such agents. Suitable cleaving agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sodium dodecyl sarcosinate, mild nonionic detergents (e.g., digitalis saponins), mild cationic detergents (e.g., N->2,3-(dioleoyloxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including but not limited to sulfobetaine (Zwittergent), 3-(3-chloroamidopropyl)dimethylammonium-1-propane sulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonium-2-hydroxy-1-propane sulfonate (CHAPSO). Organic, water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols, such as ethanol or isopropanol), or lower alkyldiols (especially C2-C4 alkyldiols such as ethylene glycol) may be used as denaturants. Phospholipids can be naturally occurring phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants, such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0178] Suitable surfactant levels can be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% of the total composition by weight. In some embodiments, the surfactant is anionic, amphoteric, nonionic, zwitterionic, cationic, or mixtures thereof. In some embodiments, the use of anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof) may be advantageous.

[0179] Useful anionic surfactants in this article include water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl group, and 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.

[0180] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having long alkyl chains containing about 8 to 18 carbon atoms, such as lauryltrimethylammonium chloride; hexadecylpyridine chloride; benzalkonium chloride; hexadecyltrimethylammonium bromide; diisobutylphenoxyethyl dimethylbenzylammonium chloride; cocoalkyltrimethylammonium nitrite; hexadecylpyridine fluoride; etc. In the compositions disclosed herein, some cationic surfactants can also act as bactericides.

[0181] Suitable nonionic surfactants that can be used in the compositions, methods, and kits of this disclosure are broadly defined as compounds produced by the condensation of an alkylene group (which is inherently hydrophilic) with an organic hydrophobic compound that may be inherently aliphatic and / or aromatic. Examples of suitable nonionic surfactants include poloxamer; sorbitol derivatives, such as sorbitol diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of fatty alcohols or alkylphenols; products produced by the condensation of ethylene oxide with the reaction products of propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides; and mixtures of these materials. These materials can be used to stabilize foams without causing excessive viscosity in consumer product compositions.

[0182] Amphoteric surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphine, and sulfone compounds, wherein the aliphatic groups can be straight-chain or branched, and one of the aliphatic substituents contains about 8 to 18 carbon atoms, and one contains an anionic water-soluble group, such as a carboxyl group, sulfonate, sulfate, phosphate, or phosphonate.

[0183] Exemplary anionic, single-chain surfactants include alkyl sulfates, alkyl sulfonates, alkylbenzene sulfonates, and saturated or unsaturated fatty acids and their salts. The portion of a cationic surfactant containing a polar head group may include, for example, quaternary ammonium, pyridine, sulfonium, and / or phosphonium groups. For example, the polar head group may include trimethylammonium. Exemplary cationic, single-chain surfactants include alkyltrimethylammonium halide, alkyltrimethyltoluenesulfonate ammonium, and N-alkylpyridine halides.

[0184] reducing agent

[0185] Lysis buffer and / or reagent compositions (e.g., dry compositions) may contain one or more reducing agents. A “reducing agent” can be a single compound or a group of compounds. As used herein, a “reducing agent,” also referred to as a “reductant,” “reducant,” or “reduction equivalent,” can refer to an element or compound that donates electrons to another substance. In particular, reducing agents are typically compounds that break disulfide bonds through reduction, thereby overcoming those tertiary protein folds and quaternary protein structures (oligomeric subunits) stabilized by disulfide bonds. Examples of suitable reducing agents include, but are not limited to, 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteine, TBP, dithioerythritol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-mercaptoacetate, sulfite, mercaptoacetic acid, and HED. In some embodiments of the methods, compositions, and kits provided herein, the lysis buffer and / or reagent compositions (e.g., dry compositions) do not contain one or more reducing agents.

[0186] reagent composition

[0187] The reagent compositions described herein (e.g., drying compositions) may be provided in a “dry form” or in a form not suspended in a liquid medium. A “dry form” of the composition may include a dried powder, a lyophilized composition, a spray-dried composition, or a precipitated composition. A “dry form” composition may contain one or more lyotropic protectants, such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Other exemplary lyotropic protectants include gelatin, dextrin, modified starch, and carboxymethyl cellulose. As used herein, the terms “lyophilized,” “lyophilized,” and “freeze-dried” refer to a process in which the material to be dried is first frozen and then the ice or freezing solvent is removed by sublimation in a vacuum environment. “Lyophilized material” refers to a lyophilized substance.

[0188] The reagent composition (e.g., a dried composition) may be freeze-dried, lyophilized, or spray-dried. The reagent composition may be heat-dried. The reagent composition may contain one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). Sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof. Polymers may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, peptides, collagen peptides, or any combination thereof. Lyophilized reagents may include poly(rA), EGTA, EDTA, Tween 80, and / or Tween 20.

[0189] Compositions that are freeze-dried, lyophilized, spray-dried, or heat-dried, or aqueous compositions used to prepare such compositions, may contain one or more of 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 F127 (polyoxyethylene-polyoxypropylene copolymer), also known as poloxamer, poloxamine, and sodium dodecyl sulfate; (iii) disaccharides, such as trehalose, sucrose, lactose, and maltose; (iv) polymers (which may have different molecular weights), such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, and albumin; and (v) amino acids, such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and γ-aminobutyric acid.

[0190] The reagent composition (e.g., a dried composition) may contain one or more protective agents and one or more amplification reagents. One or more protective agents may contain cyclodextrin compounds. Cyclodextrins (CDs) can be used to complex with lysis agents (e.g., SDS). Cyclodextrins (CDs) can be cyclic oligosaccharides resembling truncated cones with a hydrophobic lumen and a hydrophilic outer surface. The most commonly used natural cyclodextrins include 6, 7, and 8 glucose units, referred to as α, β, and γ-CDs. Natural CDs can be soluble. Chemically modified CDs, such as hydroxypropyl derivatives, will increase solubility in aqueous media by up to 50%. It is the trade name for WACKER cyclodextrin derivatives, including various α, β, and γ-CD derivatives. β-CD can form strong inclusion complexes with sodium dodecyl sulfate (SDS) (stronger than α-CD and β-CD), with a stoichiometric ratio mainly of 1:1. The binding constant of β-CD to SDS can reach 2100 M. -1 Up to 2500M -1 Within the range.

[0191] Reagent test kit

[0192] This document discloses kits for monitoring amplification reactions. In some embodiments, the kit comprises: the quality control template disclosed herein; the quality control primers disclosed herein; the signal-generating oligonucleotides disclosed herein; and / or supplementary quality control primers disclosed herein. The kit may comprise: a reagent composition containing one or more amplification reagents, the amplification reagents containing one or more components (e.g., forward primers, reverse primers) for amplifying target nucleic acid sequences under isothermal amplification conditions. In some embodiments, the quality control template, signal-generating oligonucleotides, quality control primers, supplementary quality control primers, and / or one or more components for amplification are in lyophilized or freeze-dried form and / or present in the reagent composition.

[0193] The kit may contain at least one component that provides real-time detection activity for nucleic acid amplification products and / or quality control products. Real-time detection activity may be provided by a molecular beacon. The reagent composition (e.g., a dried composition) may contain reverse transcriptase and / or reverse transcription primers.

[0194] In some embodiments, the molar ratio of one or more protective agents to one or more amplification reagents is between about 10:1 and about 1:10 (e.g., about 2:1). In some embodiments, one or more additives include Tween 20, Triton X-100, Tween 80, nonionic detergents (e.g., nonionic surfactants), or any combination thereof. In some embodiments, one or more protective agents comprise cyclodextrin compounds. In some embodiments, one or more lysis reagents constitute about 0.001% (w / v) to about 1.0% (w / v) (e.g., about 0.2% (w / v)) of the treated sample. In some embodiments, one or more lysis reagents include detergents. Detergents may include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. In some embodiments, it is advantageous that one or more protective agents are capable of isolating one or more lysis reagents, thereby preventing one or more amplification reagents from being denatured by one or more lysis reagents.

[0195] The kit may contain, 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. When amplifying a single target, the kit may contain a primer pair (forward and reverse). When amplifying multiple target sequences, the kit may contain more than one primer pair. The kit may contain a control polynucleotide, and when amplifying multiple target sequences, the kit may include more than one control polynucleotide.

[0196] Enzymes having hyperthermophilic biopolymerase activity may 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, enzymes having hyperthermophilic biopolymerase activity may include the amino acid sequence of SEQ ID NO:1.

[0197] Nucleic acid amplification products can be approximately 20 to 40 bases long. Nucleic acid amplification products may contain: (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 region sequence flanking the sequence of (1) the first primer and its reverse complement and (2) the second primer and its reverse complement, wherein the spacer region sequence is 1 to 10 bases long.

[0198] Biological entities may include one or more of the following: prokaryotic cells, eukaryotic cells, viral particles, exosomes, protoplasts, and microvesicles. Biological entities may include viruses, bacteria, fungi, protozoa, parts thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, bacteria, fungi, or protozoa. The sample nucleic acid may be derived from a virus, bacteria, fungi, or protozoa.

[0199] The kit may also include one or more components from any number of individual dishes, chambers, containers, pouches, test tubes, vials, microtiter plates, etc., or the components may be combined in various combinations within such containers. For example, the components of the kit may be present in one or more containers. In some embodiments, all components are provided in one container. In some embodiments, the enzyme (e.g., one or more polymerases and / or one or more reverse transcriptases) may be provided in a separate container from the primers. The components may be, for example, lyophilized, heat-dried, freeze-dried, or in a stable buffer. In some embodiments, one or more polymerases and / or one or more reverse transcriptases are in lyophilized or heat-dried form in a single container, and the primers are lyophilized, heat-dried, freeze-dried, or in a buffer in different containers. In some embodiments, the polymerase and / or reverse transcriptase and the primers are in lyophilized or heat-dried form in a single container.

[0200] The kit may contain vials of water or buffer solutions, such as dNTPs used in the reaction, or modified nucleotides, glassware, cuvettes, or other containers used in the reaction, or for rehydration of lyophilized or heat-dried components. For example, the buffer solution used may be suitable for both polymerase and primer annealing activities. The kit may also contain instructions for performing one or more of the methods described herein and / or descriptions of one or more components described herein. Instructions for use and / or descriptions may be in printed form and may be included in a kit insert. The kit may also contain a written description of the Internet location providing such instructions or descriptions. The kit may contain reagents for the detection method, such as reagents for FRET, lateral flow devices, test strips, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads.

[0201] Example

[0202] Some aspects of the implementation schemes discussed above are further disclosed in detail in the following embodiments, which are not intended to limit the scope of this disclosure in any way.

[0203] Example 1

[0204] Hairpin Internal Comparison (HPIC) Principle Verification

[0205] This example demonstrates the use of the methods and compositions described herein to monitor amplification reactions. Figure 1 A non-limiting exemplary embodiment of the DNA hairpin internal control assay reaction disclosed herein and tested in this embodiment is described—a two-component MB-IC hairpin method. The IC primer extends over a molecular beacon (e.g., a hairpin-shaped probe), ultimately producing two hairpin products having the same stem sequence and 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 an IC primer and a molecular beacon IC (Table 1). Figure 2 The thermal setup for the hairpin IC (HPIC) isothermal amplification reaction described in this embodiment is depicted. In the reaction described in this embodiment, the molecular beacon IC (HPIC) is detected via a HEX fluorophore, and the target (Neisseria gonorrhoeae (Ng)) is detected via a ROX fluorophore. The IC templates used are also shown in Table 1. In some embodiments, the probes (e.g., molecular beacons) provided herein contain a 5' modification (e.g., 5HEX). In some embodiments, the probes (e.g., molecular beacons) provided herein contain a 3' modification (e.g., 3IABkFQ).

[0206] Table 1: Components determined by HPIC

[0207]

[0208] The performance of the dual system for detecting hairpin IC (400 nM) and target gonococci (Ng) was evaluated in clean samples and 10% normal urine samples and in titration with HPIC template. Figures 3A-3B The sample shown is compared with one containing 10% normal urine. Figure 3B Compared to clean samples, Figure 3A Data related to target (Ng)(ROX) detection and hairpin IC detection (HEX) in ). Figure 4A The reaction of Ng alone was shown, and Figures 4B-4D The reaction was depicted with increasing levels of IC template (ICT): 0 ICT in clean samples and 10% urine samples. Figure 4B ), 50k ICT Figure 4C ) and 500k ICT ( Figure 4D These results show that HPIC (e.g., MB2-HEX) works both as a beacon for detection and as a template. The results of including additional IC templates in the mixture were examined, and it was found that adding IC templates can make the IC too strong to have accurate detection, and is not bound by any particular theory, allowing it to compete with the target (e.g., Ng) signal.

[0209] Next, the performance of the dual system of hairpin IC (400 nM) and target (Ng) detection was evaluated in samples with increased levels of inhibitory urine: 10% ( Figure 5A ), 20% Figure 5B ) and 30% Figure 5C MB hairpin IC was found to amplify detectably in samples containing 10% or 20% suppressive urine.

[0210] Figure 6A Data related to the detection of Ng in normal urine in the absence of an internal control were presented. An Ng ROX probe was used for target detection, and a Syto 82 intercalating dye was used for both specific and nonspecific product detection. Figure 6B Data related to the detection of Ng / hairpin IC in normal urine are presented, wherein the hairpin IC probe (HPIC) is detected by HEX fluorophore and the target (Ng) is detected by ROX fluorophore.

[0211] The MB hair clip IC used in this embodiment also showed similar results across different batches. Figures 7A-7B Data comparing the detection of target (Ng) (ROX) and hairpin IC (HEX) in samples from different batches containing 20% ​​inhibitory urine and hairpin IC are presented: 9.14 ( Figure 7A ) and 10.12 Figure 7B ).

[0212] In summary, this embodiment demonstrates that the hairpin IC MB can function as both an IC beacon and a template without requiring the addition of an additional IC template. The addition of the hairpin IC MB does not negatively interfere with target (Ng) detection. The hairpin IC can be fine-tuned to track matrix inhibition of target amplification. The IC response performs well in samples containing interfering substances (e.g., in 10% normal urine, 10% inhibitory urine, and 20% inhibitory urine matrices). The IC signal can increase with decreasing target (Ng) concentration. In some cases, there is a difference of approximately 1 minute between the Ng signal Td and the IC signal Td in 10% inhibitory urine, and approximately 2 minutes in 20% inhibitory urine. In a sample with 10% inhibitory urine and a 10 cp / ul target sequence, the target Td is approximately 4 minutes, and the IC signal appears at approximately 5 minutes. In a sample with 20% inhibitory urine and a 10 cp / ul target, the target Td is approximately 7 minutes, and the IC signal appears at approximately 9 minutes.

[0213] Example 2

[0214] Hairpin Internal Control (HPIC) Molecular Beacon Modification

[0215] This embodiment illustrates an exemplary modification of a signal-generating oligonucleotide (e.g., a molecular beacon) as described herein. For ease of amplification and detection, the product hybridization melting temperature (Tm) can be designed to be greater than or equal to the product hairpin Tm. The LNA-modified signal-generating oligonucleotide (e.g., a molecular beacon) in the spacer region can be used for IC product detection.

[0216] In this embodiment, the hairpin-shaped internal control target was amplified for 10 minutes in the APA reaction, while simultaneously being amplified by a molecular beacon ( Figure 9A ) and intercalating dyes ( Figure 9D The reaction was then performed. Immediately after the reaction, the reaction temperature was raised from the measurement temperature to 90°C for melt-up curve analysis. Figure 9B and Figure 9E ) and chain-resolver derivative evaluation ( Figure 9C and Figure 9F The sequences of the two molecular beacons (50 nM) and the internal control 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 position of the LNA modification on the beacons. The resulting melting temperatures of the two beacons, MB1 (red curve, upper right) and MB2 (green curve, lower right), differ by 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 (e.g., molecular beacons) provided herein contain a 3' modification (e.g., 3IABkFQ).

[0217] Table 2: Components determined by IC

[0218]

[0219] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically impractical. Those skilled in the art will understand 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 alterations are intended to fall within the scope of the subject matter defined by the appended claims.

[0220] Regarding the use of substantially any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or from singular to plural where appropriate for the context and / or application. For clarity, various singular / plural arrangements may be explicitly set forth herein. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly indicates otherwise. Unless otherwise stated, any reference to “or” herein is intended to cover “and / or.”

[0221] Those skilled in the art will understand that, in general, the terminology used herein, and especially in the appended claims (e.g., the body of the appended claims), is typically intended as “open-ended” 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.). Those skilled in the art will further understand that if a particular number of claims is intended to be presented, such intention will be explicitly stated in the claims, and if such a statement is absent, such intention does not exist. For example, to aid understanding, the appended claims may include the introductory phrases “at least one” and “one or more” to introduce the claims. However, the use of such wording should not be construed as meaning that introducing a claim statement with the indefinite article "a(a)" or "an" would limit any specific claim in a claim statement containing such an introduction to an embodiment containing only one such statement, even when the same claim includes the introductory wording "one or more" or "at least one" and indefinite articles such as "a(a)" or "an" (e.g., "a(a)" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce a claim statement. Furthermore, even if a specific number of claims is explicitly stated in the introductory claim statement, those skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (e.g., simply stating "two statements" without other modifiers implies at least two statements or two or more statements). Furthermore, in cases where conventions such as "at least one of A, B, and C" are used, this syntactic structure is generally intended to be understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, systems having a single A, having a single B, having a single C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In cases where conventions such as "at least one of A, B, or C" are used, this syntactic structure is generally intended to be understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" will include, but is not limited to, systems having a single A, having a single B, having a single C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will further understand that, in practice, any separate words and / or wording presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to take into account the possibility of including one, any, or both terms.

[0222] Furthermore, when features or aspects of this disclosure are described in terms of the Markush group, those skilled in the art will recognize that this disclosure is also described in terms of any individual member or subgroup of the Markush group.

[0223] As those skilled in the art will understand, for any and all purposes, such as providing a written description, all scopes disclosed herein also include any and all possible subscopes and combinations of subscopes. Any listed scope can be readily identified as sufficiently descriptive and such scopes can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, middle third, and upper third, etc. As those skilled in the art will also understand, all language such as “up to,” “at least,” “greater than,” “less than,” etc., includes the stated numbers and refers to a scope that can subsequently be decomposed into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes members of each individual. Thus, for example, a group having 1-3 items means a group having 1, 2, or 3 items. Similarly, a group having 1-5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0224] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting; the actual scope and spirit are indicated by the following claims.

Claims

1. A method for monitoring an amplification reaction, comprising: supply: A quality control template, the quality control template comprising: 5' subdomain; 3' subdomain; and a loop domain located between the 5' subdomain and the 3' subdomain, and 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; performing an amplification reaction on the quality control template and the quality control primer to generate a first quality control product; and The first quality control product is detected.

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

3. The method according to any one of claims 1 to 2, wherein performing an amplification reaction on the quality control template and the quality control primer to generate a first quality control product comprises: The quality control template is amplified with the quality control primers in the amplification reaction mixture under the amplification conditions to generate the first quality control product.

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

5. The method according to any one of claims 1 to 4, wherein the method further comprises: Providing an enzyme having polymerase activity, optionally the enzyme having polymerase activity is an enzyme having hyperthermophilic polymerase activity, and further optionally the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity; and / or Reverse transcriptase is provided.

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

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

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

9. The method according to any one of claims 1 to 8, wherein: 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; The first quality control product and the second quality control product have the same stem domain; and / or The first quality control product and the second quality control product comprise loop domains that are complementary to each other.

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

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

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

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

14. The method according to 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, optionally, the combined sequence of the 5' terminal domain and the 5' subdomain comprises the entire sequence of the quality control primer.

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

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

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

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

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

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

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

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

23. The method according to any one of claims 1 to 22, wherein the method further comprises: providing a signal generating oligonucleotide; performing the amplification reaction on the signal generating oligonucleotide; and The first quality control product is detected using the signal generating oligonucleotide.

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

25. The method of any one of claims 1-24, wherein 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.

26. The method of any one of claims 1-25, 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. The method of any one of claims 1-26, 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 method of any one of claims 1-27, wherein 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 the quality control primer.

29. The method according to any one of claims 1 to 28, wherein: The quality control template does not contain 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.

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

31. The method of any one of claims 1-30, 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. The method of any one of claims 1-31, wherein the amplification reaction comprises contacting a reverse transcriptase with the quality control template, thereby generating an extended quality control template, optionally wherein the extended quality control template comprises cDNA.

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

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

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

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

37. The method of any one of claims 1-36, wherein the reverse transcriptase cleaves the quality control template at the 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 a label.

38. The method of any one of claims 1-37, 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. The method according to any one of claims 1 to 38, wherein the method further comprises: Provides supplemental quality control primers; and The amplification reaction is performed on the supplemented quality control primers.

40. The method according to any one of claims 1 to 39, wherein: The signal generating oligonucleotide has a length of about 10 nucleotides to about 100 nucleotides; The quality control template has a length of about 10 nucleotides to about 100 nucleotides; The quality control primer and / or the supplemental quality control primer has a length of about 5 nucleotides to about 25 nucleotides; and / or The 5' subdomain, the 3' subdomain, the loop domain, the 5' terminal domain and / or the 3' terminal domain have a length of about 1 nucleotide to about 25 nucleotides.

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

42. The method of any one of claims 1-41, 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. The method of any one of claims 1-42, wherein the signal generating oligonucleotide comprises one or more locked nucleic acids (LNAs), optionally wherein the one or more LNAs are located within the loop domain and / or stem domain, and optionally wherein the one or more LNAs enhance the detectability of the first quality control product.

44. A method according to any one of claims 1-43, wherein the signal generating oligonucleotide is configured such that the melting temperature (Tm) of the first quality control product / signal generating oligonucleotide duplex is equal to or greater than the melting temperature (Tm) of the paired stem domain of the signal generating oligonucleotide, optionally via one or more LNA configurations located in the loop domain and / or stem domain.

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

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

47. The method according to any one of claims 1 to 46, wherein said quality control template and / or said 5' terminal domain of said signal generating oligonucleotide comprises said tag, wherein 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, wherein 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 bases and / or adenine bases, And optionally, the one or more guanine bases and / or adenine bases are capable of quenching the label when the quality control template and / or the signal generating oligonucleotide forms a hairpin structure.

48. The method according to any one of claims 1 to 47, wherein said quality control template and / or said 3' terminal domain of said signal generating oligonucleotide comprises said tag, wherein 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, wherein 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 bases and / or adenine bases, And optionally, the one or more guanine bases and / or adenine bases are capable of quenching the label when the quality control template and / or the signal generating oligonucleotide forms a hairpin structure.

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

50. The method of any one of claims 1-49, wherein the generation of the first quality control product and the second quality control product is associated with a decrease in the detected signal.

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

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

53. The method of any one of claims 1-52, wherein: When the quality control primer binds to the quality control template and / or the second quality control product and is extended with an enzyme having polymerase activity to generate a first 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 first quality control product are capable of quenching the label when the first quality control product forms a hairpin structure; and / or When the quality control primer binds to the first quality control product and is extended with 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 when the second quality control product forms a hairpin structure.

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

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

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

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

58. The method according to any one of claims 1 to 57, wherein the method comprises: performing an amplification reaction on the target nucleic acid sequence to generate a nucleic acid amplification product, and The nucleic acid amplification product is detected using a target signal generating oligonucleotide, wherein the target signal generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product.

59. The method according to any one of claims 1-58, wherein subjecting the target nucleic acid sequence to an amplification reaction capable of generating a nucleic acid amplification product comprises: The target nucleic acid sequence is amplified in the amplification reaction mixture under the amplification conditions to generate a nucleic acid amplification product.

60. The method according to any one of claims 1 to 59, comprising: contacting a sample comprising a biological entity with a lysis buffer to generate a treated sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entity to release sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of comprising the target nucleic acid sequence; and The reagent composition is contacted with the treated sample to generate the amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

61. The method of any one of claims 1-60, wherein the one or more amplification reagents comprise: Reverse transcriptase; An enzyme having a hyperthermophilic biopolymerase activity, optionally wherein the enzyme having a hyperthermophilic biopolymerase activity has a reverse transcriptase activity; Forward primer; Reverse primer; Reverse transcription primers; and / or dNTPs.

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

63. The method of any one of claims 1-62, wherein amplifying the target nucleic acid sequence comprises: A target nucleic acid sequence comprising a first strand and a second strand complementary to each other is amplified under isothermal amplification conditions, wherein the amplification comprises 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 hyperthermophilic biopolymerase activity, thereby generating the nucleic acid amplification product.

64. The method of any one of claims 1-63, wherein the nucleic acid is: double-stranded DNA; and / or A product of a reverse transcription reaction, optionally the nucleic acid is a product of a reverse transcription reaction generated from sample RNA, and optionally the amplification includes generating the nucleic acid by a reverse transcription reaction.

65. The method of any one of claims 1-64, wherein: The amplification reaction is carried out for a period of time ranging from about 5 minutes to about 60 minutes; amplifying the quality control template comprises generating 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 generating the nucleic acid amplification product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes; and / or The detecting is 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 treated sample.

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

2.

67. The method of any one of claims 1-66, 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 method of any one of claims 1-67, wherein the reagent composition is lyophilized, heat dried, and / or comprises one or more additives, wherein the one or more additives include: Tween 20, Triton X-100 and / or Tween 80; Amino acids; A sugar or sugar alcohol, optionally comprising sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof; and / or A polymer, optionally comprising polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptide, collagen peptide or any combination thereof, And optionally, contacting the reagent composition with the treated sample comprises dissolving the reagent composition in the treated sample.

69. The method of any one of claims 1-68, wherein the one or more lysis reagents comprise: about 0.001% (w / v) to about 1.0% (w / v) of said treated sample, optionally about 0.2% (w / v) of said treated sample; and / or A detergent, optionally comprising one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant and an amphoteric surfactant.

70. The method of any one of claims 1-69, wherein the method: Performed in a single reaction vessel; excluding the use of any enzyme other than the reverse transcriptase and the enzyme having hyperthermophilic biopolymerase activity; does not include the use of any enzyme other than the enzyme having hyperthermophilic biopolymerase activity; does not include thermal and / or enzymatic denaturation of the nucleic acid and / or quality control template during the amplification step; and / or Contacting the nucleic acid and / or quality control template with a single-stranded DNA binding protein is not included.

71. The method of any one of claims 1-70, wherein: The target nucleic acid sequence comprises a length of no longer than about 20 nucleotides to no longer than about 90 nucleotides, 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 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. The method of any one of claims 1-71, 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 method of any one of claims 1-72, wherein the amplifying: The amplification is performed for a period of about 5 minutes to about 60 minutes, optionally the amplification is performed for a period of about 15 minutes; and / or It is performed under isothermal amplification conditions without helicase, single-strand binding protein, nicking agent and recombinase.

74. The method according to any one of claims 1-73, wherein the enzyme having a 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 having a 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, optionally the enzyme having a hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1, optionally the enzyme having a hyperthermophilic polymerase activity has low exonuclease activity or no exonuclease activity.

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

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

77. The method of any one of claims 1-76, wherein the sample is a biological sample or an environmental sample, wherein the environmental sample is or is obtained from: a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a salt water sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof; and / or The biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof.

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

79. The method of any one of claims 1-78, wherein the amplification comprises one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (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 amplification does not comprise loop-mediated isothermal amplification (LAMP).

80. A method according to any one of claims 1-79, wherein the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) ultrasonic treatment of the treated sample; (v) ultrasonic treatment of the amplification reaction mixture; (vi) addition of ribonuclease inhibitors to the treated sample; (vii) addition of ribonuclease inhibitors to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of the one or more cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

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

82. The method of any one of claims 1-81, wherein the method comprises determining the presence, absence and / or amount of the first quality control product.

83. The method of any one of claims 1-82, wherein the presence, absence and / or amount of the signal indicates: the presence, absence and / or amount of the 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) malfunction of the apparatus in which the amplification reaction is performed; and / or (iii) sample-derived inhibition of the amplification reaction, optionally including matrix-derived inhibition; and / or The degree to which amplification of the target nucleic acid sequence is inhibited in the amplification reaction.

84. The method of any one of claims 1-83, wherein the method, the reagent composition, and / or the amplification reaction mixture comprises at least about 50,000 copies to about 10 copies of the quality control template. 12 Optionally, a comparable method of monitoring an amplification reaction employs about 20 copies to about 100 copies of an internal control template.

85. The method of any one of claims 1-84, 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 the quality control primer compared to a comparable method for monitoring an amplification reaction that does not comprise the quality control template and / or the quality control primer, optionally wherein the comparable method comprises an internal control template that is not capable of forming a hairpin structure.

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

87. The method of any one of claims 1-86, wherein the quality control template and / or the quality control primer are incapable of hybridizing to the target nucleic acid sequence.

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

89. The method of any one of claims 1-88, wherein the presence of the quality control template and / or the quality control primers 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 times compared to a comparable method wherein the quality control template and / or the quality control primers are not present in the amplification reaction mixture, optionally wherein the comparable method comprises an internal control template that is not capable of forming a hairpin structure.

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

91. A kit for monitoring an amplification reaction, comprising: A quality control template as defined in any one of claims 1 to 90; A quality control primer as defined in any one of claims 1 to 90; A signal generating oligonucleotide as defined in any one of claims 1 to 90; and / or Supplementary quality control primers as defined in any one of claims 1-90.

92. The kit according to any one of claims 91, comprising: a lysis buffer comprising one or more lysis agents capable of lysing a biological entity to release sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence, optionally wherein the one or more lysis agents comprise a detergent, and wherein the detergent comprises 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, wherein the amplification reagents comprise one or more components for amplifying the target nucleic acid sequence under isothermal amplification conditions.

93. The kit of any one of claims 91-92, wherein the one or more components for amplification comprise: (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 a hyperthermophilic biopolymerase activity, wherein the enzyme is capable of generating a nucleic acid amplification product, wherein the enzyme having a hyperthermophilic biopolymerase 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, wherein the enzyme having a hyperthermophilic biopolymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, and wherein the enzyme having a hyperthermophilic biopolymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:

1.

94. A kit according to any one of claims 91-93, wherein the quality control template, the signal generating oligonucleotide, the quality control primer, the supplemental 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 reaction mixture comprising: A quality control template as defined in any one of claims 1 to 90; A quality control primer as defined in any one of claims 1 to 90; A signal generating oligonucleotide as defined in any one of claims 1 to 90; Supplementary quality control primers as defined in any one of claims 1 to 90; target nucleic acid sequence; and / or One or more additional primers and / or one or more probes specific for the target nucleic acid sequence.

96. The reaction mixture of claim 95, wherein the one or more additional primers and / or one or more probes specific for the target nucleic acid sequence comprise: forward primer and / or 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 is capable of hybridizing to a nucleic acid amplification product generated by amplifying the target nucleic acid sequence.

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

98. The reaction mixture of any one of claims 95-97, wherein the enzyme having hyperthermophilic biopolymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof, and optionally the enzyme having hyperthermophilic biopolymerase activity comprises the amino acid sequence of SEQ ID NO: 1.

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