Archaea polymerase amplification

Abstract

The disclosure herein includes methods, compositions, and kits for detecting more than one nucleic acid sequence. The method can include amplifying a first nucleic acid sequence and a second nucleic acid sequence in an amplification reaction mixture, thereby generating a first nucleic acid amplification product and a second nucleic acid amplification product. The method may include detecting a first nucleic acid amplification product and a second nucleic acid amplification product with a first signal-generating oligonucleotide and a second signal-generating oligonucleotide, respectively, in a same optical channel.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 374,831, filed on September 7, 2022, under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Reference to a sequence listing

[0004] This application is submitted together with a sequence listing in electronic format. The sequence listing is provided as a file entitled 68EB-317352-WO, created on September 6, 2023, and is 23,010 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0005] background

[0006] field

[0007] The present disclosure generally relates to methods and compositions for amplifying (eg, isothermal amplification) nucleic acids.

[0008] Description of the Prior Art

[0009] Nucleic acid-based diagnosis can be used for rapid detection of infection, disease and / or genetic variation. For example, the identification of bacterial or viral nucleic acids in a sample can be used for diagnosing a specific type of infection. Other examples include the identification of single nucleotide polymorphisms for disease management or forensics, and the identification of genetic variation of food products indicating genetic modification. Generally, nucleic acid-based diagnostic assays require a specific portion of nucleic acid in an amplified sample. The common technique for nucleic acid amplification is polymerase chain reaction (PCR). This technology generally requires temperature cycling (i.e. thermal cycling) to be performed by denaturation (e.g., separation of chains in double-stranded DNA (dsDNA) complexes), annealing of oligonucleotide primers (short chains of complementary DNA sequences) and by polymerase extending primers along complementary targets. Such thermal cycling can be a time-consuming process that generally requires specialized agencies. Therefore, there is a demand for a faster nucleic acid amplification method that can be performed without thermal cycling. In particular, there is a demand for a multiplexed nucleic acid amplification method that can be performed without thermal cycling.

[0010] Overview

[0011] The disclosure herein includes methods for detecting more than one nucleic acid sequence. In some embodiments, the method includes: amplifying a first nucleic acid sequence and a second nucleic acid sequence in an amplification reaction mixture, thereby generating a first nucleic acid amplification product and a second nucleic acid amplification product, respectively; and detecting the first nucleic acid amplification product and the second nucleic acid amplification product with a first signal generating oligonucleotide and a second signal generating oligonucleotide, respectively, in the same optical channel. In some embodiments, the first signal generating oligonucleotide and the second signal generating oligonucleotide each comprise a label. In some embodiments, the detection includes detecting a signal of a label of the first signal generating oligonucleotide and the second signal generating oligonucleotide before amplification, during amplification, after amplification, or any combination thereof.

[0012] 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 lytic agents capable of lysing the biological entity to release sample nucleic acid contained therein; and contacting a reagent composition with the treated sample to generate an amplification reaction mixture, wherein the reagent composition contains one or more amplification reagents.

[0013] In some embodiments, the first nucleic acid sequence is a first target nucleic acid sequence. In some embodiments, the second nucleic acid sequence is a second target nucleic acid sequence. In some embodiments, the sample nucleic acid is suspected of comprising a first target nucleic acid sequence and a second target nucleic acid sequence. In some embodiments, the first nucleic acid sequence is a first target nucleic acid sequence. In some embodiments, the second nucleic acid sequence is an internal control (IC) nucleic acid sequence. In some embodiments, the sample nucleic acid is suspected of comprising a first target nucleic acid sequence. In some embodiments, the IC nucleic acid sequence is a quality control template, and wherein the second amplification product is a first quality control product. In some embodiments, detection is performed using an instrument comprising 6, 5, 4, 3, 2 or 1 optical channels. In some embodiments, the melting temperatures (Tm) of the first signal generating oligonucleotide and the second signal generating oligonucleotide differ by at least about 2°C.

[0014] In some embodiments, one or more amplification reagents include: an enzyme having a hyperthermophilic polymerase activity, optionally the enzyme having a hyperthermophilic polymerase activity has reverse transcriptase activity; two or more primer pairs, wherein each primer pair comprises a forward primer and a reverse primer; dNTPs; reverse transcriptase; and / or one or more reverse transcription primers.

[0015] In some embodiments, amplification is performed at the optimal temperature of the enzyme having the hyperthermophilic polymerase activity, optionally the optimal temperature is about 66° C. to about 68° C. In some embodiments, the first signal generating oligonucleotide has a Tm within about 1° C. of the optimal temperature of the enzyme having the hyperthermophilic polymerase activity. In some embodiments, the second signal generating oligonucleotide has a Tm that differs from the optimal temperature of the enzyme having the hyperthermophilic polymerase activity by at least about 2° C.

[0016] In some embodiments, detection includes contacting the first nucleic acid amplification product and the second nucleic acid amplification product with the first signal generating oligonucleotide and the second signal generating oligonucleotide for hybridization. In some embodiments, the first signal generating oligonucleotide and the second signal generating oligonucleotide respectively comprise a first label and a second label, optionally the first label and the second label are the same or different. In some embodiments, the first label and the second label are capable of generating a signal when the first signal generating oligonucleotide and the second signal generating oligonucleotide are hybridized with the first nucleic acid amplification product and the second nucleic acid amplification product, respectively. In some embodiments, when the first signal generating oligonucleotide and the second signal generating oligonucleotide are hybridized with the first nucleic acid amplification product and the second nucleic acid amplification product, respectively, the first label and the second label generate a first signal and a second signal, respectively. In some embodiments, the first signal and the second signal are indistinguishable. In some embodiments, the signal is fluorescence.

[0017] In some embodiments, detecting the signal of the label of the first signal generating oligonucleotide and the second signal generating oligonucleotide comprises detecting the fluorescence emitted by the first label and the second label, respectively. In some embodiments, detecting comprises: detecting the signal of the first label during amplification, optionally in real time; and detecting the signal of the second label after amplification, optionally, not detecting the signal of the second label during amplification. In some embodiments, detecting the signal of the second label after amplification comprises one or more cycles performed at the Tm of the second signal generating oligonucleotide.

[0018] The first signal generating oligonucleotide and the second signal generating oligonucleotide may each include: a 5' subdomain; a 3' subdomain; and a loop domain located between the 5' subdomain and the 3' subdomain, wherein intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, the paired stem domain of the second signal generating oligonucleotide is configured to have a melting temperature (Tm) that is at least about 2°C higher or lower than the optimal temperature of the enzyme, optionally by modifying the length of the paired domain, the GC content of the paired domain, and / or the presence of one or more chemical modifications in the paired domain.

[0019] In some embodiments, the first nucleic acid amplification product comprises: (1) the sequence of the first forward primer and its reverse complement, (2) the sequence of the first reverse primer and its reverse complement, and (3) a first spacer sequence flanked by (1) the sequence of the first forward primer and its reverse complement and (2) the sequence of the first reverse primer and its reverse complement, wherein the first spacer sequence is 1 to 10 bases long. In some embodiments, the second nucleic acid amplification product comprises: (1) the sequence of the second forward primer and its reverse complement, (2) the sequence of the second reverse primer and its reverse complement, and (3) a second spacer sequence flanked by (1) the sequence of the second forward primer and its reverse complement and (2) the sequence of the second reverse primer and its reverse complement, wherein the second spacer sequence is 1 to 10 bases long. In some embodiments, the sample nucleic acid comprises a first nucleic acid comprising a first target nucleic acid sequence and a second nucleic acid comprising a second target nucleic acid sequence.

[0020] In some embodiments, amplifying a first target nucleic acid sequence comprises: amplifying a first target nucleic acid sequence comprising a first chain and a second chain that are complementary to each other under isothermal amplification conditions, wherein amplifying comprises contacting a first nucleic acid comprising the first target nucleic acid sequence with: i) a first forward primer and a first reverse primer, wherein the first forward primer is capable of hybridizing with a sequence of the first chain of the first target nucleic acid sequence, and the first reverse primer is capable of hybridizing with a sequence of the second chain of the first target nucleic acid sequence; and ii) an enzyme having a hyperthermophilic polymerase activity, thereby generating a first nucleic acid amplification product; and wherein amplifying a second target nucleic acid sequence comprises: amplifying a second target nucleic acid sequence comprising a first chain and a second chain that are complementary to each other under isothermal amplification conditions, wherein amplifying comprises contacting a second nucleic acid comprising the second target nucleic acid sequence with: i) a second forward primer and a second reverse primer, wherein the second forward primer is capable of hybridizing with a sequence of the first chain of the second target nucleic acid sequence, and the second reverse primer is capable of hybridizing with a sequence of the second chain of the second target nucleic acid sequence; and ii) an enzyme having a hyperthermophilic polymerase activity, thereby generating a second nucleic acid amplification product.

[0021] The first nucleic acid and the second nucleic acid can be double-stranded DNA. In some embodiments, the first nucleic acid and the second nucleic acid are products of reverse transcription reaction, optionally, the first nucleic acid and the second nucleic acid are products of reverse transcription reaction generated from sample ribonucleic acid, and optionally, step (c) includes generating the first nucleic acid and the second nucleic acid by reverse transcription reaction. In some embodiments, the sample nucleic acid includes sample ribonucleic acid, and wherein the method includes contacting the sample ribonucleic acid with reverse transcriptase and / or reverse transcription primer to generate the first cDNA and the second cDNA.

[0022] In some embodiments, amplifying the first target nucleic acid sequence and the second target nucleic acid sequence comprises: (c1) contacting the sample ribonucleic acid with a reverse transcriptase, a first reverse transcription primer and / or a second reverse transcription primer to generate a first cDNA and a second cDNA; (c2) contacting the first cDNA and the second cDNA with an enzyme having a hyperthermophilic biopolymerase activity to generate a first double-stranded DNA (dsDNA) and a second dsDNA, respectively, wherein the first dsDNA and the second dsDNA comprise the first target nucleic acid sequence and the second target nucleic acid sequence, respectively, and wherein the first target nucleic acid sequence and the second target nucleic acid sequence comprise a first strand and a second strand that are complementary to each other; and (c3) amplifying the first target nucleic acid sequence under isothermal amplification conditions. nucleic acid sequence and a second target nucleic acid sequence, wherein amplifying comprises contacting the first dsDNA and the second dsDNA with: (i) a first forward primer and a first reverse primer, wherein the first forward primer is capable of hybridizing to a sequence of a first strand of the first target nucleic acid sequence, and the first reverse primer is capable of hybridizing to a sequence of a second strand of the first target nucleic acid sequence; and (ii) a second forward primer and a second reverse primer, wherein the second forward primer is capable of hybridizing to a sequence of a first strand of the second target nucleic acid sequence, and the second reverse primer is capable of hybridizing to a sequence of a second strand of the second target nucleic acid sequence; and (iii) an enzyme having hyperthermophilic biopolymerase activity, thereby generating a first nucleic acid amplification product and a second nucleic acid amplification product, respectively.

[0023] In some embodiments, the first amplification product and the second amplification product are generated during the first amplification reaction (first amplification subreaction) and the second amplification reaction (second amplification subreaction), respectively, and optionally the first amplification product and the second amplification product are generated separately in time. In some embodiments, the amplification reaction includes: a first amplification reaction performed at a first temperature; and a second amplification reaction performed at a second temperature, wherein the first amplification reaction is performed before the second amplification reaction, wherein the first amplification reaction and the second amplification reaction are each at least about 2 minutes, optionally 5 minutes, and wherein the second temperature is at least 2°C higher than the first temperature, optionally the first temperature is 66°C and the second temperature is 70°C.

[0024] In some embodiments, the first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence are shorter than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence. In some embodiments, the first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence have a lower Tm than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence. In some embodiments, the first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence are present at a concentration lower than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence.

[0025] The first signal generating oligonucleotide and / or the second signal generating oligonucleotide may include one or more phosphorothioate linkages and / or one or more locked nucleic acids. In some embodiments, the first signal generating oligonucleotide and / or the second signal generating oligonucleotide are TaqMan detection probe oligonucleotides, 3' minor groove binder probe oligonucleotides, hairpin detection probe oligonucleotides (e.g., molecular beacons) or molecular torch (molecular torch) detection probe oligonucleotides. In some embodiments, the labeling includes a quenchable labeling, and optionally the quenchable labeling is a fluorophore; and / or the first signal generating oligonucleotide and / or the second signal generating oligonucleotide include a quencher.

[0026] In some embodiments, the method includes determining the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, determining the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample includes determining the presence, absence and / or amount of dsDNA and / or nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, the presence, absence and / or amount of the first signal and the second signal respectively indicate the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, the presence, absence and / or amount of the first signal and the second signal respectively indicate the presence, absence and / or amount of dsDNA and / or nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, amplifying the first nucleic acid sequence and / or the second nucleic acid sequence includes generating the first nucleic acid amplification product and / or the second nucleic acid amplification product at a detectable level within about 20 minutes, about 15 minutes or about 10 minutes.

[0027] In some embodiments, wherein the method does not comprise an intercalating dye; and / or detecting the first nucleic acid amplification product and the second nucleic acid amplification product does not comprise detecting a signal of an intercalating dye. In some embodiments, the melting temperatures of the first amplification product and the second amplification product are the same, and wherein the melting temperatures of the first signal generating oligonucleotide and the second signal generating oligonucleotide are different. In some embodiments, the melting temperatures of the first signal generating oligonucleotide and the second signal generating oligonucleotide differ by at least about 2°C.

[0028] In some embodiments, detecting the first nucleic acid amplification product and the second nucleic acid amplification product in the same optical channel includes a melting curve analysis (MCA). In some embodiments, MCA is performed at least about 1 minute after the amplification step. In some embodiments, MCA includes: incubating the first nucleic acid amplification product and the second nucleic acid amplification product at a series of increasing temperatures (optionally from the starting temperature to the final temperature); and detecting the signal of the label of the first signal generating oligonucleotide and the second signal generating oligonucleotide at the series of increasing temperatures, thereby generating a melting curve. In some embodiments, the starting temperature is at least about 50°C, optionally, the starting temperature is the optimal temperature of an enzyme with hyperthermophilic biopolymerase activity; and / or the final temperature is at least about 80°C, optionally 90°C. In some embodiments, the temperature conversion from the starting temperature to the final temperature is a linear function of time, optionally the linear conversion is at least 0.05°C per second. In some embodiments, MCA includes deriving a negative derivative (-dF / dt vs.T) relative to the temperature signal intensity. In some embodiments, the signal derived from the first signal generating oligonucleotide can be distinguished from the signal derived from the second signal generating oligonucleotide or its negative first-order derivative in the melting curve.

[0029] In some embodiments, the presence, absence, and / or amount of a signal at a first melting temperature in a melting curve indicates the presence, absence, and / or amount of a first amplified product. In some embodiments, the presence, absence, and / or amount of a signal at a second melting temperature in a melting curve indicates the presence, absence, and / or amount of a second amplified product. In some embodiments, the melting temperature corresponds to the highest level of the negative derivative (-dF / dT) of fluorescence with temperature (T) relative to temperature (T), and optionally also corresponds to a temperature within 1°C-4°C of the highest level.

[0030] In some embodiments, the first melting temperature corresponds to the melting temperature (Tm) of the first amplification product / first signal generating oligonucleotide duplex and / or the melting temperature (Tm) of the paired stem domain of the first signal generating oligonucleotide. In some embodiments, the second melting temperature corresponds to the melting temperature (Tm) of the second amplification product / second signal generating oligonucleotide duplex and / or the melting temperature (Tm) of the paired stem domain of the second signal generating oligonucleotide. In some embodiments, the first melting temperature differs from the second melting temperature by at least about 2°C. In some embodiments, the first signal generating oligonucleotide and / or the second signal generating oligonucleotide comprises one or more locked nucleic acids (LNAs), optionally one or more LNAs are located in the loop domain, and optionally one or more LNAs increase the difference between the first melting temperature and the second melting temperature. In some embodiments, the first signal generating oligonucleotide and / or the second signal generating oligonucleotide are configured so that the first melting temperature differs from the second melting temperature by at least about 2°C, optionally via one or more LNAs located in the loop domain.

[0031] In some embodiments, the method includes: providing: a quality control template, the quality control template comprising: a 5' subdomain; a 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; performing an amplification reaction on the quality control template and the quality control primer that is capable of generating a first quality control product; and detecting the first quality control product.

[0032] In some embodiments, the amplification reaction is performed in an amplification reaction mixture under amplification conditions (optionally isothermal amplification conditions). In some embodiments, performing an amplification reaction on a quality control template and a quality control primer that can generate a first quality control product comprises: amplifying the quality control template with a quality control primer in an amplification reaction mixture under amplification conditions, thereby generating a first quality control product. In some embodiments, the amplification reaction comprises a reverse transcription reaction.

[0033] The method may include: providing an enzyme with polymerase activity, optionally the enzyme with polymerase activity is an enzyme with hyperthermophilic polymerase activity, optionally the enzyme with hyperthermophilic polymerase activity has reverse transcriptase activity; and / or providing a reverse transcriptase. 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 to 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 a first quality control product for hybridization, and extending the quality control primer hybridized to the first quality control product with an enzyme with polymerase activity, thereby generating a second quality control product. The amplification reaction may include: contacting a quality control primer with a second quality control product for hybridization, and extending the quality control primer hybridized to the second quality control product with an enzyme with polymerase activity, thereby generating a first quality control product.

[0034] The first quality control product and the second quality control product may include a 5' subdomain and a 3' subdomain that can form 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 include loop domains that are complementary to each other. In some embodiments, the amplification reaction includes linear amplification and / or exponential amplification of the first quality control product and the second quality control product. In some embodiments, the 5' subdomain includes a sequence of at least a portion of a quality control primer. In some embodiments, both the first quality control product and the second quality control product are capable of forming a hairpin structure. In some embodiments, the quality control template includes a 5' terminal domain located at 5' of the 5' subdomain, and / or the quality control template includes a 3' terminal domain located at 3' of the 3' subdomain. In some embodiments, the 5' terminal domain of the quality control template includes at least a portion of the sequence of the quality control primer, and optionally, the combined sequence of the 5' terminal domain and the 5' subdomain includes the entire sequence of the quality control primer.

[0035] In some embodiments, detecting the first quality control product comprises detecting the first quality control product with a second signal generating oligonucleotide, optionally the second signal generating oligonucleotide is capable of hybridizing with the first quality control product. In some embodiments, detecting comprises contacting the first quality control product with the second signal generating oligonucleotide for hybridization. In some embodiments, the second 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. In some embodiments, the second signal generating oligonucleotide comprises a quencher, optionally the quencher is capable of quenching the label. In some embodiments, detecting comprises contacting the first quality control product with the second signal generating oligonucleotide for hybridization. In some embodiments, the label is capable of generating a second signal when the second signal generating oligonucleotide hybridizes with the first quality control product; and / or the label generates a second signal when the second signal generating oligonucleotide hybridizes with the first quality control product, optionally the second signal is fluorescence. In some embodiments, detecting the first quality control product comprises detecting a second signal generated by the label of the second signal generating oligonucleotide, optionally the label is a fluorophore and the second signal is fluorescence. In some embodiments, detecting includes detecting a labeled second signal before the amplification reaction, during the amplification reaction, after the amplification reaction, or any combination thereof.

[0036] In some embodiments, the method further comprises: providing a second signal generating oligonucleotide; performing an amplification reaction on the second signal generating oligonucleotide; and detecting the first quality control product with the second signal generating oligonucleotide. In some embodiments, the quality control template is the second 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) a means for detecting the first quality control product. In some embodiments, the second signal generating oligonucleotide is capable of (i) detecting the first quality control product and (ii) serving as a template for the synthesis driven by the quality control primer for the first quality control product.

[0037] In some embodiments, the 5' terminal domain of the quality control template comprises: one or more RNA nucleotides; and / or a sequence of at least a portion of a quality control primer. In some embodiments, the quality control template does not comprise a 3' terminal domain; and / or the 3' end of the quality control template is complementary to the 5' end of the 5' subdomain of the quality control template. In some embodiments, the reverse transcriptase is capable of using one or more RNA nucleotides of the 5' terminal domain of the quality control template as a template to extend the 3' end of the quality control template, thereby generating an extended quality control template. In some embodiments, the 3' end of the extended quality control template comprises a sequence complementary to at least a portion of the quality control primer. In some embodiments, the amplification reaction comprises contacting the reverse transcriptase with the quality control template, thereby generating an extended quality control template, optionally 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 a reverse transcriptase and / or an enzyme having polymerase activity, thereby generating a first quality control product.

[0038] In some embodiments, the quality control template is a second signal generating oligonucleotide, wherein the second 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. In some embodiments, the second 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. In some embodiments, the amplification reaction includes: contacting the quality control primer with the quality control template for hybridization, and extending the quality control primer hybridized with the quality control template with a reverse transcriptase, thereby generating a first quality control product, optionally the reverse transcriptase comprises RNase H activity. In some embodiments, the reverse transcriptase cuts 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 comprising a label. In some embodiments, detecting the first quality control product includes detecting a second signal generated by the first cleavage product comprising a label, optionally, the label is a fluorophore and the second signal is fluorescence. In some embodiments, the method further comprises: providing supplemental quality control primers; and performing an amplification reaction on the supplemental quality control primers.

[0039] In some embodiments, the second signal generating oligonucleotide comprises one or more locked nucleic acids (LNAs), optionally one or more LNAs are located in the loop domain, and optionally one or more LNAs enhance the detectability of the first quality control product. In some embodiments, the second signal generating oligonucleotide is configured such that the melting temperature (Tm) of the first quality control product / second signal generating oligonucleotide duplex is equal to or greater than the melting temperature (Tm) of the paired stem domain of the second signal generating oligonucleotide, optionally via one or more LNAs located in the loop domain.

[0040] In some embodiments, providing a quality control primer, a quality control template, and / or a second signal generating oligonucleotide comprises providing a reagent composition comprising a quality control primer, a quality control template, and / or a second signal generating oligonucleotide. In some embodiments, performing an amplification reaction on the quality control primer, the quality control template, and / or the second signal generating oligonucleotide comprises contacting the reagent composition with the treated sample to generate an amplification reaction mixture.

[0041] 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 second signal indicates the presence, absence, and / or amount of a first quality control product. In some embodiments, the presence, absence, and / or amount of a second signal indicates the presence, absence, and / or amount of one or more interfering components in an amplification reaction mixture. In some embodiments, the presence, absence, and / or amount of a second signal indicates: (i) the integrity of one or more amplification reagents in an amplification reaction mixture; (ii) a malfunction of an instrument in which an amplification reaction is performed; and / or (iii) inhibition of a sample source of an amplification reaction, optionally wherein the inhibition of a sample source includes inhibition of a matrix source. In some embodiments, the presence, absence, and / or amount of a second signal indicates the extent to which amplification of a first target nucleic acid sequence in an amplification reaction is inhibited.

[0042] In some embodiments, 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. In some embodiments, the reagent composition is lyophilized, heat dried and / or comprises one or more additives, wherein the one or more additives comprise: Tween 20, Triton X-100 and / or Tween 80; amino acids; sugars or sugar alcohols, optionally sugars or sugar alcohols include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, cyclodextrin, mannitol or any combination thereof; and / or polymers, optionally polymers include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides or any combination thereof, optionally contacting the reagent composition with the treated sample comprises dissolving the reagent composition in the treated sample.

[0043] The one or more lytic agents may include: about 0.001% (w / v) to about 1.0% (w / v) of the treated sample, optionally about 0.2% (w / v) of the treated sample; and / or a detergent, optionally the detergent includes one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. In some embodiments, the method: is performed in a single reaction vessel; does not include the use of any enzyme other than a reverse transcriptase and an enzyme having hyperthermophilic biopolymerase activity; does not include the use of any enzyme other than an enzyme having hyperthermophilic biopolymerase activity; thermally and / or enzymatically denatures the first and second nucleic acids during amplification; and / or contacts the first and second nucleic acids with a single-stranded DNA binding protein.

[0044] The length of the first signal generating oligonucleotide and / or the second signal generating oligonucleotide can be about 10 nucleotides to about 100 nucleotides. The length of the forward primer and / or the reverse primer can be about 5 nucleotides to about 25 nucleotides. In some embodiments, the length of the 5' subdomain, the 3' subdomain and / or the ring domain is about 1 nucleotide to about 25 nucleotides. In some embodiments, the first nucleic acid sequence and / or the second nucleic acid sequence comprises a length of not longer than about 20 nucleotides to not longer than about 90 nucleotides, and optionally the first nucleic acid sequence and / or the second nucleic acid sequence comprises a length of about 30 nucleotides. In some embodiments, the first forward primer, the second forward primer, the first reverse primer, the second reverse primer, the first reverse transcription primer and / or the second reverse transcription primer are about 8 to 16 bases long; the first nucleic acid amplification product and / or the second nucleic acid amplification product are about 20 to 40 bases long; and / or the first spacer sequence and / or the second spacer sequence respectively include a portion of the first nucleic acid sequence and / or the second nucleic acid sequence, and optionally the first spacer sequence and / or the second spacer sequence are 1 to 10 bases long. In some embodiments, isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, optionally about 56°C to about 67°C.

[0045] In some embodiments, amplification is performed for a period of about 5 minutes to about 60 minutes, optionally for a period of about 15 minutes; and / or is performed under isothermal amplification conditions without helicase, single-strand binding protein, cleavage agent-free, and recombinase. In some embodiments, amplification is performed using a method selected from the group consisting of: archaeal polymerase amplification (APA), polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA), optionally PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0046] In some embodiments, 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.

[0047] In some embodiments, the sample ribonucleic acid is simultaneously contacted with a reverse transcriptase and an enzyme having a hyperthermophilic polymerase activity, optionally the sample ribonucleic acid is simultaneously contacted with a reverse transcriptase, an enzyme having a hyperthermophilic polymerase activity, a first forward primer and a second forward primer and a first reverse primer and a second reverse primer, also optionally the sample ribonucleic acid is simultaneously contacted with a reverse transcriptase, an enzyme having a hyperthermophilic polymerase activity, a first forward primer and a second forward primer, a first reverse primer and a second reverse primer and a first reverse transcription primer and a second reverse transcription primer.

[0048] In some embodiments, the sample nucleic acid comprises sample RNA and / or sample deoxyribonucleic acid, and optionally, the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, 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 first target nucleic acid sequence and / or the second target nucleic acid sequence is a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoa, and optionally the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoa.

[0049] The virus can be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus. In some embodiments, the bacteria include Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, In some embodiments, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum.In some embodiments, the protozoa includes one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.

[0050] In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the environmental sample is the following, 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 freshwater sample, a wastewater sample, a salt water sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is the following, 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. In some embodiments, more than one target nucleic acid sequence is specific to two or more different organisms, and optionally two or more different organisms include one or more of SARS-CoV-2, influenza A virus, influenza B virus, and / or influenza C virus.

[0051] In some embodiments, amplification does not include 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 amplification does not include loop-mediated isothermal amplification (LAMP).

[0052] Amplification may include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR and IMDA, optionally the amplification does not include LAMP.

[0053] 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 cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal and / or enzymatic denaturation of the sample nucleic acid prior to and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0054] In some embodiments, the suspected sample nucleic acid contains a third target nucleic acid sequence, and wherein the method includes: (c) amplifying the third target nucleic acid sequence in an amplification reaction mixture, thereby generating a third nucleic acid amplification product; and (d) detecting the third nucleic acid amplification product with a third signal generating oligonucleotide, wherein the third signal generating oligonucleotide contains a label, wherein the detection includes detecting the signal of the label of the third signal generating oligonucleotide before, during, after, or any combination thereof, and wherein the first signal generating oligonucleotide, the second signal generating oligonucleotide, and the third signal generating oligonucleotide are detectable using the same optical channel, and wherein the melting temperatures (Tm) of the first signal generating oligonucleotide, the second signal generating oligonucleotide, and the third signal generating oligonucleotide differ from each other by at least about 2°C, optionally the first signal generating oligonucleotide, the second signal generating oligonucleotide, and the third signal generating oligonucleotide contain the same label.

[0055] The disclosure herein includes a kit. In some embodiments, the kit comprises: a first forward primer and a first reverse primer disclosed herein; a second forward primer and a second reverse primer disclosed herein; a first signal generating oligonucleotide, a second signal generating oligonucleotide, and / or a third signal generating oligonucleotide disclosed herein; a quality control template disclosed herein, a quality control primer disclosed herein; a signal generating oligonucleotide disclosed herein; and / or a supplemental quality control primer disclosed herein.

[0056] The kit may include: a lysis buffer, the lysis buffer including 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 containing a target nucleic acid sequence, optionally, the one or more lysis agents include a detergent, and wherein the detergent includes one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit may include: a reagent composition, the reagent composition including one or more amplification reagents, the amplification reagents including one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1A-1B A non-limiting exemplary schematic diagram of an isothermal amplification reaction provided herein is shown.

[0059] Figure 2 Depicted are non-limiting exemplary embodiments of the DNA hairpin internal control assay disclosed herein. IC Primer, internal control primer; HpIC1 MB2, hairpin internal control probe; HpIC1 P1, hairpin internal control product 1; HpIC1 P2, hairpin internal control product 2.

[0060] Figure 3A-Figure 3F Depicted are data relating to the detection of Group A Streptococcus (GAS) and Neisseria gonorrhoeae (NG) via singleplex-based and duplex-based hairpin probe detection. Figure 3A and Figure 3D The real-time amplification and detection of GAS in single (red curve) and duplex (green curve) reactions and NG in single (blue curve) and duplex (green curve) reactions are shown. After the reaction, the reaction temperature was raised from the assay temperature to 90°C for melting curve analysis ( Figure 3B and Figure 3E ) and melting derivative evaluation ( Figure 3C and Figure 3F ). 500cp Ng indicates only Ng reaction. 500cp GAS indicates only GAS reaction. 500cp Ng / GAS indicates Ng-GAS dual reaction.

[0061] Figure 4A-4C Depicts data related to amplification of the same reaction as in Example 1 and real-time detection by a nonspecific fluorescent dye (syto 61) in the CY5 channel ( Figure 4A ), followed by melting curve analysis ( Figure 4B ) and melting derivative evaluation ( Figure 4C ). 500cp Ng indicates Ng-only reaction. 500cp GAS indicates GAS-only reaction. 500cp Ng / GAS indicates Ng-GAS duplex reaction.

[0062] Figure 5A-5F Depicted is the interaction with the hairpin probes HpIC1b MB1 and HpIC1b MB2 ( Figure 5A-Figure 5C ) and intercalating dyes ( Figure 5D-5F ) amplified internal control. The hairpin internal control target was amplified in the APA reaction for 10 minutes, while the hairpin probe ( Figure 5A ) and intercalating dyes ( Figure 5D ) for detection. After the reaction, the reaction temperature was immediately raised from the measurement temperature to 90°C for melting curve analysis ( Figure 5B and Figure 5E ) and melting derivative evaluation ( Figure 5C and Fig. 5F ).

[0063] Figure 6A-6F Data associated with a Neisseria gonorrhoeae / internal control duplex reaction are depicted. In the presence of an internal control, Neisseria gonorrhoeae genomic DNA was amplified at 67°C for 10 minutes while the target in the ROX channel was detected by a hairpin probe ( Fig. 6A ) and the internal control in the HEX channel ( Fig.6D After the reaction, the reaction temperature was immediately raised from the measurement temperature to 90°C for melting curve analysis ( Figure 6B and Fig. 6E ) and melting derivative evaluation ( Figure 6C and Fig. 6F ). DETAILED DESCRIPTION

[0064] Reference is made to the accompanying drawings forming a part of this document in the following detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context otherwise indicates. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the accompanying drawings can be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form a part of the present disclosure.

[0065] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

[0066] 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 the present disclosure belongs. 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 the present disclosure, the following terms are defined below.

[0067] The disclosure herein includes methods for detecting more than one nucleic acid sequence. In some embodiments, the method includes: amplifying a first nucleic acid sequence and a second nucleic acid sequence in an amplification reaction mixture, thereby generating a first nucleic acid amplification product and a second nucleic acid amplification product, respectively; and detecting the first nucleic acid amplification product and the second nucleic acid amplification product with a first signal generating oligonucleotide and a second signal generating oligonucleotide, respectively, in the same optical channel. In some embodiments, the first signal generating oligonucleotide and the second signal generating oligonucleotide each comprise a label. In some embodiments, the detection includes detecting a signal of a label of the first signal generating oligonucleotide and the second signal generating oligonucleotide before amplification, during amplification, after amplification, or any combination thereof.

[0068] The disclosure herein includes a kit. In some embodiments, the kit comprises: a first forward primer and a first reverse primer disclosed herein; a second forward primer and a second reverse primer disclosed herein; a first signal generating oligonucleotide, a second signal generating oligonucleotide, and / or a third signal generating oligonucleotide disclosed herein; a quality control template disclosed herein, a quality control primer disclosed herein; a signal generating oligonucleotide disclosed herein; and / or a supplemental quality control primer disclosed herein.

[0069] Archaeal polymerase amplification multiplex

[0070] In some embodiments, multiplexing compositions and methods are provided, using probes that melt at different temperatures for assays based on Archaeal polymerase amplification (APA). Disclosed herein are multiplexing strategies for molecular assays comprising APA, using probes that melt at temperatures different from the optimal enzyme temperature. The methods and compositions provided herein can be used to multiplex targets or add internal controls to APA reactions.

[0071] For APA-based assays, multiplexing is difficult. However, the compositions and methods provided herein achieve multiplexing capabilities by adding cycles at different temperatures after the APA amplification run (typically performed at 68°C). In some embodiments, additional probes (specific for additional targets) are added to the reaction mixture and optimized to melt at a temperature different from the optimal APA reaction temperature. Therefore, a single optical channel can be used to detect more than one different target.

[0072] In some multiplexing embodiments described herein, an internal control (IC, e.g., hairpin IC) is detected in the same optical device but only after the standard APA reaction occurs. In some embodiments, the reaction is designed to amplify and detect the pathogenic target at 68°C, while the internal control sequence is amplified at the same temperature, but the internal control sequence is not detected. After the amplification run, the temperature can be set to the optimal temperature for the IC-specific beacon (e.g., 57°C, 60°C, or any other temperature). In some embodiments, the temperature for IC-specific probe detection can be set to be higher than the assay temperature, such as, for example, in the range of 75°C to 80°C. If the IC is amplified, fluorescence will be detected at this single temperature; if amplification does not occur, it will not be detected.

[0073] Without being bound by any particular theory, in some embodiments of the multiplexed compositions and methods provided herein, the only limit to the number of detectable targets is the number of different probes that can be designed within a particular temperature range.

[0074] The methods and compositions provided herein increase the multiplexing capabilities of platforms for using a limited number of optical channels. In addition, the disclosed methods and compositions can reduce competition between different probes at the optimal APA temperature. Multiplexed assays using enzymes that work without temperature cycling (at a specific temperature) can employ the compositions and methods provided herein. In some embodiments, the optimal melting temperature of the probe is adjusted by modifying the sequence and / or adding chemical modifications.

[0075] The melting temperature (Tm) of the APA assay product (amplicon) can be designed to be close to the assay temperature, for example, at ~67°C-68°C. The length of the APA amplicon can be about 23-35 nucleotides in length and can be limited by the melting temperature and can be designed to be close to the reaction temperature for optimal amplification. The limitation of the Tm and length of the APA amplicon can be that a high Tm or long amplicon results in no amplification, while a low Tm or short amplicon results in poor specificity / interference.

[0076] In some embodiments, specific detection can only be achieved through hairpin probes, rather than through intercalating dyes. The use of hairpin probes (e.g., molecular beacons) can provide additional levels of assay specificity, since there can be specific detection of any given amplicon, and the Tm can be designed to have a span of up to 15°C-20°C to support detection in multiplexing. However, in some embodiments, intercalating dyes cannot be used for specific detection such as in melting curve analysis in PCR, because intercalating dyes detect but cannot distinguish specific amplification signals from nonspecific amplification signals.

[0077] Currently available APA assays are designed to have amplicon Tm close to the assay Tm. In some embodiments, due to the rapid kinetics of APA amplification, poor resolution in melting curves in APA can be observed and expected. For example, in PCR / qPCR, the resolution of temperature in melting curve analysis can be 2.5°C, while in APA it is 5°C-10°C. In some embodiments of the compositions and methods provided herein, include adding a temperature step for amplification (e.g., 2 reaction temperature steps from 66°C to 70°C, 5 minutes each), the use of shorter / lower primer concentrations / low Tm probes for low Tm target detection, and a stepwise increase for high Tm target / probe detection. In some embodiments, these methods can improve the multiplexing capabilities of APA.

[0078] In some embodiments, a method for detecting more than one nucleic acid sequence is provided. In some embodiments, the method includes: amplifying a first nucleic acid sequence and a second nucleic acid sequence in an amplification reaction mixture, thereby generating a first nucleic acid amplification product and a second nucleic acid amplification product, respectively; and detecting the first nucleic acid amplification product and the second nucleic acid amplification product with a first signal generating oligonucleotide and a second signal generating oligonucleotide in the same optical channel, respectively. In some embodiments, the first signal generating oligonucleotide and the second signal generating oligonucleotide each comprise a label. Detection may include detecting the signal of the label of the first signal generating oligonucleotide and the second signal generating oligonucleotide before amplification, during amplification, after amplification, or any combination thereof.

[0079] 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 acid contained therein; and contacting a reagent composition with the treated sample to generate an amplification reaction mixture, wherein the reagent composition contains one or more amplification reagents.

[0080] The first nucleic acid sequence can be a first target nucleic acid sequence. The second nucleic acid sequence can be a second target nucleic acid sequence. It can be suspected that the sample nucleic acid contains the first target nucleic acid sequence and the second target nucleic acid sequence. The first nucleic acid sequence can be a first target nucleic acid sequence. The second nucleic acid sequence can be an internal control (IC) nucleic acid sequence. It can be suspected that the sample nucleic acid contains the first target nucleic acid sequence. The IC nucleic acid sequence can be a quality control template, and the second amplification product can be a first quality control product. Detection can be performed using an instrument comprising 6, 5, 4, 3, 2 or 1 optical channels. The melting temperatures (Tm) of the first signal generating oligonucleotide and the second signal generating oligonucleotide can differ by at least about 2°C.

[0081] The one or more amplification reagents may include: an enzyme having hyperthermophilic polymerase activity, optionally the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity; two or more primer pairs, wherein each primer pair comprises a forward primer and a reverse primer; dNTPs; reverse transcriptase; and / or one or more reverse transcription primers.

[0082] Amplification can be performed at an optimal temperature of the enzyme having hyperthermophilic polymerase activity, optionally the optimal temperature is about 67° C. to about 68° C. The first signal generating oligonucleotide can have a Tm within about 1° C. of the optimal temperature of the enzyme having hyperthermophilic polymerase activity. The second signal generating oligonucleotide can have a Tm that differs from the optimal temperature of the enzyme having hyperthermophilic polymerase activity by at least about 2° C.

[0083] Detection may include: contacting the first nucleic acid amplification product and the second nucleic acid amplification product with the first signal generating oligonucleotide and the second signal generating oligonucleotide for hybridization. The first signal generating oligonucleotide and the second signal generating oligonucleotide may respectively include a first label and a second label, and optionally the first label and the second label are the same or different. The first label and the second label may be capable of generating a signal when the first signal generating oligonucleotide and the second signal generating oligonucleotide hybridize with the first nucleic acid amplification product and the second nucleic acid amplification product, respectively.

[0084] In some embodiments, when the first signal generating oligonucleotide and the second signal generating oligonucleotide are hybridized with the first nucleic acid amplification product and the second nucleic acid amplification product, respectively, the first label and the second label generate the first signal and the second signal, respectively. The first signal and the second signal may be indistinguishable. The signal may be fluorescence. In some embodiments, detecting the signal of the label of the first signal generating oligonucleotide and the second signal generating oligonucleotide includes detecting the fluorescence emitted by the first label and the second label, respectively. Detection may include: detecting the signal of the first label during amplification, optionally in real time; and detecting the signal of the second label after amplification, optionally, not detecting the signal of the second label during amplification. In some embodiments, detecting the signal of the second label after amplification includes one or more cycles performed at the Tm of the second signal generating oligonucleotide.

[0085] In some embodiments, the first signal generating oligonucleotide and the second signal generating oligonucleotide each comprise: a 5' subdomain; a 3' subdomain; and a loop domain located between the 5' subdomain and the 3' subdomain, wherein intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. Optionally, the paired stem domain of the second signal generating oligonucleotide can be configured to have a melting temperature (Tm) that is at least about 2°C higher or lower than the optimal temperature of the enzyme, by modifying the length of the paired domain, the GC content of the paired domain, and / or the presence of one or more chemical modifications in the paired domain.

[0086] The first nucleic acid amplification product may include: (1) the sequence of the first forward primer and its reverse complement, (2) the sequence of the first reverse primer and its reverse complement, and (3) a first spacer sequence flanked by (1) the sequence of the first forward primer and its reverse complement and (2) the sequence of the first reverse primer and its reverse complement, wherein the first spacer sequence is 1 to 10 bases long. The second nucleic acid amplification product may include: (1) the sequence of the second forward primer and its reverse complement, (2) the sequence of the second reverse primer and its reverse complement, and (3) a second spacer sequence flanked by (1) the sequence of the second forward primer and its reverse complement and (2) the sequence of the second reverse primer and its reverse complement, wherein the second spacer sequence is 1 to 10 bases long. The sample nucleic acid may include: a first nucleic acid including a first target nucleic acid sequence and a second nucleic acid including a second target nucleic acid sequence.

[0087] In some embodiments, amplifying a first target nucleic acid sequence comprises: amplifying a first target nucleic acid sequence comprising a first chain and a second chain that are complementary to each other under isothermal amplification conditions, wherein amplifying comprises contacting a first nucleic acid comprising the first target nucleic acid sequence with: i) a first forward primer and a first reverse primer, wherein the first forward primer is capable of hybridizing with a sequence of the first chain of the first target nucleic acid sequence, and the first reverse primer is capable of hybridizing with a sequence of the second chain of the first target nucleic acid sequence; and ii) an enzyme having a hyperthermophilic polymerase activity, thereby generating a first nucleic acid amplification product; and amplifying a second target nucleic acid sequence comprises: amplifying a second target nucleic acid sequence comprising a first chain and a second chain that are complementary to each other under isothermal amplification conditions, wherein amplifying comprises contacting a second nucleic acid comprising the second target nucleic acid sequence with: i) a second forward primer and a second reverse primer, wherein the second forward primer is capable of hybridizing with a sequence of the first chain of the second target nucleic acid sequence, and the second reverse primer is capable of hybridizing with a sequence of the second chain of the second target nucleic acid sequence; and ii) an enzyme having a hyperthermophilic polymerase activity, thereby generating a second nucleic acid amplification product.

[0088] The first nucleic acid and the second nucleic acid can be double-stranded DNA. The first nucleic acid and the second nucleic acid can be the product of a reverse transcription reaction, optionally, the first nucleic acid and the second nucleic acid are the products of a reverse transcription reaction generated from a sample RNA, and optionally, step (c) includes generating the first nucleic acid and the second nucleic acid by a reverse transcription reaction. The sample nucleic acid can include sample RNA, and wherein the method includes contacting the sample RNA with a reverse transcriptase and / or a reverse transcription primer to generate a first cDNA and a second cDNA.

[0089] Amplifying the first target nucleic acid sequence and the second target nucleic acid sequence may include: (c1) contacting the sample RNA with a reverse transcriptase, a first reverse transcription primer and / or a second reverse transcription primer to generate a first cDNA and a second cDNA; (c2) contacting the first cDNA and the second cDNA with an enzyme having a hyperthermophilic biopolymerase activity to generate a first double-stranded DNA (dsDNA) and a second dsDNA, respectively, wherein the first dsDNA and the second dsDNA respectively comprise the first target nucleic acid sequence and the second target nucleic acid sequence, and wherein the first target nucleic acid sequence and the second target nucleic acid sequence comprise a first strand and a second strand that are complementary to each other; and (c3) amplifying the first target nucleic acid sequence under isothermal amplification conditions. The invention relates to a method for amplifying a first dsDNA and a second target nucleic acid sequence, wherein amplifying comprises contacting the first dsDNA and the second dsDNA with: (i) a first forward primer and a first reverse primer, wherein the first forward primer is capable of hybridizing to a sequence of a first strand of the first target nucleic acid sequence, and the first reverse primer is capable of hybridizing to a sequence of a second strand of the first target nucleic acid sequence; and (ii) a second forward primer and a second reverse primer, wherein the second forward primer is capable of hybridizing to a sequence of a first strand of the second target nucleic acid sequence, and the second reverse primer is capable of hybridizing to a sequence of a second strand of the second target nucleic acid sequence; and (iii) an enzyme having hyperthermophilic biopolymerase activity, thereby generating a first nucleic acid amplification product and a second nucleic acid amplification product, respectively.

[0090] The first amplification product and the second amplification product may be generated during the first amplicon reaction and the second amplicon reaction, respectively, and optionally the first amplification product and the second amplification product are generated separately in time. The amplification reaction may include: a first amplicon reaction performed at a first temperature; and a second amplicon reaction performed at a second temperature. The amplification reaction may include three or more amplicon reactions, wherein each sub-reaction is performed at a different temperature. The first amplicon reaction may be performed before the second amplicon reaction. The first amplicon reaction and the second amplicon reaction may each be at least about 2 minutes, optionally 5 minutes. The second temperature may be at least 2°C higher than the first temperature, optionally, the first temperature is 66°C, and the second temperature is 70°C. The first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence may be shorter than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence. The first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence may have a lower Tm than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence. The first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence can be present at a lower concentration than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence.

[0091] The first signal generating oligonucleotide and / or the second signal generating oligonucleotide may comprise one or more phosphorothioate linkages, one or more 2'O-methyl modified nucleic acids and / or one or more locked nucleic acids. The first signal generating oligonucleotide and / or the second signal generating oligonucleotide may be a TaqMan detection probe oligonucleotide, a hairpin probe (e.g., a molecular beacon) detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. In some embodiments, the label comprises a quenchable label, and optionally the quenchable label is a fluorophore; and / or the first signal generating oligonucleotide and / or the second signal generating oligonucleotide comprises a quencher.

[0092] In some embodiments, the method includes determining the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, determining the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample includes determining the presence, absence and / or amount of dsDNA and / or nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, the presence, absence and / or amount of the first signal and the second signal respectively indicate the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, the presence, absence and / or amount of the first signal and the second signal respectively indicate the presence, absence and / or amount of dsDNA and / or nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence in the sample. In some embodiments, amplifying the first nucleic acid sequence and / or the second nucleic acid sequence includes generating the first nucleic acid amplification product and / or the second nucleic acid amplification product at a detectable level within about 20 minutes, about 15 minutes or about 10 minutes.

[0093] In some embodiments, the method does not include an intercalating dye; and / or detecting the first nucleic acid amplification product and the second nucleic acid amplification product does not include detecting a signal of an intercalating dye. In some embodiments, the melting temperatures of the first amplification product and the second amplification product are the same, and wherein the melting temperatures of the first signal generating oligonucleotide and the second signal generating oligonucleotide are different. The melting temperatures of the first signal generating oligonucleotide and the second signal generating oligonucleotide may differ by at least about 2°C.

[0094] Melting curve analysis can be performed by incubating the nucleic acid amplification product at a series of increasing temperatures. The term "melting temperature" as used herein can refer to the temperature at which the maximum discrete melting step occurs. In some embodiments, the melting temperature corresponds to the highest level of the negative derivative (-dF / dT) of the temperature change of fluorescence with respect to temperature (T). In some embodiments, detecting the first nucleic acid amplification product and the second nucleic acid amplification product in the same optical channel includes a melting curve analysis (MCA). MCA can be performed at least about 1 minute after the amplification step. MCA can include: incubating the first nucleic acid amplification product and the second nucleic acid amplification product at a series of increasing temperatures (optionally from the starting temperature to the final temperature); and detecting the signal of the label of the first signal generating oligonucleotide and the second signal generating oligonucleotide at the series of increasing temperatures, thereby generating a melting curve. In some embodiments, the starting temperature is at least about 50°C, optionally, the starting temperature is the optimal temperature of an enzyme with hyperthermophilic biopolymerase activity; and / or the final temperature is at least about 80°C, optionally 90°C. The temperature conversion from the starting temperature to the final temperature can be a linear function of time, optionally the linear conversion is at least 0.05°C per second. In some embodiments, MCA includes deriving the negative derivative of signal intensity with respect to temperature (-dF / dt vs. T). In some embodiments, the signal derived from the first signal generating oligonucleotide is distinguishable in the melting curve from the signal derived from the second signal generating oligonucleotide or its negative first derivative.

[0095] In some embodiments, the presence, absence and / or amount of the signal at the first melting temperature in the melting curve indicates the presence, absence and / or amount of the first amplified product. In some embodiments, the presence, absence and / or amount of the signal at the second melting temperature in the melting curve indicates the presence, absence and / or amount of the second amplified product. In some embodiments, the melting temperature corresponds to the highest level of the negative derivative (-dF / dT) of the temperature change of fluorescence with temperature (T) relative to temperature (T), and optionally also corresponds to the temperature within 1°C-4°C of the highest level. In some embodiments, the first melting temperature corresponds to the melting temperature (Tm) of the first amplified product / first signal generating oligonucleotide duplex and / or the melting temperature (Tm) of the paired stem domain of the first signal generating oligonucleotide. In some embodiments, the second melting temperature corresponds to the melting temperature (Tm) of the second amplified product / second signal generating oligonucleotide duplex and / or the melting temperature (Tm) of the paired stem domain of the second signal generating oligonucleotide. The first melting temperature can differ from the second melting temperature by at least about 2°C.

[0096] In some embodiments, the first signal generating oligonucleotide and / or the second signal generating oligonucleotide comprises one or more locked nucleic acids (LNAs), optionally one or more LNAs are located in the loop domain, and optionally one or more LNAs increase the difference between the first melting temperature and the second melting temperature. The first signal generating oligonucleotide and / or the second signal generating oligonucleotide can be configured so that the first melting temperature differs from the second melting temperature by at least about 2°C, optionally via one or more LNAs located in the loop domain.

[0097] The sample nucleic acid may be suspected of containing a third target nucleic acid sequence, and the method may include: (c) amplifying the third target nucleic acid sequence in an amplification reaction mixture, thereby generating a third nucleic acid amplification product; and (d) detecting the third nucleic acid amplification product with a third signal generating oligonucleotide, wherein the third signal generating oligonucleotide contains a label, wherein the detection includes detecting a signal of the label of the third signal generating oligonucleotide before, during, after, or any combination thereof, and wherein the first signal generating oligonucleotide, the second signal generating oligonucleotide, and the third signal generating oligonucleotide are detectable using the same optical channel, and wherein the melting temperatures (Tm) of the first signal generating oligonucleotide, the second signal generating oligonucleotide, and the third signal generating oligonucleotide differ from each other by at least about 2°C, optionally the first signal generating oligonucleotide, the second signal generating oligonucleotide, and the third signal generating oligonucleotide contain the same label.

[0098] Hairpin internal control

[0099] The disclosure provided herein includes methods, compositions, kits, and reaction mixtures that, in some embodiments, can use archaeal polymerase amplification ("APA") to isothermally amplify a region of interest within a target nucleotide template for the purpose of real-time analyte detection, and simultaneously monitor or evaluate the amplification reaction (e.g., an internal control ("IC") assay). The methods, compositions, reaction mixtures, and kits provided herein can overcome the challenges posed by competition for target amplification and address the above-mentioned needs in the art by exploiting the stability of the hairpin structure to reduce non-specific interactions with the primary target amplification. Advantages of the hairpin IC systems, methods, compositions, reaction mixtures, and kits provided herein can include reduced assay complexity and reduced unwanted interactions with target amplification. Unexpectedly, in some embodiments, when double-stranded with a hairpin IC, the target amplification shows an improvement in low copy detection compared to a target-only assay.

[0100] Disclosed herein are methods, compositions, reaction mixtures, and kits for internal control assays that can be duplexed with specific target assays and can report the integrity of core reagents in the reaction, instrument failure, and / or sample inhibition in the absence of specific signals from target amplification. In some embodiments, the internal control assay is designed to utilize APA to simultaneously amplify a DNA target and an internal control template for real-time detection under isothermal conditions.

[0101] The disclosed internal control method can achieve simultaneous amplification of specific targets and internal controls (IC) by utilizing the characteristic structural stability of stem-loop hairpins. Different from the currently available IC methods in which linear DNA is used as IC templates, hairpin-shaped templates (e.g., quality control templates) are used in some embodiments of the methods and compositions provided herein. Due to the complementary nature of the hairpin stems, in some embodiments, the amplification of the hairpin IC target only requires a single primer complementary to the 3' end of the stem. IC primers (e.g., quality control primers) can be extended on the hairpin template to generate a first round of hairpin products (e.g., first quality control products) complementary to the IC template. Subsequent repeated extensions of the IC template driven by a single IC primer can generate two hairpin products with a stem structure and a complementary loop sequence. The generated IC product can be detected by a signal generating oligonucleotide (e.g., a second signal generating oligonucleotide, a probe, a hairpin probe (e.g., a molecular beacon)). In some embodiments, the signal generating oligonucleotide (e.g., a hairpin probe) is modified with LNA to enhance the detectability of the hairpin product. As described herein, a hairpin probe is a probe comprising a nucleic acid sequence capable of forming a hairpin structure, such as a hairpin-shaped probe. The hairpin probe can include a hairpin structure having, for example, a blunt end, a 5' overhang, or a 3' overhang. In some embodiments, the hairpin probe has a hairpin shape with a blunt end. In some embodiments, the hairpin probe has a hairpin shape with a 5' overhang. In some embodiments, the molecular beacon comprises a nucleic acid sequence capable of forming a hairpin structure having a blunt end (i.e., a blunt-ended stem).

[0102] The IC methods described herein can be extended to RNA IC assays comprising IC primers, signal generating oligonucleotides (e.g., hairpin probes (e.g., molecular beacons)) and / or hairpin-shaped IC templates. The IC template can contain an RNA segment at the 5' end and a hairpin-shaped DNA segment with an RT primer sequence at the 3' end in the stem region. In the presence of a reverse transcriptase, the RT primer in the stem region of the hairpin can extend on the RNA segment of the IC template to generate cDNA that is also complementary to the IC primer. Subsequent amplification driven by a single IC primer can generate a hairpin-shaped product complementary in the loop region, which can be detected by a signal generating oligonucleotide (e.g., a hairpin probe (e.g., a molecular beacon)). In some embodiments, the signal generating oligonucleotide (e.g., a hairpin probe) is modified with LNA.

[0103] The methods, compositions, reaction mixtures and kits disclosed herein advantageously employ a hairpin-based IC method that can allow strong internal control amplification without the risk of competing with target amplification. In some embodiments, only a single IC primer is required. In some embodiments, a high concentration of IC primers can be used without affecting target amplification. In some embodiments, a high IC template copy number can be used without hindering target amplification, for example, using 50,000 to 500,000 copies, compared to 20-100 copies of IC targets commonly used in other IC systems to reduce competition with target amplification. Without being bound by any particular theory, fewer primer-dimers and mispriming can occur due to the use of a single primer and a hairpin-shaped template and product.

[0104] In some embodiments, the internal control assay comprises a single primer, a hairpin-shaped internal control template, and / or a signal generating oligonucleotide (e.g., a hairpin probe (e.g., a molecular beacon)) for detecting amplified hairpin products. In some embodiments, the internal control assay comprises a single primer and a signal generating oligonucleotide (e.g., a hairpin probe) that serves as both a template and a detector.

[0105] In some embodiments, the signal generating oligonucleotide (e.g., a hairpin probe (e.g., a molecular beacon)) comprises a fluorophore at the 5' end and a quencher at the 3' end. In some embodiments, the signal generating oligonucleotide (e.g., a hairpin probe) can be replaced by a hairpin DNA probe without a quencher to detect hybridization with a hairpin amplification product. In the absence of IC amplification, the fluorescence of a hairpin probe (e.g., a molecular beacon) with a fluorophore attached at the 5' end can be quenched by a guanine base in a complementary stem. Without being bound by any particular theory, upon hybridization, conformational reorganization can occur, resulting in an increase in fluorescence intensity.

[0106] The basic technical principles for the design of the internal control based on hairpins provided herein can include the nearest neighbor (NN) theory for predicting DNA thermodynamics using energy values ​​and the secondary structure dynamics of DNA hairpins. Without being bound by any particular theory, the mechanism of hairpin amplification and its advantage as an internal control can be related to the thermodynamics of the hairpin stem-loop structure.

[0107] Other embodiments of the methods and compositions provided herein include probe-free formats based on hairpin internal controls. In this method, a single IC primer and a hairpin template labeled with a fluorophore at the 5' end can be the only two components necessary for IC amplification and detection. The 5' end of the primer can contain one or more cytosine bases adjacent to the fluorophore. The labeled primer can exponentially copy the hairpin template to produce a hairpin product, wherein the 5' end fluorophore is quenched due to the proximity of the guanine base via light-induced electron transfer.

[0108] Three-component IC assay

[0109] In some embodiments, the RNA hairpin IC assay includes an IC primer, a signal generating oligonucleotide (e.g., a hairpin probe (e.g., a molecular beacon)) and / or a hairpin IC template. The IC template may include an RNA segment at the 5' end and a hairpin DNA segment having an RT primer sequence at the 3' end in the stem region. In the presence of a reverse transcriptase, the RT primer in the stem region of the hairpin may extend on the RNA segment of the IC template, generating a cDNA that is also complementary to the IC primer. Without being bound by any particular theory, subsequent amplification may be driven entirely by a single IC primer to form a pan-handle-shaped product. For ease of amplification and detection, the product hybridization melting temperature (Tm) may be designed to be greater than or equal to the product hairpin Tm. In some embodiments, a signal generating oligonucleotide (e.g., a hairpin probe (e.g., a molecular beacon)) modified with a locked nucleic acid (LNA) in the spacer region may be used for IC product detection. In some embodiments, a fluorescent dye may be used for amplified IC product detection.

[0110] The DNA hairpin IC assay may comprise a hairpin-shaped IC template, a single IC primer, and / or a hairpin probe (e.g., a molecular beacon). In some embodiments, the IC primer may be extended on the IC template to generate two "pot-handle" products that are complementary in the loop (spacer) region. Without being bound by any particular theory, subsequent repeated extensions of the IC primer on the pot-handle product may 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, a signal generating oligonucleotide modified with LNA (e.g., a hairpin probe (e.g., a molecular beacon)) may be used for IC product detection. In some embodiments, fluorescent dyes may be used for detection of amplified IC products.

[0111] 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, templates, and probes (e.g., hairpin probes (e.g., molecular beacons)) for DNA or RNA IC determinations. In the RNA IC determinations provided herein, the RT primer can be embedded in the chimeric IC template. In some embodiments, the combination of low IC primer concentrations and the formation of a "pot-handle" structure can further reduce nonspecific interactions with the target being measured.

[0112] Two-component IC assay

[0113] In some embodiments, the hairpin IC method comprises a single IC primer and a signal generating oligonucleotide (eg, a hairpin probe (eg, a molecular beacon)) for both IC amplification and detection ( Figure 2 ). The sequence of the signal generating oligonucleotide (e.g., a hairpin probe) can include part or all of the IC primer, the spacer region of the IC template, and 4-5 nucleotides adjacent to the IC spacer and complementary to the 3' end of the IC primer. The IC primer can hybridize with the beacon and can generate a first round extension product whose 3' end is complementary to the IC primer. Subsequent extension of the IC primer can generate a "pot-handle" shaped internal control product. Without being 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 an IC beacon modified with, for example, LNA to enhance the detectability of the pot-handle product.

[0114] The two-component IC method based on hairpin described herein can be extended to RNA IC determination. In this case, the signal generating oligonucleotide (e.g., hairpin probe (e.g., molecular beacon)) can include some RNA bases in the loop region. The RT primer 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 in the reverse transcriptase, resulting in the release of fluorescent signal. Depending on the beacon design, in some embodiments, IC primers can be used to improve exponential amplification.

[0115] Advantageously, the method can have minimal primer-related interactions with the target being assayed relative to alternative methods of assay monitoring. In some embodiments, a DNA IC assay has only two IC components: primers and hairpin probes (e.g., molecular beacons) used for both IC amplification and detection as internal controls. For a two-component RNA IC assay provided herein, an RT primer and a signal-generating oligonucleotide (e.g., hairpin probe) containing an RNA segment in the loop region can be the minimum components, with optional IC primers being employed in some embodiments.

[0116] Methods for Monitoring Amplification Reactions

[0117] The content disclosed herein includes a method for monitoring an amplification reaction. In some embodiments, the method includes providing: a quality control template, the quality control template comprising: a 5' subdomain; a 3' subdomain; and a loop domain between the 5' subdomain and the 3' subdomain, wherein the intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the method includes providing: a quality control primer that can hybridize with at least a portion of the 3' subdomain. In some embodiments, the method includes: performing an amplification reaction capable of generating a first quality control product on the quality control template and the quality control primer. 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 capable of generating a first quality control product on the quality control template and the quality control primer includes: amplifying the quality control template with a quality control primer in the amplification reaction mixture under amplification conditions, thereby generating a first quality control product. The amplification reaction can include a reverse transcription reaction.

[0118] The method may include providing an enzyme with polymerase activity (e.g., an enzyme with hyperthermophilic polymerase activity). The method may include providing a reverse transcriptase. In some embodiments, the enzyme with hyperthermophilic polymerase 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 with an enzyme with polymerase activity, thereby generating a first quality control product. The amplification reaction may include contacting a quality control primer with a 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. The amplification reaction may include contacting a quality control primer with a 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. The first quality control product and the second quality control product may include a 5' subdomain and a 3' subdomain of a stem domain capable of forming a pair. In some embodiments, the first quality control product and the second quality control product have the same stem domain. The first quality control product and the second quality control product can include loop domains that are complementary to each other. The amplification reaction can include linear amplification and / or exponential amplification of the first quality control product and the second quality control product. The 5' subdomain can include the sequence of at least a portion of the quality control primer. Both the first quality control product and the second quality control product can be able to form a hairpin structure. As used herein, the term "hairpin structure" should be given its ordinary meaning, and should also refer to the double helix region formed by base pairing between adjacent, reverse, complementary sequences in a single strand of RNA or DNA. The quality control template can include a 5' terminal domain located at 5' of the 5' subdomain, and / or the quality control template can include a 3' terminal domain located at 3' of the 3' subdomain. The 5' terminal domain of the quality control template can include at least a portion of the sequence of the quality control primer. The combined sequence of the 5' terminal domain and the 5' subdomain can include the entire sequence of the quality control primer.

[0119] Detecting the first quality control product may include detecting the first quality control product with 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 comprise a quencher, a label, or both. The label may comprise a quenchable label (e.g., a fluorophore). The quenchable label may be, for example, a fluorophore. As used herein, the term "fluorophore" shall be given its ordinary meaning and also refers to any reporter group whose presence can be detected by its 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), Pyronine 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 corresponding fluorophores in nanometers). In some embodiments, the fluorophore is Cy5 TM In some embodiments, the fluorophore is hexachlorofluorescein (HEX).

[0120] The signal generating oligonucleotide may comprise a quencher. The quencher may be capable of quenching the label. The quenching may 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 be achieved at up to FRET also depends on the relative orientation of the donor-acceptor spectral overlap and the transition dipole moments of the donor and acceptor. The quenching of the fluorophore can also occur due to the formation of a non-fluorescent complex or a non-fluorescent molecule between one fluorophore and another fluorophore. This mechanism is called "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 believed that a quencher portion is not required in order to observe detectable changes in fluorescence, and the proximal-base quenching effects are sufficient to produce detectable fluorescence shifts to allow 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.

[0121] Detection can include contacting the first quality control product with a signal generating oligonucleotide for hybridization. The label can be 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. The signal can be fluorescence. Detection of the first quality control product can include detecting a signal generated by the label of the signal generating oligonucleotide. The label can be a fluorophore, and the signal can be fluorescence. Detection can include detecting the signal of the label before the amplification reaction, during the amplification reaction, after the amplification reaction, or any combination thereof.

[0122] 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 with the signal generating oligonucleotide. The quality control template may be a 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.

[0123] In some embodiments, the 5' terminal domain of the quality control template comprises: one or more RNA nucleotides; and / or a sequence of at least a portion of a quality control primer. In some embodiments, the quality control template does not comprise a 3' terminal domain. The 3' end of the quality control template may be complementary to the 5' end of the 5' subdomain of the quality control template. In some embodiments, the reverse transcriptase may be able to use one or more RNA nucleotides of the 5' terminal domain of the quality control template as a template to extend the 3' end of the quality control template, thereby generating an extended quality control template. The 3' end of the extended quality control template may comprise a sequence complementary to at least a portion of the quality control primer. The amplification reaction may comprise contacting the reverse transcriptase with the quality control template, thereby generating an extended quality control template. The extended quality control template may comprise cDNA. In some embodiments, the amplification reaction 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 a reverse transcriptase and / or an enzyme having polymerase activity, thereby generating a first quality control product.

[0124] The quality control template can be a signal generating oligonucleotide. The signal generating oligonucleotide can include a label. The loop domain can include one or more RNA nucleotides. The label can include a quenchable label (e.g., a fluorophore). The signal generating oligonucleotide can include a quencher. The label can be located in the 3' terminal domain, and the quencher can be located in the 5' terminal domain, and / or the label can be located in the 5' terminal domain, and the quencher can be located in the 3' terminal domain. The amplification reaction can include: contacting the quality control primer with the quality control template for hybridization, and extending the quality control primer hybridized with the quality control template with a reverse transcriptase, thereby generating a first quality control product. The reverse transcriptase can include RNase H activity. In some embodiments, the reverse transcriptase cuts 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 comprising a label. Detecting the first quality control product can include detecting a signal generated by the first cleavage product comprising a label. Detecting the first quality control product can include detecting the signal of the label. The first cleavage product can include a label. The amount of the detected signal can indicate the absence, presence or amount of the first cleavage product. The absence, presence or amount of the first cleavage product may indicate the absence, presence or amount of the first quality control product. The label may be a fluorophore and the signal may be fluorescence. The method may include: providing a supplemental quality control primer; and performing an amplification reaction on the supplemental quality control primer.

[0125] The length of the signal generating oligonucleotide may be from about 10 nucleotides to about 100 nucleotides. The length of the quality control template may be from about 10 nucleotides to about 100 nucleotides. The length of the quality control primer and / or the supplementary quality control primer may be from about 5 nucleotides to about 25 nucleotides. The length of the 5' subdomain, the 3' subdomain, the loop domain, the 5' terminal domain and / or the 3' terminal domain may be from about 1 nucleotide to about 25 nucleotides. The signal generating oligonucleotide, the quality control template and / or the quality control primer may comprise one or more thiophosphate linkages and / or one or more locked nucleic acids. The signal generating oligonucleotide may be a TaqMan detection probe oligonucleotide, a hairpin probe detection probe oligonucleotide (e.g., a molecular beacon) or a molecular torch detection probe oligonucleotide.

[0126] The signal generating oligonucleotide may comprise one or more LNAs. One or more LNAs may be located within the 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., via one or more modified and / or modified bases, such as LNAs located in the loop domain). Modified and modified bases may include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite TM , Adenylation, Azide (NHS ester), Digoxigenin (NHS ester), Cholesterol-TEG, I-Linker TM , amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link TM amino modifiers), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dibiotin, PC biotin, desthiobiotin-TEG), thiol modifications (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, reversed dT, reversed dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinosine, Locked nucleic acid (LNA), 5-nitroindole, 2'-O-methyl RNA base, hydroxymethyl dC, UNA non-locked nucleic acid (e.g., UNA-A, UNA-U, UNA-C, UNA-G), iso-dC, iso-dG, fluorine C, fluorine U, fluorine A, fluorine G); phosphorothioate bond modification (e.g., phosphorothioated DNA base, phosphorothioated RNA base, phosphorothioated 2'-O-methyl base, phosphorothioated LNA base); and click chemistry modification. In some embodiments, the modified and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, phosphorothioate linkages, 3' phosphate groups, spacer bases (e.g., C3 spacer bases or other spacer bases). One or more modified nucleotides can include spacers, abasic sites, unmethylated RNA bases, 2'-O-methylated nucleotides, and any combination thereof.

[0127] In some embodiments, the signal generating oligonucleotide does not include a dye capable of quenching a label. In some embodiments, the signal generating oligonucleotide does not include a portion capable of quenching a label other than the nucleotides of the signal generating oligonucleotide. The 5' terminal domain of the quality control template and / or the signal generating oligonucleotide may include a label. The 5' terminal domain and / or the 5' subdomain of the quality control template and / or the signal generating oligonucleotide may include one or more cytosine bases. The 3' terminal domain and / or the 3' subdomain of the quality control template and / or the signal generating oligonucleotide may include one or more guanine bases. One or more guanine bases may be capable of quenching a label when the quality control template and / or the signal generating oligonucleotide form a hairpin structure. The 3' terminal domain of the quality control template and / or the signal generating oligonucleotide may include a label. The 3' terminal domain and / or the 3' subdomain of the quality control template and / or the signal generating oligonucleotide may include one or more cytosine bases. The 5' terminal domain and / or 5' subdomain of the quality control template and / or signal generating oligonucleotide may comprise one or more guanine bases. The one or more guanine bases may be capable of quenching the label when the quality control template and / or signal generating oligonucleotide forms a hairpin structure.

[0128] Detecting the first quality control product may include detecting a reduction in the amount of a signal generated by a label of a quality control primer. The label may be a fluorophore, and the signal may be fluorescence. The generation of the first quality control product and the second quality control product may be associated with a decrease in the detected signal. The 5' end of the quality control primer may include a label, and the quality control primer may include one or more cytosine bases adjacent to the label. The 3' terminal domain and / or 3' subdomain of the quality control template, the first quality control product, and / or the second quality control product may include one or more guanine 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 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 the quality control primer binds to the first quality control product and is extended by an enzyme having polymerase activity to generate a second quality control product, one or more guanine bases present in the 3' terminal domain and / or the 3' subdomain of the second quality control product can be capable of quenching the label when the second quality control product forms a hairpin structure. Detecting the first quality control product can include contacting the first quality control product with a fluorescent dye.

[0129] Providing a quality control primer, a quality control template, and / or a signal generating oligonucleotide may include providing a reagent composition comprising a quality control primer, a quality control template, and / or a signal generating oligonucleotide. Performing an amplification reaction on the quality control primer, the quality control template, and / or the signal generating oligonucleotide may include contacting the reagent composition with the treated sample to generate an amplification reaction mixture. The method may include detecting a target nucleic acid sequence in the sample. The method may include: performing an amplification reaction capable of generating a nucleic acid amplification product on the target nucleic acid sequence. The method may include: detecting the nucleic acid amplification product with a target signal generating oligonucleotide, wherein the target signal generating oligonucleotide is capable of hybridizing with the nucleic acid amplification product. Performing an amplification reaction capable of generating a nucleic acid amplification product on the target nucleic acid sequence may include amplifying the target nucleic acid sequence in an amplification reaction mixture under amplification conditions, thereby generating a nucleic acid amplification product. The method may include: 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 the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence. The method may include: contacting a reagent composition with the treated sample to generate an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0130] One or more amplification reagents may include: reverse transcriptase; an enzyme with hyperthermophilic polymerase activity (e.g., an enzyme with hyperthermophilic polymerase activity and reverse transcriptase activity); a forward primer; a reverse primer; a reverse transcription primer; and / or dNTPs. The sample nucleic acid may include a nucleic acid containing a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence includes: 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 includes contacting the nucleic acid comprising the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing 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 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 a sample ribonucleic acid (e.g., amplification includes generating nucleic acids by a reverse transcription reaction). The amplification reaction may be performed for a period of about 5 minutes to about 60 minutes. In some embodiments, amplifying the quality control template can include generating a first quality control product and / or a second quality control product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes.

[0131] In some embodiments, the method: is performed in a single reaction vessel; does not include the use of any enzyme other than a reverse transcriptase and an enzyme with a thermophilic polymerase activity; does not include the use of any enzyme other than an enzyme with a thermophilic polymerase activity; does not include thermal and / or enzymatic denaturation of the nucleic acid and / or quality control template during the amplification step; and / or does not include contacting the nucleic acid and / or quality control template with a single-stranded DNA binding protein.

[0132] In some embodiments, the methods, reagent compositions and / or amplification reaction mixtures do not include: a template other than the quality control template that is capable of generating a first quality control product; a probe other than the signal generating oligonucleotide that is capable of detecting the first quality control product; a double-stranded template that is capable of generating a first quality control product; a linear template that is capable of generating a 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.

[0133] The 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 a 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 an amplification reaction mixture. The presence, absence and / or amount of a signal may indicate: (i) the integrity of one or more amplification reagents in an amplification reaction mixture; (ii) a malfunction of an instrument in which an amplification reaction is performed; and / or (iii) inhibition of a sample source of an amplification reaction (e.g., inhibition of a matrix source). The presence, absence and / or amount of a signal may indicate the extent to which amplification of a target nucleic acid sequence is inhibited in an amplification reaction.

[0134] Method, reagent composition and / or amplification reaction mixture can comprise the quality control template of at least about 50,000 copies to about 500,000 copies.The comparable method of monitoring amplification reaction can adopt the internal control template of about 20 copies to about 100 copies.In some embodiments, the method, reagent composition and / or amplification reaction mixture comprise the quality control template and / or quality control primer of more copy number at least about 1.1 times (for example, 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 between these values ​​or scope) compared with the comparable method of monitoring the amplification reaction that does not comprise quality control template and / or quality control primer.In some embodiments, the comparable method comprises the internal control template that can not form hairpin structure. In some embodiments, a comparable method for monitoring an amplification reaction that does not include a quality control template and / or a quality control primer suppresses amplification of a target nucleic acid sequence and / or detection of a nucleic acid amplification product by at least about 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 an amount or range between any two of these values) more than the methods disclosed herein. In some embodiments, the quality control template and / or the quality control primer are not capable of hybridizing to the target nucleic acid sequence. In some embodiments, the presence of the quality control template and / or the quality control primer does not suppress amplification of a target nucleic acid sequence and / or detection of a nucleic acid amplification product. The presence of a quality control template and / or a quality control primer in the amplification reaction mixture can improve amplification of a target nucleic acid sequence and / or detection of a nucleic acid amplification product by at least about 1.1 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 a number or range between any two of these values) compared to a comparable method in which the quality control template and / or the quality control primer are not present in the amplification reaction mixture. The number of mispriming events and / or the generation of primer-dimers can be reduced by at least about 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 a number or range between any two of these values) compared to a comparable method in which an amplification reaction not containing a quality control template and / or a quality control primer is monitored.

[0135] The disclosure herein includes a reaction mixture. In some embodiments, the reaction mixture comprises: a quality control template disclosed herein; a quality control primer disclosed herein; a signal generating oligonucleotide disclosed herein; a supplemental quality control primer disclosed herein; a target nucleic acid sequence; and / or one or more additional primers and / or one or more probes specific to the target nucleic acid sequence. The reaction mixture may comprise one or more of an enzyme having polymerase activity, dNTPs, and a buffer.

[0136] In some embodiments, quality control primers are provided. The quality control primers can include 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 a number or range between any two of these values) identical sequences to SEQ ID NO: 3 or SEQ ID NO: 9. The quality control primers can include sequences with 1, 2, 3, 4 or more mismatches or universal nucleotides relative to SEQ ID NO: 3 or SEQ ID NO: 9. In some embodiments, quality control templates (e.g., hairpin internal control (HPIC) molecular beacons) are provided. The quality control template may comprise a sequence that is 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 a number or range between any two of these values) identical to SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 11. The quality control template may comprise a sequence having 1, 2, 3, 4, or more mismatches or universal nucleotides relative to SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 11. The quality control primer and quality control template may comprise one or more modifications (e.g., thiophosphorylated DNA bases, LNA). The quality control template may comprise a 5' modification (e.g., 5HEX) and / or a 3' modification (e.g., 3IABkFQ). The IC assay components provided herein can be employed in multiplex assays (eg, in conjunction with assays for detecting C. trachomatis and / or N. Gonorrhea). Exemplary HPIC assay components are shown in Table 1.

[0137] Table 1: HPIC Assay Components

[0138]

[0139] Amplifying the target nucleic acid sequence can include generating nucleic acid amplification products and / or quality control products at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes. Detection can be carried out in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time when the reagent composition contacts the sample being processed.

[0140] The lysis buffer may include 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). The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample nucleic acid may include cell RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination 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; forward primer; reverse primer; reverse transcription primer. The reagent composition may be freeze-dried, heat-dried, and / or include 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. Sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof. Polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptide, collagen peptide or any combination thereof. Contacting the reagent composition with the treated sample can include dissolving the reagent composition in the treated sample. In some embodiments, one or more lytic reagents include: 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 detergent (e.g., one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant and amphoteric surfactant).

[0141] In some embodiments, the method: is performed in a single reaction vessel; does not include the use of any enzyme other than reverse transcriptase and an enzyme with thermophilic polymerase activity; does not include the use of any enzyme other than an enzyme with thermophilic polymerase activity; does not include thermal and / or enzymatic denaturation of the nucleic acid during the amplification step; and / or does not include contacting the nucleic acid with a single-stranded DNA binding protein.

[0142] The target nucleic acid sequence may include a length of 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 sequence may include a portion of the target nucleic acid sequence. The spacer 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, 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., a period of about 15 minutes). Amplification may be performed under isothermal amplification conditions without helicase, single-stranded binding protein, cutting agent, and recombinase. Amplification may be performed using a method selected from PCR, LAMP, SDA, replicase-mediated amplification, immune amplification, NASBA, 3SR, RCA, and TMA. PCR may be real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0143] The enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. The enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1. The enzyme with hyperthermophilic polymerase activity can be a polymerase comprising 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. The sample ribonucleic acid can be contacted with a reverse transcriptase and an enzyme with hyperthermophilic polymerase activity at the same time. The sample ribonucleic acid can be contacted with a reverse transcriptase, an enzyme with hyperthermophilic polymerase activity, and a forward primer and a reverse primer at the same time. The sample ribonucleic acid can be contacted with a reverse transcriptase, an enzyme with hyperthermophilic polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer at the same time.

[0144] In some embodiments, amplification includes and / or does not include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, amplification does not include LAMP.

[0145] 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 cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal and / or enzymatic denaturation of the sample nucleic acid prior to and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0146] 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, during the main enzymatic reaction step where amplification occurs, the temperature deviation of the isothermal amplification reaction does not exceed 10°C, for example, not more than 5°C or not more than 2°C. According to the method of isothermal amplification of nucleic acids, different enzymes can be used for amplification. Isothermal amplification compositions and methods are described in the PCT application published as WO2017176404, the contents of which are incorporated herein by reference in their entirety.

[0147] In some embodiments, the methods and components described herein include storage-stable lysis buffer. In some embodiments, the lysis buffer resists the formation of precipitation for a period of time under storage conditions (e.g., storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer resists precipitation are described in International Application No. PCT / US23 / 61980, entitled "NON-OPAQUELYTIC BUFFER COMPOSITION FORMULATIONS" and filed on February 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0148] Some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote chain separation and / or promote chain melting) other than acid and / or low pH conditions. Compositions, kits, and methods for nucleic acid detection in which nucleic acid chains dissociate under low pH conditions (e.g., via contact with an acidic lysis buffer) to promote 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 on February 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0149] In some embodiments, the methods and compositions described herein may include a lysis buffer and / or a reagent composition. A lysis buffer comprising a lysis agent and a reducing agent and a reagent composition comprising an amplifying agent and one or more protective agents (e.g., cyclodextrin compounds) capable of isolating the lysis agent are described in International Application No. PCT / US22 / 21015, entitled "ISOTHERMAL AMPLIFICATION OFPATHOGENS" and filed on March 18, 2022, the contents of which are incorporated herein by reference in their entirety.

[0150] In some embodiments, the methods and compositions described herein may include a signal generating oligonucleotide comprising one or more polymerase terminators (e.g., protected signal generating oligonucleotides). Compositions, kits, and methods for nucleic acid detection in which protected signal generating oligonucleotides can reduce non-specific 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” and filed on September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0151] In some embodiments, some embodiments of the methods and compositions described herein may be employed in conjunction with the systems, methods, compositions, and kits for monitoring amplification reactions described in U.S. Provisional Patent Application No. 63 / 374,835, entitled “HAIRPIN INTERNAL CONTROL FOR ISOTHERMAL NUCLEIC ACID AMPLIFICATION,” filed on September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0152] In some embodiments, some embodiments of the methods and compositions described herein may be employed 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” and filed on September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0153] Nucleic Acids, Subjects, Samples, and Nucleic Acid Processing

[0154] Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" may be used interchangeably herein. These terms refer to nucleic acids of any composition, such as DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), etc.), RNA (e.g., messenger RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs and / or non-natural backbones, etc.), RNA / DNA hybrids and polyamide nucleic acids (polyamide nucleic acids). Acids, PNAs), all of which may be in single-stranded or double-stranded form, and unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be or may be derived from a plasmid, a bacteriophage, an autonomously replicating sequence (ARS), a centromere, an artificial chromosome, a chromosome, or other nucleic acids that can replicate or be replicated in vitro or in a host cell, a cell, a nucleus, a mitochondria, or the cytoplasm of a cell. Unless specifically limited, 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 conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, interspecies homologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as explicitly indicated sequences. In particular, degenerate codon substitutions may be obtained by generating a third position in which one or more selected (or all) codons are mixed. The term nucleic acid can be used interchangeably with locus, gene, cDNA and mRNA encoded by a gene. As an equivalent, the term can also include derivatives, variants and analogs of RNA or DNA synthesized from nucleotide analogs, single-stranded ("sense" or "antisense", "positive" strand or "negative" strand, "forward" reading frame or "reverse" reading frame, "forward" strand or "reverse" strand) and double-stranded polynucleotides. The term "gene" means a DNA segment involved in producing a polypeptide chain. ; and generally include regions before and after the coding region (leader and trailer) that participate in the transcription / translation of the gene product and the regulation of transcription / translation, 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 base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed in terms of the number of bases.

[0155] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid can be referred to as a target sequence, a target polynucleotide and / or a target polynucleotide sequence, and can include a double-stranded nucleic acid molecule and a single-stranded nucleic acid molecule. The target nucleic acid can be, for example, DNA or RNA. When the target nucleic acid is an RNA molecule, the molecule can be, for example, double-stranded, single-stranded, or the RNA molecule can include a single-stranded target sequence. When the target nucleic acid is double-stranded, the target nucleic acid generally includes a first strand and a second strand. The first strand and the second strand can be referred to as a forward strand and a reverse strand, and are generally complementary to each other. When the target nucleic acid is single-stranded, a complementary strand can be produced, for example, by polymerization and / or reverse transcription, so that the target nucleic acid is double-stranded and has a first / forward strand and a second / reverse strand.

[0156] The target sequence can refer to the sense strand or antisense strand of the nucleic acid sequence, and can also refer to the sequence present in the target nucleic acid, amplified copy or amplified product of the original target sequence. The target sequence can be a subsequence in a larger polynucleotide. For example, the target sequence can be a short sequence (for example, 20 to 50 bases) in a nucleic acid fragment, a chromosome, a plasmid, which is targeted for amplification. In some embodiments, the target sequence can refer to a sequence complementary to an oligonucleotide (for example, a primer) for amplifying nucleic acid in a target nucleic acid. Therefore, the target sequence can refer to the entire sequence targeted for amplification, or can refer to a subsequence of oligonucleotide binding in a target nucleic acid. Amplified product can be a larger molecule comprising the target sequence and at least one other sequence or other nucleotides. The amplified product can be a length approximately the same as the target sequence, for example, a length identical to the target sequence. The amplified product can include the target sequence or be composed of the target sequence.

[0157] The length of the target sequence and / or guanine cytosine (GC) concentration (percentage) can depend in part on the temperature at which the amplification reaction is run, and the 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 length of the target sequence 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%.

[0158] Target nucleic acid can include, for example, genomic nucleic acid, plasmid nucleic acid, mitochondrial nucleic acid, cellular nucleic acid, extracellular nucleic acid, bacterial nucleic acid and viral nucleic acid. In some embodiments, target nucleic acid can include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, gene, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA or synthetic oligonucleotide. Genomic nucleic acid can include any nucleic acid from any genome, for example, animal, plant, insect, virus and bacterial genome (for example, the genome present in spore). In some embodiments, genomic target nucleic acid is in a specific genomic locus or more than one genomic locus. Genomic locus can include any one or combination of open reading frame DNA, non-transcribed DNA, intron sequence, exon sequence, promoter sequence, enhancer sequence, flanking sequence or any sequence considered to be related to a given genomic locus.

[0159] The target sequence may comprise one or more repeat elements (e.g., multiple repeats, inverted repeats, palindromes, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence is present in the sample nucleic acid (e.g., in a nucleic acid fragment, chromosome, genome, plasmid) as a repeat element (e.g., multiple repeats, inverted repeats, palindromes, tandem repeats, microsatellites, minisatellites, etc.). For example, the target sequence may appear multiple times as a repeat element, and one, some, or all occurrences of the target sequence in the repeat element may be amplified using the methods described herein (e.g., using a single pair of primers). In some embodiments, the target sequence is present in the sample nucleic acid (e.g., in a nucleic acid fragment, chromosome, genome, plasmid) as a repeat and / or homolog within the species.

[0160] The target nucleic acid may include microRNA. MicroRNA, miRNA or small temporal RNAs (stRNA) are short (e.g., about 21 to 23 nucleotides long) and single-stranded RNA sequences involved in gene regulation. MicroRNA can interfere with the translation of messenger RNA and is partially complementary to messenger RNA. The target nucleic acid may include microRNA precursors, such as primary transcripts (pri-miRNA) and pre-miRNA stem-loop structure RNAs that are further processed into miRNA. The target nucleic acid may include short interfering RNA (siRNA), which is a short (e.g., about 20 to 25 nucleotides long) and at least partially double-stranded RNA molecule involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0161] The nucleic acid used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, from the sample separation obtained from the subject. 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 humans or non-human animals can be selected, including but not limited to mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (for example, cattle), equines (for example, horses), caprines and ovines (for example, sheep, goats), swine (for example, pigs), camelids (for example, camels, llamas, alpacas), monkeys, apes (for example, gorillas, chimpanzees), ursids (for example, bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales and sharks. Subject can be male or female, and subject can be any age (for example, embryo, fetus, infant, child, adult).

[0162] Sample or test sample can be any sample separated or obtained from a subject or part thereof. The non-limiting examples of sample include fluid or tissue 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, spinal fluid, lavage fluid (e.g., bronchoalveolar, stomach, peritoneum, catheter, ear, arthroscopy), serum, plasma, urine, aspirate (aspirate), biopsy sample, abdominal puncture sample, cell (e.g., blood cell) or its part (e.g., mitochondria, nucleus, extract, etc.), female reproductive tract washings, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, hard tissue (e.g., liver, spleen, kidney, lung or ovary), etc. or its combination. The term blood includes any fraction of whole blood, blood products or blood, such as serum, plasma, buffy coat or the analog of conventional definition. Plasma refers to the whole blood fraction produced by centrifugation of blood treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after the blood sample has coagulated. Fluid or tissue samples are usually collected according to the standard protocols usually followed in hospitals or clinics. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) is usually collected and can be stored according to standard procedures before or after preparation.

[0163] Samples can include samples 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 of spores (e.g., without the need for lysis). Samples can be isolated from any material suspected of containing the target sequence, such as from a subject described above. In some embodiments, the target sequence is present in air, plants, soil, or other materials suspected of containing biological organisms.

[0164] Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method can be used to isolate, extract and / or purify nucleic acids from a biological sample, including DNA preparation methods known in the art, as well as various commercially available reagents or kits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep TM Blood DNA Isolation Kit (Promega, Madison, Wis.), GFX TM Genomic blood DNA purification kit (Amersham, Piscataway, NJ), etc. or a combination thereof. US Patent No. 7,888,006 provides a DNA purification method and does not disclose the compositions (eg, lysis buffer, protective agent) and methods provided herein.

[0165] In some embodiments, a cell lysis procedure is performed. Cell lysis can be performed before starting the amplification reaction described herein (for example, to release DNA and / or RNA from cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical (for example, detergents, hypotonic solutions, enzymatic procedures, etc., or combinations thereof), physical (for example, French press, ultrasonic treatment, etc.) or electrolytic lysis methods. For example, chemical methods typically use lysing agents to destroy cells and extract nucleic acids from cells, and then treat with chaotropic salts. In some embodiments, cell lysis includes the use of detergents (for example, ions, non-ions, anions, zwitterions). In some embodiments, cell lysis includes the use of ionic detergents (for example, sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), deoxycholate, cholate, sodium lauryl sarcosine (sarkosyl)). Physical methods such as grinding after freezing / thawing, 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 an alternative phenol-chloroform-free procedure involving three solutions may 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; a second solution may contain 0.2 N NaOH and 1% SDS; and a third solution may contain, for example, 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and compositions described herein.

[0166] Nucleic acid can be provided for carrying out the methods described herein without processing samples containing nucleic acid. For example, nucleic acid can be provided for carrying out the amplification methods described herein without prior nucleic acid purification. In some embodiments, the target sequence is amplified directly from the sample (for example, without performing any nucleic acid extraction, separation, purification and / or partial purification steps). In some embodiments, after processing a sample containing nucleic acid, nucleic acid is provided for carrying out the methods described herein. For example, nucleic acid can be extracted, separated, purified or partially purified from a sample. The term "isolated" generally refers to nucleic acid taken out from its original environment (for example, if it is naturally present, it is a natural environment, if it is exogenously expressed, it is a host cell), and thus changed from its original environment by human intervention (for example, "by human hands"). The term "isolated nucleic acid" can refer to nucleic acid taken out from a subject (for example, a human subject). Isolated nucleic acid can provide non-nucleic acid components (for example, proteins, lipids, carbohydrates) less than the amount of components present in the source sample. The composition comprising isolated nucleic acid can be free of about 50% to greater than 99% of non-nucleic acid components. The composition comprising the nucleic acid of separation can be free of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% non-nucleic acid components. The term "purified" generally refers to that the non-nucleic acid components (e.g., protein, lipid, carbohydrate) provided by the nucleic acid are less than the amount of the non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. The composition comprising the nucleic acid of purification can be free of other non-nucleic acid components by about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%.

[0167] Nucleic acids can be provided for performing the methods described herein without modifying the nucleic acids. Modifications can include, for example, denaturation, digestion, nicking, melting, incorporation and / or ligation of heterologous sequences, addition of epigenetic modifications, addition of labels (e.g., radioactive labels 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.

[0168] The method disclosed herein for detecting a target nucleic acid sequence (single-stranded or double-stranded DNA and / or RNA) in a sample can detect a target nucleic acid sequence (e.g., DNA or RNA) with a high degree of sensitivity. In some embodiments, the method can be used to detect a target DNA / RNA present in a sample containing more than one RNA / DNA (including a target RNA / DNA and more than one non-target RNA / DNA), wherein the target RNA / DNA is present in a sample at a concentration of 10, 20, 25, 50, 100, 500, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 80000, 90000, 100000, 15 ... 3 pcs, 5×10 3 10 4 pcs, 5×10 4 10 5 pcs, 5×10 5 10 6 or 10 7 One or more copies of each non-target DNA / RNA are present. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" should be given their ordinary meanings, and should also refer to DNA, or RNA, or a combination of DNA and RNA.

[0169] 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 ordinary meaning, and should also describe the minimum amount of target RNA / DNA that must be present in a sample in order to detect. 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 embodiments, the disclosed methods have a detection threshold of 5nM or less, 1nM or less, 0.5nM or less, 0.1nM or less, 0.05nM or less, 0.01nM or less, 0.005nM or less, 0.001nM or less, 0.0005nM or less, 0.0001nM or less, 0.00005nM or less, 0.00001nM or less, 10pM or less, 1pM or less, 500fM or less, 250fM or less, 100fM or less, 50fM or less, 500aM (attomolar) or less, 250aM or less, 100aM or less, 50aM or less, 10aM or less, or 1aM or less. In some embodiments, the disclosed compositions or methods exhibit detection sensitivity of attomole / liter (aM), femtomolar / liter (fM), picomolar / liter (pM), and / or nanomolar / liter (nM).

[0170] The sample can include sample nucleic acids (e.g., more than one sample nucleic acid). The term "more than one" is used herein to mean two or more. Thus, in some embodiments, the sample includes 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 very sensitive means of detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, the sample includes 5, 10, 20, 25, 50, 100, 500, 1000 or more) sample nucleic acids that are different in sequence from each other. 3 5×10 3 10 4 5×10 4 10 5 5×10 5 10 6 Species or 10 7 In some embodiments, the sample comprises 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.).

[0171] The term "sample" as used herein should be given its ordinary meaning and should include any sample containing RNA and / or DNA (e.g., in order to determine whether a target DNA and / or target RNA is present in a RNA and / or DNA population). The sample can be a biological sample or an environmental sample. The sample can be derived from any source, for example, the sample can be a synthetic combination of purified DNA and / or RNA; the sample can be a cell lysate, a cell lysate enriched in DNA / RNA, or DNA / RNA isolated and / or purified from a cell lysate. The sample can be from a patient (e.g., for diagnostic purposes). The sample can be from permeabilized cells, cross-linked cells, tissue sections, or a combination thereof. The sample can be from a tissue prepared by cross-linking followed by delipidation and adjustment to form a uniform refractive index. The sample can contain a target nucleic acid (e.g., a target DNA / RNA) and more than one non-target DNA / RNA. In some embodiments, the target DNA / RNA is present in 10, 20, 25, 50, 100, 500, 10 3 pcs, 5×10 3 10 4 pcs, 5×10 4 10 5 pcs, 5×105 10 6 or 10 7 One copy of each non-target DNA / RNA is present in the sample.

[0172] Samples about patients include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, as well as samples that have been manipulated in any way after their acquisition (such as by treatment with reagents), washed or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA). Samples may include the following or are the following: biological samples, including but not limited to clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), and also tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, 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.), for example, samples containing RNA obtained from such cells (e.g., cell lysates or other cell extracts containing RNA). In some embodiments, 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 freshwater sample, a wastewater sample, a saltwater sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof.

[0173] The source of the sample can be (or suspected to be) pathological cells, fluids, tissues or organs; or normal (non-lesion) cells, fluids, tissues or organs. In some embodiments, the source of the sample is (or suspected to be) pathogen-infected cells, tissues or organs. For example, the source of the sample can be an individual who may be infected or may not be infected, and the sample can be any biological sample collected from the individual (for example, blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, stool sample, cerebrospinal fluid, fine needle aspiration, swab sample (for example, oral swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal mucus, tears, buffy coat, mucosal sample, epithelial cell sample (for example, epithelial cell scraping) etc.), and its culture. The sample can be a cell-free liquid sample or a liquid sample comprising cells. Pathogens can be viruses, fungi, worms, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites, etc. "Herms" include roundworms, heartworms, and phytophagous nematodes (Nematoda), flukes (Trematoda), acanthocephala, and tapeworms (Cestoda). Protozoan infections include Giardia spp., Trichomonas spp. infections, African trypanosomiasis, amoebic dysentery, babesiosis, balantidium dysentery, 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.albicans). Pathogenic viruses include, for example, immunodeficiency viruses (e.g., HIV); influenza virus; dengue fever; West Nile virus; herpes virus; yellow fever virus; hepatitis C virus; hepatitis A virus; hepatitis B virus; papillomavirus, etc. Pathogenic viruses can include DNA viruses such as: papovaviruses (e.g., HPV, polyomavirus); hepadnavirus; 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., thymovirus, avian adenovirus, ichtadenovirus, mammalian adenovirus, sialidase adenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, capripox virus, pseudovaccinia, bovine papular stomatitis virus; tanapox virus, yaba monkey tumor virus (yaba monkey tumor virus)); virus); Molluscum contagiosum virus (MCV); Parvovirus (e.g., adeno-associated virus (AAV), Parvovirus B19, Human Bocavirus, Buffavirus, 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, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, and the like.disease spirochetes), Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, human serum parvovirus-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, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria sp. (e.g., Eimeria tenella), Onchocerca volvulus, Leishmania sp. (e.g., Leishmania tropicala), Streptococcus pneumonia, Pneumocystis carinii, Trichophyton rubrum, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp.), SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), herpes simplex virus, herpes virus 6, herpes virus 7, JC virus, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, human enterovirus, Hantavirus, Legionella dumoffii, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsiirickettsii), Ehrlichia sp. (e.g., Ehrlichia chaffeensis), Borrelia burgdorferi, Yersinia pestis, Chlamydia pneumoniae, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma sp. (e.g., Mycoplasma arthritidis), M.hyorhinis, M.orale, M.arginini, Acholeplasma laidlawii, M.salivarium, and M.pneumoniae.

[0174] Amplification

[0175] Provided herein is a method for amplifying nucleic acid. In some embodiments, nucleic acid is amplified using a suitable amplification method. Nucleic acid amplification generally relates to the enzymatic synthesis of nucleic acid amplicon (copy), which comprises a sequence complementary to the nucleotide sequence amplified. In some embodiments, amplification method is carried out in a single container, a single chamber and / or a single volume (i.e., a continuous volume). In some embodiments, amplification method and detection method (e.g., detection method described herein) are carried out in a single container, a single chamber and / or a single volume (i.e., a continuous volume).

[0176] The terms "amplify," "amplification," "amplification reaction," or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification sometimes refers to an "exponential" increase in a target nucleic acid. "Amplification" may also refer to a linear increase in the number of target nucleic acids, but is distinct from a single, single primer extension step. In some embodiments, a limited amplification reaction, also referred to as pre-amplification, may be performed. Pre-amplification is a method in which a limited amount of amplification occurs because a small number of cycles, such as 10 cycles, are performed. Pre-amplification may allow some amplification, but stops amplification before the exponential phase and typically produces about 500 copies of the desired nucleotide sequence. The use of pre-amplification may limit inaccuracies associated with depleted reactants in certain amplification reactions, and may also reduce amplification biases due to the nucleotide sequence or material abundance of the target. In some embodiments, a primer extension may be performed as a prelude to linear or exponential amplification.

[0177] A general description of amplification process is presented herein. Primers (e.g., oligonucleotides described herein) contact with target nucleic acid, and for example, complementary sequences anneal or hybridize to each other. Primers can anneal with target nucleic acid at or near the sequence of interest (e.g., adjacent, contiguous, etc.). Primers annealed with target can be referred to as primer-target hybrids, hybridized primer-targets or primer-target duplexes. When referring to a nucleotide sequence of interest, the term near or adjacent to the distance (e.g., base number) or region between one or more nucleotides (e.g., nucleotide sequence) of the primer end and the target. Typically, proximity is a range of about 1 nucleotide to about 50 nucleotides from a nucleotide or nucleotide sequence of interest (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 nucleotides). In some embodiments, a group 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 a nucleotide or nucleotide sequence of interest, and produce an amplified product. In some embodiments, the primers are annealed within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended by a polymerase along the target (i.e., template strand) to produce a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplified 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.

[0178] The components of the 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 multiple amplification, etc.), nucleic acid targets (e.g., target nucleic acids from samples), one or more polymerases, nucleotides (e.g., dNTPs, etc.), and suitable buffers (e.g., buffers 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 agents.

[0179] Nucleic acid amplification can be carried out in the presence of natural nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derived nucleotides. Natural nucleotides generally refer to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid or uridylic acid. Derived nucleotides are generally nucleotides different from natural nucleotides. Ribonucleotide triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T or U. Monomeric nucleotide subunits may be represented herein as A, G, C, T or U, without particular reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs may be used, such as analogs containing detectable labels (e.g., fluorescent or colorimetric labels). For example, nucleic acid amplification can be carried out in the presence of labeled dNTPs, such as radioactive labels, 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, or fluorescent dyes. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, for example, heat-activated dNTPs (e.g., CleanAmp from TriLink TM dNTP).

[0180] One or more amplification reagents can include non-enzyme components and enzyme components.Non-enzyme components can include, for example, primers, nucleotides, buffer, salt, reducing agent, detergent and ion.In some embodiments, non-enzyme components do not include protein (for example, nucleic acid binding protein), enzyme or protein with enzymatic activity, for example, polymerase, reverse transcriptase, helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nickase, recombinase etc.In some embodiments, enzyme component is made up of polymerase or is made up of polymerase and reverse transcriptase.Therefore, such enzyme component will exclude other proteins (for example, nucleic acid binding protein and / or protein with enzymatic activity), for example, helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nickase, recombinase etc.

[0181] In some embodiments, the amplification conditions include enzymatic activity (e.g., enzymatic activity provided by a polymerase or enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include enzymatic activity provided by enzymes other than polymerases and / or reverse transcriptases, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, etc. The polymerase activity and reverse transcriptase activity can be provided by separate enzymes or separate enzyme types (e.g., polymerase and reverse transcriptase), or by a single enzyme or enzyme type (e.g., polymerase).

[0182] The amplification of nucleic acid can include the PCR of non-thermal cycle type. In some embodiments, the amplification of nucleic acid includes isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process carried out at a constant temperature. Terms such as isothermal conditions, isothermally and constant temperature generally refer to reaction conditions in which the reaction temperature remains constant during the amplification reaction process. Isothermal amplification conditions do not generally include thermal cycling (i.e., circulating between higher temperatures and lower temperatures) components in the amplification process. When amplified under isothermal conditions, the reaction can be maintained at a substantially constant temperature, which means that the temperature can be maintained at a temperature inaccurately. For example, in the isothermal amplification process, due to, for example, environment or variables based on equipment, small fluctuations of temperature (such as ± 1 ° C to 5 ° C) may occur. Generally, the whole reaction volume is maintained at a substantially constant temperature, and the isothermal reaction herein does not generally include the amplification conditions that depend on the temperature gradient generated in the reaction vessel and / or the temperature cycle based on convection.

[0183] Isothermal amplification reaction herein can be carried out at a substantially constant temperature. In some embodiments, isothermal amplification reaction herein is carried out at a temperature of about 55 ° C to a temperature of about 75 ° C, for example, at the following temperature or at about the following temperature: 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 any two of these values ​​between the quantity or scope. In some embodiments, temperature element (for example, 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 of substantially constant temperature.

[0184] Amplification process herein can be carried out for a certain time length, for example until detectable nucleic acid amplification product and / or quality control product are generated. Nucleic acid amplification product and / or quality control product can be detected by any suitable detection process and / or detection process described herein. Amplification process can be carried out for a time length of about 20 minutes or less time or about 10 minutes or less time. For example, amplification process can be carried out in about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 minutes or in any two of these values, quantity or scope.

[0185] In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, during the amplification step (and / or other steps), the nucleic acid target can be amplified without exposure to agents or conditions that promote strand separation. In some embodiments, during the amplification step (and / or other steps), the nucleic acid target can be amplified without exposure to agents or conditions that promote unwinding. Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote unwinding can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemicals, proteins (e.g., enzymatic agents), etc.

[0186] In some embodiments, the methods disclosed herein do not include thermal denaturation of nucleic acids (e.g., heating a solution containing nucleic acids to a high temperature, such as, for example, a temperature higher than 75°C, 80°C, 90°C, or 95°C or higher) or protein-based (e.g., enzymatic) denaturation. Protein-based (e.g., enzymatic) denaturation can include contacting nucleic acids 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 include 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 include intercalating agents, alkylating agents, and / or chemicals, such as formamide, glycerol, urea, dimethyl sulfoxide (DMSO) or N,N,N-trimethylglycine (betaine). In some embodiments, disclosed methods do not include contacting nucleic acid with a denaturant (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions for denaturing nucleic acid (e.g., promoting chain separation and / or promoting unwinding). In some embodiments, the amplification step (e.g., step (c)) does not include agents and / or conditions for denaturing nucleic acid (e.g., promoting chain separation and / or promoting unwinding) other than polymerase (e.g., hyperthermophilic biopolymerase). In some embodiments, methods and compositions provided herein do not include agents and / or conditions for denaturing nucleic acid (e.g., promoting chain separation and / or promoting unwinding) other than polymerase (e.g., hyperthermophilic biopolymerase) and / or low pH conditions (e.g., contacting with one or more acids).

[0187] The nucleic acid target can be amplified without exposure to agents or conditions that promote chain separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-stranded binding proteins) or any combination thereof. For example, the nucleic acid target can be amplified without exposure to a helicase, which includes but is not limited to DNA helicases and RNA helicases. Amplification conditions that do not include the use of a helicase are amplification conditions without a helicase.

[0188] The nucleic acid target can be amplified without being exposed to a recombinase, and the recombinase includes but is not limited to Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, T4 uvsX. In some embodiments, the nucleic acid target is amplified without being exposed to a recombinase auxiliary protein, such as a recombinase loading factor (e.g., T4 uvsY). The nucleic acid target can be amplified without being exposed to a nucleic acid binding protein (e.g., a single-stranded binding protein or a single-stranded DNA binding protein (SSB)), such as T4gp32. In some embodiments, the nucleic acid target is amplified without being exposed to a topoisomerase. The nucleic acid target can be amplified under exposure to or without being exposed to an agent or condition that destabilizes the nucleic acid. As used herein, the term "destabilization" should be given its ordinary meaning, and should also refer to the destruction of the overall organization and geometric orientation of nucleic acid molecules (e.g., double helix structures) by one or more of the tilting, rolling, twisting, sliding and flipping effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, 17 pages). Destabilization does not generally refer to the unwinding or separation (e.g., denaturation) of nucleic acid chains. Nucleic acid destabilization can be achieved by, for example, 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 include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, nucleic acid targets are amplified without exposure to ligases and / or RNA replicases.

[0189] In some embodiments, the nucleic acid target can be amplified without cutting or digestion. For example, the nucleic acid target can be amplified without being pre-exposed to one or more cleavage agents, and the intact nucleic acid is amplified. In some embodiments, the nucleic acid target is amplified without being exposed to one or more cleavage agents during amplification. In some embodiments, the nucleic acid target is amplified without being exposed to one or more cleavage agents after amplification. Amplification conditions that do not include the use of cleavage agents may be referred to herein as cleavage-free amplification conditions. The term "cleavage agent" generally refers to an agent that can cut nucleic acids at one or more specific or non-specific sites, sometimes a chemical substance or an enzyme. Specific cleavage agents are generally cut specifically according to a specific nucleotide sequence at a specific site. Cleavage agents may include endonucleases (e.g., restriction endonucleases, nickases, etc.); exonucleases (DNA enzymes, RNA enzymes (e.g., RNAse H), 5' to 3' exonucleases (e.g., exonucleases II), 3' to 5' exonucleases (e.g., exonucleases I) and poly (A) specific 3' to 5' exonucleases); and chemical cleavage agents.

[0190] Nucleic acid target can be amplified without restriction endonuclease and / or nickase. In some embodiments, nucleic acid is amplified without being exposed to restriction endonuclease and / or nickase in advance. In some embodiments, nucleic acid is amplified without being exposed to restriction endonuclease and / or nickase during amplification. In some embodiments, nucleic acid is amplified without being exposed to restriction endonuclease and / or nickase after amplification. Nucleic acid target can be amplified without exonuclease treatment. Exonuclease includes, for example, DNA enzyme, RNA enzyme (for example, RNA enzyme H), 5' to 3' exonuclease (for example, exonuclease II), 3' to 5' exonuclease (for example, exonuclease I) and multi (A) specific 3' to 5' exonuclease. In some embodiments, nucleic acid is amplified without exonuclease treatment before, during and / or after amplification. Exonuclease-free amplification conditions are not included. In some embodiments, nucleic acid is amplified without DNA enzyme treatment and / or RNA enzyme treatment. In some embodiments, the nucleic acid is amplified in the absence of RNase H treatment.

[0191] The nucleic acid of amplification can be referred to as nucleic acid amplification product or amplicon in this article.In some embodiments, amplification product comprises naturally occurring nucleotide, non-naturally occurring nucleotide, nucleotide analog etc. and aforementioned combination.Amplification product usually has the nucleotide sequence identical or substantially identical with the sequence (for example, target sequence) or its complement in sample nucleic acid.The nucleotide sequence of "substantially identical" in amplification product usually will have the sequence identity (for example, about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity) of height with the nucleotide sequence amplified or its complement, and variation is the result of polymerase infidelity (infidelity) or other variables sometimes.

[0192] In some embodiments, nucleic acid amplification product comprises polynucleotides that are continuously complementary or substantially identical with the target sequence in sample nucleic acid.Continuous complementation generally refers to the nucleotide sequence in the first chain, for example, wherein each ordered base (for example, read from 5' to 3') is paired with the corresponding ordered base in the second chain, and there is no gap, other sequence or unpaired base in the sequence considered to be continuous complementary. In other words, continuous complementation generally refers to the corresponding continuous base complementation of all continuous bases of nucleotide sequence in the first chain and nucleotide sequence in the second chain.Continuous complementary sequence sometimes has a length of about 5 to about 25 continuous bases, for example, a length of about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 continuous bases, or any two of these values.In some embodiments, nucleic acid amplification product is composed of polynucleotides that are continuously complementary or substantially identical with the target sequence in sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequences that are not continuously complementary or substantially identical to the target sequence (e.g., at the 5' and / or 3' ends, or within the product), for example, additional sequences incorporated into the amplification product by tailing primers or ligation, and / or additional sequences that provide a cleavage agent recognition site (e.g., a nickase recognition site). Typically, unless the target sequence includes a tandem repeat sequence, the amplification product does not include a product in the form of a tandem repeat sequence.

[0193] The nucleic acid amplification product may comprise a sequence that is complementary or substantially identical to one or more primers used in the amplification reaction. In some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is continuously complementary or identical to a first primer sequence, and a second nucleotide sequence that is continuously complementary or identical to a second primer sequence.

[0194] Nucleic acid amplification products can include spacer sequences. As described herein, the spacer sequence in the amplified product is a sequence (1 or more bases) that is continuously complementary or substantially identical to a portion of the target sequence in the sample nucleic acid, and the flank is a sequence that is complementary or substantially identical to one or more primers used in the amplification reaction in the amplified product. The spacer sequence flanking the sequence in the amplified product is generally located between the first sequence (complementary or substantially identical to the first primer) and the second sequence (complementary or substantially identical to the second primer). Therefore, the amplified product generally includes the first sequence, followed by the spacer sequence, followed by the second sequence. The spacer sequence is generally not complementary or substantially identical to the sequence in the primer. The spacer sequence can be the following or can include the following: about 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases. In some embodiments, the nucleic acid amplification product consists of or consists essentially of: a first nucleotide sequence that is continuously complementary or identical to the first primer sequence, a second nucleotide sequence that is continuously complementary or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence (e.g., at the 5' end and / or the 3' end; or within the product), which is not continuously complementary or identical to the first primer sequence and the second primer sequence, and is not part of the spacer sequence, for example, additional sequences 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 include additional sequences (e.g., at the 5' end and / or the 3' end; or within the product), which is not continuously complementary or identical to the first primer sequence and the second primer sequence, and is not part of the spacer sequence, for example, additional sequences incorporated into the amplification product by tailing or circularizing primers, ligation, or other mechanisms. However, in such embodiments, 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), for example, introduced into the product by error or contamination during the amplification process.

[0195] The length of the nucleic acid amplification product can be up to 50 bases, including 10, 15, 20, 25, 30, 35, 40, 45, 50 bases, or a number or range of bases between any two of these values. In some embodiments, the nucleic acid amplification products of a given target sequence have the same length or substantially the same length (e.g., within 1 to 10 bases). Therefore, the nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel), and generally do not produce multiple signals indicating multiple lengths (e.g., a ladder or tail on an electrophoresis gel). For multiple reactions, the nucleic acid amplification products of different target sequences can have different lengths.

[0196] The methods and components described herein can be used for multiple 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, multiple amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in the sample. For example, the amplification step can include the multiple amplification of two or more target nucleic acid sequences, and the detection step can include the multiple detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. Two or more target nucleic acid sequences can be specific to two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A virus, influenza B virus and / or influenza C virus). Multiple amplification can also refer to amplifying one or more sequences present in multiple samples simultaneously or in a stepwise manner. For example, multiple 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, an amplification reaction is prepared to detect at least two target sequences, but only one target sequence is present in the sample being tested, so that although both sequences can be amplified, only one sequence is amplified. In some embodiments, when there are two target sequences, the amplification reaction results in the amplification of the two target sequences. Multiple amplification reactions can result in the amplification of one, some or all target sequences, wherein appropriate primers and enzymes are included. In some embodiments, an amplification reaction is prepared to detect two sequences with a pair of primers, wherein one sequence is a target sequence, and one sequence is a control sequence (e.g., a synthetic sequence that can be amplified by the same primers as the target sequence and has a spacer base or sequence different from the target). In some embodiments, an amplification reaction is prepared to detect multiple groups of sequences with corresponding primer pairs, wherein each group of sequences includes a target sequence and a control sequence.

[0197] Primers

[0198] Nucleic acid amplification is usually carried out in the presence of one or more primers. Primers are usually characterized as oligonucleotides, which include nucleotide sequences that can hybridize or anneal with target nucleic acids at or near (e.g., adjacent to) a specific region of interest (i.e., a target sequence). For example, a primer can allow the specific determination of a target nucleic acid nucleotide sequence or the detection of a target nucleic acid or its characteristics (e.g., the presence or absence of a sequence). Primers can be naturally occurring or synthetic. The term specific, or specificity, usually refers to the combination or hybridization of a molecule with another molecule, such as a primer for a target polynucleotide. That is, specific or specific refers to the recognition, contact and formation of a stable complex between two molecules, in contrast, any one of the two molecules with the recognition, contact or complex formation of other molecules is much less. The term annealing or hybridization usually refers to the formation of a stable complex between two molecules. When referring to primers, the terms primer, oligomer or oligonucleotide can be used interchangeably herein.

[0199] Primers can be designed and synthesized using a suitable method, and can have any length suitable for hybridizing with the target sequence and performing amplification processes described herein. Primers are generally designed according to the sequence in the target nucleic acid. Primer length in some embodiments can be from about 5 to about 30 bases, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length. Primers can include naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides) or mixtures thereof. Modified and modified bases can include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite TM , Adenylation, Azide (NHS ester), Digoxigenin (NHS ester), Cholesteryl-TEG, I-Linker TM , amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link TM amino modifications), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylations (e.g., biotin, biotin(azide), biotin-dT, biotin-TEG, dibiotin, PC-biotin, desthiobiotin-TEG), thiol modifications (e.g., thiol modifier C3 SS, dithiol, thiol modifier C6 SS)); fluorophores (e.g., Freedom TM Dye, Alexa Dye, LI-COR ATTO TM 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 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-bromo-dU, deoxyuridine, reverse dT, reverse dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinosine, Locked nucleic acids (LNA's), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), iso-dC, iso-dG, fluoro-C, fluoro-U, fluoro-A, fluoro-G); phosphorothioate (PS) bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'-O-methyl bases, phosphorothioated LNA bases); and click chemistry modifications. In some embodiments, the modified and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS connections, 3' phosphate groups, spacer bases (such as, C3 spacer bases or other spacer bases). For example, a primer may contain one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is typically a post-transcriptional modification of RNA found in tRNA and other small RNAs. Primers comprising 2'-O-methyl RNA bases can be directly synthesized. For example, this modification can increase the Tm of RNA: RNA duplexes and provide stability in the presence of single-stranded ribonucleases and DNA enzymes. 2'-O-methyl RNA bases can be included in primers, for example, to increase stability and binding affinity to the target sequence. In some embodiments, primers can include one or more phosphorothioate (PS) connections (for example, PS key modifications). PS keys replace non-bridging oxygen in the primer phosphate backbone with sulfur atoms. This modification usually makes the internucleotide bond resist nuclease degradation. For example, PS keys can be introduced between about the last 3 to 5 nucleotides of the 5'-end or 3'-end of the primer to inhibit exonuclease degradation. In some embodiments, PS keys included in the entire primer can help reduce the attack of endonucleases. Primers can, for example, include 3' phosphate groups. In some cases, 3' phosphorylation can inhibit degradation by certain 3'-exonucleases, and can be used to block extension by DNA polymerase. In some embodiments, the primer comprises one or more spacer bases (e.g., one or more C3 spacers). The C3 spacer phosphoramidite can be incorporated into the interior or 5' end of the primer. For example, multiple C3 spacers can be added to either end of the primer to introduce a long hydrophilic spacer arm for attaching a fluorophore or other pendant group.

[0200] Primers can include DNA bases, RNA bases or both, wherein one or more DNA bases and RNA bases are modified or unmodified. For example, primers can be a mixture of DNA bases and RNA bases. Primers can be composed of DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, primers are composed of unmodified DNA bases. In some embodiments, primers are composed of modified DNA bases. Primers can be composed of RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, primers are composed of unmodified RNA bases. In some embodiments, primers are composed of modified RNA bases. In some embodiments, primers do not include RNA bases. In some embodiments, primers do not include DNA bases. In some embodiments, primers do not include cutting agent recognition sites (e.g., do not include nickase recognition sites). In some embodiments, primers do not include tails (e.g., do not include tails containing nickase recognition sites).

[0201] In some embodiments, all or part of the primer sequence can be complementary or substantially complementary to the target nucleic acid.Substantially complementary about sequence generally refers to nucleotide sequences that will hybridize to each other.The stringency of hybridization conditions can be changed to tolerate different numbers of sequence mispairings.The target sequence and primer sequence can be, for example, 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 to each other.The primer substantially complementary to the target nucleic acid sequence is also generally identical with the complement of the target nucleic acid sequence (that is, 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, e.g., 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.

[0202] In some embodiments, primer includes a pair of primers. A pair of primers can include a forward primer and a reverse primer (for example, a primer combined with the sense and antisense strands of the target nucleic acid). In some embodiments, primers are composed of a pair of primers (that is, a forward primer and a reverse primer). Therefore, in some embodiments, a pair of primers is used to amplify the target sequence, and other primers or oligonucleotides are not included in the amplification of the target sequence (for example, the amplification reaction component does not include other primer pairs of a given target sequence, does not include nested primers, does not include a buffer primer (bumper primer), does not include oligonucleotides other than primers, does not include probes, etc.). In some embodiments, primers are composed of a pair of primers. In some embodiments, amplification reaction can include other primer pairs for amplifying different target sequences, such as in multiple amplification. In some embodiments, primers are composed of a pair of primers, however, in some embodiments, amplification reaction can include other primers, oligonucleotides or probes for detection process, and this detection process is not considered to be a part of 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, primers that amplify a first target sequence are considered a primer set, and primers that amplify a second target sequence are considered a different primer set.

[0203] Nucleic acid described herein (e.g., amplification product, sample nucleic acid, target nucleic acid sequence) can include a first strand and a second strand that are complementary to each other. Amplification reaction components can include or consist of a first primer (first oligonucleotide) complementary to a target sequence in a first strand (e.g., sense strand, forward strand) of a sample nucleic acid and a second primer (second oligonucleotide) complementary to a target sequence in a second strand (e.g., antisense strand, reverse strand) of a sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) includes a first polynucleotide that is continuously complementary to a target sequence in a first strand of a sample nucleic acid, and the second primer (second oligonucleotide) includes a second polynucleotide that is continuously complementary to a target sequence in a second strand of a sample nucleic acid. The continuous complementarity of primer-target generally refers to a nucleotide sequence in a primer, wherein each base is paired with a corresponding ordered base in a target sequence in order, and a gap in the sequence, an additional sequence, or an unpaired base is not considered to be continuously complementary. In some embodiments, the primer does not include any additional sequence that is not continuously complementary to the target sequence (e.g., at the 5' and / or 3' end, or within the primer), for example, an additional sequence present in a tailing primer or a circularizing primer, and / or an additional sequence that provides a cleavage agent recognition site (e.g., a nickase recognition site). In some embodiments, the amplification reaction components do not include primers containing additional sequences (i.e., sequences other than the sequence that is continuously complementary to the target sequence), for example, tailing primers, circularizing primers, primers that can form a stem-loop structure, a hairpin structure, and / or additional sequences that provide a cleavage agent recognition site (e.g., a nickase recognition site), etc.

[0204] In some embodiments, the primer may comprise modifications such as one or more inosines, abasic sites, LNAs, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifiers that alter primer binding properties. In some embodiments, the primer may comprise a detectable molecule or entity (e.g., a fluorophore, a radioisotope, a colorimetric agent, a particle, an enzyme, etc.).

[0205] Polymerase

[0206] Amplification reaction components (e.g., one or more amplification reagents) may include one or more polymerases. Polymerases are proteins that can specifically incorporate nucleotides to extend primer molecules (e.g., amplification primers described herein) for nucleic acid target sequences (e.g., primers annealing thereto) to extend the 3' hydroxyl termini of primer molecules. Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that are active at elevated reaction temperatures (e.g., higher than 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C). Hyperthermophilic polymerases may be referred to as hyperthermophilic biopolymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, polymerases may incorporate about 1 to about 50 nucleotides in a single synthesis, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in a single synthesis, or the quantity or range between any two of these values.

[0207] The amplification reaction components may include one or more DNA polymerases selected from the group consisting of: 9°N DNA polymerase; 9°Nm TM DNA polymerase; 90N D family DNA polymerase; Therminator TM DNA polymerase; Therminator TM IIDNA polymerase; Therminator TM IIIDNA polymerase; Therminator TM gamma DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo); T4 DNA polymerase; T7 DNA polymerase; Deep VentR TM (Exo)DNA polymerase; Deep VentR TM DNA polymerase; DyNAzyme TMEXT DNA; DyNAzyme TM II hot start DNA polymerase; Phusion TM High-fidelity DNA polymerase; DNA polymerase; (Exo)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.

[0208] In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermally stable at high temperatures). 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 components may include one or more hyperthermophilic DNA polymerases from archaea (e.g., hyperthermophilic DNA polymerases from Thermococcus or hyperthermophilic DNA polymerases from Thermococcaceae archaea). In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components comprise one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0209] In some embodiments, the amplification reaction components include hyperthermophilic organism DNA polymerase or its functional fragment. Functional fragments usually retain one or more functions of full-length polymerase, such as the ability of polymerized DNA (for example, in amplification reaction). In some cases, functional fragments perform functions (for example, polymerized DNA in amplification reaction) at least about 50%, at least about 75%, at least about 90%, at least about 95% of the level of full-length polymerase functional level. The level of polymerase activity can be, for example, assessed using a detectable nucleic acid amplification method, such as described herein. In some embodiments, the amplification reaction components include hyperthermophilic organism DNA polymerase, which includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or the functional fragment of SEQID NO:1 or SEQ ID NO:2.

[0210] In some embodiments, the amplification reaction components (e.g., one or more amplification reagents) comprise a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophilic biopolymerase or a functional fragment thereof. In some embodiments, the amplification reaction components comprise a polymerase comprising an amino acid sequence at least about 90%, 95%, or 99% identical to an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a functional fragment thereof.

[0211] The polymerase may have reverse transcription capability. In such an embodiment, the amplification reaction may amplify the RNA target in a single step without using a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase capability include Bst (large fragment), 9°N DNA polymerase, 9°Nm TM DNA polymerase, Therminator TM 、Therminator TM II, etc. The amplification reaction components may include one or more independent reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction may include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0212] 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, the polymerase without exonuclease activity or with low exonuclease activity comprises one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, compared with unmodified polymerase, the modified polymerase with low exonuclease activity can have 10% or less exonuclease activity, for example, compared with unmodified polymerase, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% exonuclease activity. 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 has no or low single-strand-dependent exonuclease activity, and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of the polymerase include one or more amino acid substitutions 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.

[0213] Detection and quantification

[0214] The methods described herein may include detecting and / or quantifying nucleic acid amplification products and / or quality control products. For example, amplification products (e.g., signal generating oligonucleotides) may be detected and / or quantified by any suitable detection and / or quantification methods described herein. Non-limiting examples of detection and / or quantification methods include hairpin probes (e.g., molecular beacons) (e.g., real-time, endpoints), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), embedding / 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 TM), molecular inversion probe (MIP) technology from Affymetrix, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and LNA probes, AlphaScreen, SNPstream, genetic bit analysis (GBA), multiplex microsequencing, SNaPshot, GOOD assay, microarray microsequencing, array primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed incorporation (TDI), colorimetric oligonucleotide ligation assay (OLA), sequence-encoded OLA, microarray ligation, ligase chain reaction, padlock probes, invader assay (invader In some embodiments, the detection of nucleic acid amplification products includes the use of real-time detection methods (i.e., detection and / or continuous monitoring of products during the amplification process). In some embodiments, the detection of nucleic acid amplification products and / or quality control products includes the use of endpoint detection methods (i.e., detection of products after completion or cessation of the amplification process). Nucleic acid detection methods can also be used using labeled nucleotides directly incorporated into the target sequence or in a probe containing a target complementary sequence. Such labels can be radioactive and / or fluorescent in nature and can be distinguished in any manner discussed herein. In some embodiments, the quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, the detection method can be used in conjunction with the measurement of signal intensity and / or the generation (or reference) of a standard curve and / or a lookup table for quantitative nucleic acid amplification products and / or quality control products.

[0215] Detection of nucleic acid amplification products and / or quality control products can include the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule, wherein the detectable properties of the molecule are detectable under certain conditions, so that the molecule can 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 speed of passing through a known size opening. Molecular beacons for detecting nucleic acid molecules can be, for example, hairpin-shaped oligonucleotides, one end of which contains a fluorophore, and the other end contains a quenching dye. The loop of the hairpin can contain a probe sequence complementary to the target sequence, and the stem is formed by annealing of the complementary arm sequence on either side of the probe sequence. Fluorophores and quenching molecules can be covalently linked at the opposite ends of each arm. Under conditions that prevent oligonucleotides from hybridizing with their complementary targets, or when the molecular beacon is free in solution, the fluorescent molecule and the quenching molecule approach each other, thereby preventing FRET. When the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product and / or a quality control product), hybridization can occur, and the ring structure is converted into a stable, more rigid conformation, resulting in the separation of the fluorophore and the quencher molecule, thereby generating fluorescence. Due to the specificity of the probe, the generation of fluorescence is usually entirely due to the synthesis of the expected amplification product. In some cases, the molecular beacon probe sequence hybridizes with a sequence in the amplification product, and the sequence in the amplification product is identical or complementary to the sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes with a sequence in the amplification product, and the sequence in the amplification product is not identical or complementary to the sequence in the target nucleic acid (e.g., hybridizing with a sequence added to the amplification product by tailing an amplification primer or connecting). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with fluorophores of different colors and different target sequences, so that several products can be detected simultaneously in the same reaction (e.g., in a multiplex reaction). For quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each amplification cycle, and because unhybridized molecular beacons are dark, there is no need to separate the probe-target hybrid to quantitatively determine the amount of amplified product. The signal generated is proportional to the amount of amplified product. Detection using molecular beacons can be done in real time or as an end-point detection method.

[0216] Detection of nucleic acid amplification products and / or quality control products may include the use of lateral flow. The use of lateral flow generally includes the use of lateral flow devices, including but not limited to test strip assays and thin layer chromatography plates with various suitable coatings. Fixed in the flow path are various binding reagents for the sample, binding partners, or conjugates involving binding partners for the sample and the signal generating system.

[0217] Detection of nucleic acid amplification products and / or quality control products may include the use of FRET, which is an energy transfer mechanism between two chromophores of a donor and an acceptor molecule. In short, the donor fluorophore molecule is excited at a specific excitation wavelength. When the donor molecule returns to its ground state, the subsequent emission of the donor molecule can transfer the excitation energy to the acceptor molecule by a long-range dipole-dipole interaction. The emission intensity of the acceptor molecule can be monitored and varies with the distance between the donor and the acceptor, the overlap of the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be used for quantitative molecular dynamics, for example, in the DNA-DNA interactions described for molecular beacons. In order to monitor the generation of a specific product, the probe can be labeled with a donor molecule on one end and with an acceptor molecule on the other end. Probe-target hybridization causes the distance or orientation of the donor and the acceptor to change, and FRET changes are observed.

[0218] Detection of nucleic acid amplification products and / or quality control products can 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 will emit fluorescence whose degree of polarization is 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 constrained and cannot rotate between the time when the light is absorbed and emitted. When a free tracer compound (i.e., not bound to a nucleic acid) is excited by linearly polarized light, it rotates much faster than the corresponding tracer-nucleic acid conjugate, and the molecule is more randomly oriented, so the emitted light is depolarized. Therefore, fluorescence polarization provides a quantitative method for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0219] Detection of nucleic acid amplification products and / or quality control products can include the use of surface capture, such as by fixing specific oligonucleotides to the surface to achieve, thereby producing a biosensor that is both highly sensitive and selective. Example surfaces that can be used to attach probes include gold and carbon. Detection of nucleic acid amplification products and / or quality control products can include the use of 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN). For example, TAQMAN probes are specific hydrolysis probes that can increase quantitative amplification methods (e.g., quantitative PCR). The TAQMAN probe principle relies on 1) the 5' to 3' exonuclease activity of Taq polymerase to cut dual-labeled probes during hybridization with complementary target sequences and 2) fluorophore-based detection. The fluorescent signal generated allows the accumulation of amplified products to be quantitatively measured during the exponential phase of amplification, and the TAQMAN probe can significantly increase the specificity of detection.

[0220] Detection of nucleic acid amplification products and / or quality control products may include the use of intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes that exhibit enhanced fluorescence when bound to DNA or RNA). The dyes may include DNA or RNA intercalating fluorophores, including but not limited to 82. Acridine orange, ethidium bromide, Hoechst dye, Propidium iodide, I (asymmetric cyanine dyes), II, TOTO (thiazole orange dimer) and YOYO (oxazole 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, the detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260nm) to concentration. The direct measurement of nucleic acids can be converted to concentration using the BeerLambert law, which uses the measured path length and extinction coefficient to associate absorbance with concentration. 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 amount of nucleic acids, and can be used to provide rapid analysis of complex mixtures. After amplification, the sample can be ionized, and the ions produced are separated in an electric field and / or magnetic field according to their mass-to-charge ratio, and the detector measures the mass-to-charge ratio of the ions. Mass spectrometry includes, for example, MALDI, MALDI-TOF and electrospray. These methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS).Mass spectrometry (eg, matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can have high throughput due to high-speed signal acquisition and automated analysis off solid surfaces.

[0221] Detecting nucleic acid amplification products and / or quality control products can include the use of nucleic acid sequencing. The entire sequence or partial sequence of the amplified product can be determined, and the determined nucleotide sequence can be referred to as a read. For example, a linear amplification product can be directly analyzed without further amplification (e.g., by using a single molecule sequencing method). In some embodiments, the linear amplification product is further amplified and then analyzed (e.g., using a ligation sequencing or pyrophosphate sequencing method). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, sequencing based on reversible terminators, ligation sequencing, pyrophosphate sequencing, synthetic sequencing, single molecule sequencing, multiple sequencing, solid phase single nucleotide sequencing, and nanopore sequencing. Detecting nucleic acid amplification products and / or quality control products can include the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., company).

[0222] Lysis buffer

[0223] Lysis Agent

[0224] As disclosed herein, the lysing agent may include a detergent. The detergent may include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The anionic surfactant may include NH4 + , K + 、Na + or Li + As a counter ion. Cationic surfactants may include I - Br - or Cl - as a counter ion.

[0225] The lysing agent provided herein can serve as a denaturant. "Denaturing agent" or "denaturant" used herein should be given its common meaning, and include any compound or material that will cause the reversible unfolding of the protein. The intensity of the denaturing agent or denaturant will be determined by the characteristics and concentration of the specific denaturing agent or denaturant. Suitable denaturing agents or denaturants include chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids or two or more such agents. Suitable chaotropes 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., digitonin), mild cationic detergents (e.g., N->2,3-(dioleyloxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including but not limited to sulfobetaines (Zwittergent), 3-(3-chloroamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-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 alkane diols (especially C2-C4 alkandiols such as ethylene glycol) may be used as denaturants. The phospholipids may be naturally occurring phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol, or synthetic phospholipid derivatives or variants, such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0226] 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% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, the use of anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof) can be advantageous.

[0227] Anionic surfactants useful herein 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.

[0228] Suitable cationic surfactants can be broadly defined as derivatives of fatty quaternary ammonium compounds having long alkyl chains containing about 8 to 18 carbon atoms, such as lauryl trimethyl ammonium chloride; cetyl pyridinium chloride; benzalkonium chloride; cetyl trimethyl ammonium bromide; diisobutyl phenoxyethyl dimethyl benzyl ammonium chloride; coconut alkyl trimethyl ammonium nitrite; cetyl pyridinium fluoride; etc. In the compositions disclosed herein, certain cationic surfactants may also act as bactericides.

[0229] Suitable nonionic surfactants that can be used in the compositions, methods and kits of the present disclosure can be broadly defined as compounds produced by the condensation of alkylene oxide groups (hydrophilic in nature) with organic hydrophobic compounds that can be aliphatic and / or aromatic in nature. Examples of suitable nonionic surfactants include poloxamers; sorbitan derivatives, such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of 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 foam without causing excessive viscosity to the consumer product composition.

[0230] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphine, and sulfone compounds in which the aliphatic radical can be straight or branched chain and one of the aliphatic substituents contains from about 8 to 18 carbon atoms and one contains an anionic water-solubilizing group such as carboxy, sulfonate, sulfate, phosphate, or phosphonate.

[0231] Exemplary anionic, single-chain surfactants include alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates and saturated or unsaturated fatty acids and salts thereof. The portion containing the polar head group in the cationic surfactant 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 alkyl trimethyl ammonium halides, alkyl trimethyl toluene sulfonate ammonium and N-alkyl pyridinium halides.

[0232] reducing agent

[0233] Lysis buffer and / or reagent composition (for example, dry composition) can include one or more reducing agents. "Reducing agent" can be a compound or a group of compounds. As used herein, "reducing agent (reducing agent)", also referred to as "reducing agent (reductant)", "reducing substance" or "reduction equivalent", can refer to an element or compound that provides electrons to another substance. In particular, a reducing agent is a compound that destroys a disulfide bond by reduction, thereby overcoming those tertiary protein folding and quaternary protein structures (oligomeric subunits) stabilized by disulfide bonds. The example of a suitable reducing agent includes but is not limited to 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythritol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, sulfite, thioglycolic acid and HED. In some embodiments of the method, composition and kit provided herein, lysis buffer and / or reagent composition (for example, dry composition) do not include one or more reducing agents.

[0234] Reagent composition

[0235] The reagent compositions described herein (e.g., dry compositions) can be provided in a "dry form" or in a form that is not suspended in a liquid medium. The "dry form" of the composition can include a dry powder, a freeze-dried composition, a spray-dried or precipitated composition. The "dry form" composition can contain one or more lyoprotectants, such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly (ethylene glycol) or poly (propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch and carboxymethyl cellulose. As used herein, the terms "lyophilization", "freeze-dried" and "freeze drying" refer to the process of first freezing the material to be dried and then removing the ice or freezing solvent by sublimation in a vacuum environment. "Lyophilate" refers to a freeze-dried substance.

[0236] The reagent composition (for example, dry composition) can be frozen or lyophilized or spray-dried. The reagent composition can be heat dried. The reagent composition can include one or more additives (for example, amino acid, polymer, sugar or sugar alcohol). Sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof. Polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptide, collagen peptide or any combination thereof. Freeze-dried reagent can include poly-rA, EGTA, EDTA, Tween 80 and / or Tween 20.

[0237] The frozen or lyophilized or spray-dried or heat-dried composition or the aqueous composition used to prepare the frozen or lyophilized or spray-dried composition 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 lauryl sulfate. (iii) disaccharides such as trehalose, sucrose, lactose and maltose. (iv) polymers (which may have different MW) such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll and albumin. (v) amino acids such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine and γ-aminobutyric acid.

[0238] The reagent composition (e.g., a dried composition) may include one or more protective agents and one or more amplification reagents. One or more protective agents may include cyclodextrin compounds. Cyclodextrin (CD) can be used for complexing with a lysing agent (e.g., SDS). Cyclodextrin (CD) can be a truncated cone-like cyclic oligosaccharide with a hydrophobic inner cavity 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-CD. Natural CD may have solubility. Chemically modified CDs such as hydroxypropyl derivatives increase solubility in aqueous media by up to 50%. It is the trade name of WACKER cyclodextrin derivatives, including various α, β and γ-CD derivatives. β-CD can form a strong inclusion complex with sodium dodecyl sulfate (SDS) (stronger than α-CD and β-CD), and the stoichiometric ratio is mainly 1:1. The binding constant of β-CD and SDS can be found at 2100M -1 Up to 2500M -1 within the range.

[0239] Reagent test kit

[0240] The disclosure herein includes a kit. In some embodiments, the kit comprises: a first forward primer and a first reverse primer disclosed herein; a second forward primer and a second reverse primer disclosed herein; a first signal generating oligonucleotide, a second signal generating oligonucleotide, and / or a third signal generating oligonucleotide disclosed herein; a quality control template disclosed herein, a quality control primer disclosed herein; a signal generating oligonucleotide disclosed herein; and / or a supplemental quality control primer disclosed herein.

[0241] The kit may comprise: 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, one or more lysis agents comprising a detergent, and wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant and a zwitterionic surfactant.

[0242] The kit may include: 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.

[0243] The kit may include one or more components for monitoring the amplification reaction. In some embodiments, the kit includes: a quality control template disclosed herein; a quality control primer disclosed herein; a signal generating oligonucleotide disclosed herein; and / or a supplementary quality control primer disclosed herein. The kit may include: a reagent composition, the reagent composition including one or more amplification reagents, the amplification reagents including one or more components (e.g., forward primers, reverse primers) for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the quality control template, the signal generating oligonucleotide, the quality control primer, the supplementary quality control primer and / or one or more components for amplification are in a lyophilized or freeze-dried form and / or are present in a reagent composition.

[0244] The kit may include: at least one component that provides real-time detection activity for nucleic acid amplification products and / or quality control products. The real-time detection activity may be provided by a hairpin probe (e.g., a molecular beacon). The reagent composition (e.g., a dried composition) may include a reverse transcriptase and / or a reverse transcription primer.

[0245] 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 include cyclodextrin compounds. In some embodiments, one or more lysing agents constitute about 0.001% (w / v) to about 1.0% (w / v) (e.g., about 0.2% (w / v)) of the sample processed. In some embodiments, one or more lysing agents 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 can isolate one or more lysing agents, thereby preventing one or more amplification reagents from being denatured by one or more lysing agents.

[0246] The kit may include, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, as described herein. In the case of amplifying one target, a pair of primers (forward and reverse) may be included in the kit. In the case of amplifying multiple target sequences, more than one primer pair may be included in the kit. The kit may include a control polynucleotide, and in the case of amplifying multiple target sequences, more than one control polynucleotide may be included in the kit.

[0247] The enzyme 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, the enzyme having hyperthermophilic biopolymerase activity may include the amino acid sequence of SEQ ID NO: 1.

[0248] The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may comprise: (1) a sequence of a first primer and its reverse complement, (2) a sequence of a second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length.

[0249] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a viral particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoa. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoa.

[0250] Test kit can also include one or more components in any number of independent vessels, chambers, containers, packets, test tubes, vials, microtiter plates, etc., or components can be combined in such containers in various combinations. For example, the components of test kit can be present in one or more containers. In some embodiments, all components are provided in a container. In some embodiments, enzyme (for example, one or more polymerases and / or one or more reverse transcriptases) can be provided in a container separated from primers. Component can be, for example, freeze-dried, heat-dried, lyophilized or in a stable buffer. In some embodiments, one or more polymerases and / or one or more reverse transcriptases are in a single container in a lyophilized form or a heat-dried form, and primers are freeze-dried, heat-dried, lyophilized or in a buffer in different containers. In some embodiments, polymerase and / or reverse transcriptase and primers are in a single container in a lyophilized form or a heat-dried form.

[0251] The kit may include, for example, dNTPs used in the reaction, or modified nucleotides, vessels, cuvettes or other containers for the reaction, or vials of water or buffer for rehydrating lyophilized or heat-dried components. For example, the buffer used may be suitable for both polymerase and primer annealing activity.

[0252] The kit may also include instructions for performing one or more of the methods described herein and / or a description of one or more of the components described herein. The instructions and / or description may be in printed form and may be included in a kit insert. The kit may also include a written description of an Internet location that provides such instructions or descriptions.

[0253] The kit can include reagents for a detection method, such as reagents for FRET, lateral flow devices, test strips, fluorescent dyes, colloidal gold particles, latex particles, hairpin probes (eg, molecular beacons), or polystyrene beads.

[0254] Example

[0255] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended in any way to limit the scope of the disclosure.

[0256] Example 1

[0257] Group A Streptococcus (GAS) and Neisseria gonorrhoeae (NG) testing

[0258] Singleplex and Duplex Hairpin Probe Detection

[0259] This example demonstrates GAS and NG genomic DNA amplification and hairpin probe detection of GAS and NG amplicons in two fluorescence channels in real time for 10 minutes. After the reaction, the reaction temperature was gradually increased from the assay temperature (67°C) to 90°C.

[0260] Figure 3A and Figure 3D Real-time amplification and detection of GAS in singleplex (red curve) and duplex (green curve) reactions and NG in singleplex (blue curve) and duplex (green curve) reactions are shown, respectively. Figure 3B and Figure 3E Their corresponding melting curves are shown, and Figure 3C and Figure 3F The corresponding melting derivatives are shown. The melting temperatures of GAS and NG hairpin probes differ by 2°C, which in some embodiments is insufficient for differentiation of amplicon detection if the same fluorophore is used for the beacon and in a single fluorescence channel.

[0261] Example 2

[0262] Group A Streptococcus (GAS) and Neisseria gonorrhoeae (NG) testing

[0263] Single and Dual Intercalating Dye Detection

[0264] This example demonstrates the amplification of the same reaction as in Example 1 and the real-time detection by a nonspecific fluorescent dye (syto61) in the CY5 channel ( Figure 4A ), followed by melting curve analysis ( Figure 4B ) and melting derivative evaluation ( Figure 4C ). The derivative of the melting curves indicated that the amplicons of GAS and NG had comparable melting temperatures.

[0265] These results illustrate one of the characteristics of the APA assay: the Tm of the amplicons of the reaction is close to the reaction temperature, which indicates in some embodiments that nonspecific intercalating fluorescent dyes cannot be used to distinguish APA amplicons in a single fluorescent channel, and unlike in PCR / qPCR reactions, melting curve analysis cannot be used for multiplexed detection of APA reactions.

[0266] Example 3

[0267] Hairpin Internal Control (HPIC) Hairpin Probe Modification and Detection

[0268] This example demonstrates exemplary modifications of signal generating oligonucleotides (e.g., hairpin probes) as described herein. This example provides proof of principle for multiplexed amplification and detection of APA amplicons in a single fluorescent channel using hairpin probes designed to have differences in Tm for detecting more than one target with comparable amplicon Tm. 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. Signal generating oligonucleotides (e.g., hairpin probes) modified with LNA in the spacer region can be used for IC product detection.

[0269] In this example, the hairpin-shaped internal control target was amplified in the APA reaction for 10 minutes and detected by the hairpin probe (FIG. 9A) and the intercalating dye (FIG. 9D). After the reaction, the reaction temperature was immediately raised from the assay temperature to 90°C for melting curve analysis (FIG. 9B and 9E) and melting derivative evaluation (FIG. 9C and 9F). The sequences of the two hairpin probes (50nM) and the internal control primer (500nM) are shown in Table 2. The two hairpin probe 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 beacon. The resulting melting temperature difference of the two beacons MB1 (red curve, upper right) and MB2 (green curve, lower right) is 9°C, which may be sufficient for subsequent decoupling of the fluorescence signals for the detection of the two targets by melting curve analysis in a single fluorescence channel.

[0270] The results show that multiplexed amplification and detection of APA amplicons in a single fluorescent channel is feasible by using hairpin probes designed to have substantial differences in Tm for detecting more than one target with comparable amplicon Tm. In some embodiments, the probes provided herein (e.g., molecular beacons) comprise a 5' modification (e.g., 5HEX). In some embodiments, the probes provided herein (e.g., molecular beacons) comprise a 3' modification (e.g., 3IABkFQ).

[0271] Table 2: IC Assay Components

[0272]

[0273] Example 4

[0274] Neisseria gonorrhoeae / internal control duplex reaction

[0275] The HPIC assay components shown in Table 1 were used in the N. gonorrhoeae / internal control duplex reactions. Figure 6A-6FData associated with a Neisseria gonorrhoeae / internal control duplex reaction are depicted. Neisseria gonorrhoeae genomic DNA was amplified at 67°C for 10 minutes in the presence of an internal control while the target in the ROX channel was detected by a hairpin probe ( Fig. 6A ) and the internal control in the HEX channel ( Fig.6D After the reaction, the reaction temperature was immediately raised from the measurement temperature to 90°C for melting curve analysis ( Figure 6B and Fig. 6E ) and melting derivative evaluation ( Figure 6C and Fig. 6F ).

[0276] In at least some previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0277] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art may convert from the plural to the singular and / or from the singular to the plural where appropriate for the context and / or application. For clarity, various singular / plural arrangements may be expressly 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 clearly indicates otherwise. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0278] Those skilled in the art will understand that, in general, the terms used herein, and especially in the appended claims (e.g., the bodies of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "includes but is not limited to", etc.). Those skilled in the art will further understand that if a specific number of an introduced claim statement is intended, such an intent will be explicitly stated in the claim, and in the absence of such a statement, no such intent is present. For example, as an aid to understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim statements. However, the use of such words should not be interpreted as meaning that introduction of a claim statement by the indefinite article "a" or "an" will limit any particular claim containing such introduced claim statement to embodiments containing only one such statement, even when the same claim includes the introductory words "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim statements. In addition, even if a particular number of introduced claim statements is explicitly stated, one skilled in the art will recognize that such a statement should be interpreted to mean at least the stated number (e.g., merely stating "two statements" without other modifiers means at least two statements or two or more statements). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, generally such syntactic structure is intended so that one skilled in the art will understand the meaning of the convention (e.g., “a system having at least one of A, B, and C” would include but is not limited to systems having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to “at least one of A, B, or C, etc.” is used, generally such syntactic structure is intended so that one skilled in the art will understand the meaning of the convention (e.g., “a system having at least one of A, B, or C” would include but is not limited to systems having A alone, having B alone, having C alone, 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 appreciate that, in fact, any disjunctive words and / or phrases presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one, either or both terms.

[0279] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0280] As will be understood by those skilled in the art, for any and all purposes, such as in providing written description, all scopes disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges of the scope.Any listed scope can be easily identified as fully describing and enabling the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As non-limiting examples, each scope discussed herein can be easily decomposed into lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", etc. include stated numbers, and refer to the scope that can be subsequently decomposed into sub-ranges as discussed above.Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles.Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0281] 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 purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method for detecting more than one nucleic acid sequence, the method comprising: amplifying a first nucleic acid sequence and a second nucleic acid sequence in an amplification reaction mixture to generate a first nucleic acid amplification product and a second nucleic acid amplification product, respectively; and detecting the first nucleic acid amplification product and the second nucleic acid amplification product using a first signal generating oligonucleotide and a second signal generating oligonucleotide, respectively, in the same optical channel, wherein the first signal generating oligonucleotide and the second signal generating oligonucleotide each comprise a label, and Wherein the detecting comprises detecting the signal of the label of the first signal generating oligonucleotide and the second signal generating oligonucleotide before the amplifying, during the amplifying, after the amplifying, or any combination thereof.

2. The method according to claim 1, 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 A reagent composition is contacted with the treated sample to generate the amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

3. The method according to any one of claims 1 to 2, wherein the first nucleic acid sequence is a first target nucleic acid sequence, wherein the second nucleic acid sequence is a second target nucleic acid sequence, and Wherein the sample nucleic acid is suspected to contain the first target nucleic acid sequence and the second target nucleic acid sequence.

4. The method according to any one of claims 1 to 3, wherein the first nucleic acid sequence is a first target nucleic acid sequence, wherein the second nucleic acid sequence is an internal control (IC) nucleic acid sequence, and Wherein the sample nucleic acid is suspected to contain the first target nucleic acid sequence.

5. The method according to any one of claims 1 to 4, wherein: The IC nucleic acid sequence is a quality control template, and wherein the second amplification product is a first quality control product; The detecting is performed using an instrument comprising 6, 5, 4, 3, 2 or 1 optical channels; and / or The first signal generating oligonucleotide and the second signal generating oligonucleotide have melting temperatures (Tm) that differ by at least about 2°C.

6. The method of any one of claims 1-5, wherein the one or more amplification reagents comprise: An enzyme having a hyperthermophilic biopolymerase activity, optionally wherein the enzyme having a hyperthermophilic biopolymerase activity has a reverse transcriptase activity; two or more primer pairs, wherein each primer pair comprises a forward primer and a reverse primer; dNTPs; Reverse transcriptase; and / or One or more reverse transcription primers.

7. The method according to any one of claims 1 to 6, wherein the amplification is performed at an optimal temperature for the enzyme having hyperthermophilic biopolymerase activity, optionally the optimal temperature is about 66°C to about 68°C.

8. The method according to any one of claims 1 to 7, wherein: The first signal generating oligonucleotide has a Tm within about 1°C of the optimal temperature of the enzyme having hyperthermophilic biopolymerase activity; and The second signal generating oligonucleotide has a Tm that is at least about 2°C different from the temperature optimum of the enzyme having hyperthermophilic biopolymerase activity.

9. The method of any one of claims 1-8, wherein the detecting comprises contacting the first nucleic acid amplification product and the second nucleic acid amplification product with the first signal generating oligonucleotide and the second signal generating oligonucleotide, respectively, for hybridization.

10. The method according to any one of claims 1 to 9, wherein: The first signal generating oligonucleotide and the second signal generating oligonucleotide comprise a first label and a second label, respectively, and optionally the first label and the second label are the same or different; The first label and the second label are capable of generating a signal when the first signal generating oligonucleotide and the second signal generating oligonucleotide hybridize with the first nucleic acid amplification product and the second nucleic acid amplification product, respectively; and / or The first label and the second label generate a first signal and a second signal when the first signal generating oligonucleotide and the second signal generating oligonucleotide hybridize with the first nucleic acid amplification product and the second nucleic acid amplification product, respectively. Optionally, the first signal and the second signal are indistinguishable, Also optionally, the signal is fluorescence.

11. The method of any one of claims 1-10, wherein detecting the signals of the labels of the first signal generating oligonucleotide and the second signal generating oligonucleotide comprises detecting fluorescence emitted by the first label and the second label, respectively.

12. The method according to any one of claims 1 to 11, wherein the detecting comprises: detecting a signal from said first label during said amplification, optionally in real time; and The signal of the second label is detected after the amplification, and optionally, the signal of the second label is not detected during the amplification.

13. The method of any one of claims 1-12, wherein detecting the signal of the second label after the amplification comprises one or more cycles at the Tm of the second signal generating oligonucleotide.

14. The method of any one of claims 1-13, wherein the first signal generating oligonucleotide and the second signal generating oligonucleotide each comprise: 5' subdomain; 3' subdomain; and a loop domain located between the 5' subdomain and the 3' subdomain, Wherein intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain.

15. A method according to any one of claims 1-14, wherein the paired stem domain of the second signal generating oligonucleotide is configured to have a melting temperature (Tm) that is at least about 2°C higher or lower than the enzyme optimal temperature, optionally by modifying the length of the paired domain, the GC content of the paired domain and / or the presence of one or more chemical modifications in the paired domain.

16. The method according to any one of claims 1 to 15, wherein: The first nucleic acid amplification product comprises: (1) the sequence of the first forward primer and its reverse complement, (2) the sequence of the first reverse primer and its reverse complement, and (3) a first spacer sequence flanked by (1) the sequence of the first forward primer and its reverse complement and (2) the sequence of the first reverse primer and its reverse complement, wherein the first spacer sequence is 1 to 10 bases long; and and The second nucleic acid amplification product comprises: (1) the sequence of the second forward primer and its reverse complement, (2) the sequence of the second reverse primer and its reverse complement, and (3) a second spacer sequence flanked by (1) the sequence of the second forward primer and its reverse complement and (2) the sequence of the second reverse primer and its reverse complement, wherein the second spacer sequence is 1 to 10 bases in length.

17. The method according to any one of claims 1 to 16, wherein the sample nucleic acid comprises a first nucleic acid comprising the first target nucleic acid sequence and a second nucleic acid comprising the second target nucleic acid sequence.

18. The method according to any one of claims 1 to 17, Wherein amplifying the first target nucleic acid sequence comprises: amplifying a first target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplifying comprises contacting a first nucleic acid comprising the first target nucleic acid sequence with: i) a first forward primer and a first reverse primer, wherein the first forward primer is capable of hybridizing to a sequence of the first strand of the first target nucleic acid sequence, and the first reverse primer is capable of hybridizing to a sequence of the second strand of the first target nucleic acid sequence; and ii) an enzyme having hyperthermophilic biopolymerase activity, thereby generating the first nucleic acid amplification product; and Wherein amplifying the second target nucleic acid sequence comprises: amplifying a second target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplifying comprises contacting a second nucleic acid comprising the second target nucleic acid sequence with: i) a second forward primer and a second reverse primer, wherein the second forward primer is capable of hybridizing to a sequence of the first strand of the second target nucleic acid sequence, and the second reverse primer is capable of hybridizing to a sequence of the second strand of the second target nucleic acid sequence; and ii) an enzyme having hyperthermophilic biopolymerase activity, thereby generating the second nucleic acid amplification product.

19. The method of any one of claims 1-18, wherein the first nucleic acid and the second nucleic acid are double-stranded DNA.

20. The method according to any one of claims 1-19, wherein the first nucleic acid and the second nucleic acid are products of a reverse transcription reaction, optionally, the first nucleic acid and the second nucleic acid are products of a reverse transcription reaction generated from sample ribonucleic acid, and optionally, step (c) comprises generating the first nucleic acid and the second nucleic acid by a reverse transcription reaction.

21. The method according to any one of claims 1-20, wherein the sample nucleic acid comprises sample ribonucleic acid, and wherein the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate a first cDNA and a second cDNA.

22. The method of any one of claims 1-21, wherein amplifying the first target nucleic acid sequence and the second target nucleic acid sequence comprises: (c1) contacting the sample RNA with a reverse transcriptase, a first reverse transcription primer and / or a second reverse transcription primer to generate a first cDNA and a second cDNA; (c2) contacting the first cDNA and the second cDNA with an enzyme having a hyperthermophilic biopolymerase activity to generate a first double-stranded DNA (dsDNA) and a second dsDNA, respectively, wherein the first dsDNA and the second dsDNA comprise the first target nucleic acid sequence and the second target nucleic acid sequence, respectively, and wherein the first target nucleic acid sequence and the second target nucleic acid sequence comprise a first strand and a second strand that are complementary to each other; and (c3) amplifying the first and second target nucleic acid sequences under isothermal amplification conditions, wherein the amplification comprises contacting the first and second dsDNA with: (i) a first forward primer and a first reverse primer, wherein the first forward primer is capable of hybridizing to a sequence of the first strand of the first target nucleic acid sequence, and the first reverse primer is capable of hybridizing to a sequence of the second strand of the first target nucleic acid sequence; and (ii) a second forward primer and a second reverse primer, wherein the second forward primer is capable of hybridizing to a sequence of the first strand of the second target nucleic acid sequence, and the second reverse primer is capable of hybridizing to a sequence of the second strand of the second target nucleic acid sequence; and (iii) the enzyme having hyperthermophilic biopolymerase activity, thereby generating the first nucleic acid amplification product and the second nucleic acid amplification product, respectively.

23. The method according to any one of claims 1-22, wherein the first amplification product and the second amplification product are generated during a first amplicon reaction and a second amplicon reaction, respectively, optionally the first amplification product and the second amplification product are generated separately in time.

24. The method according to any one of claims 1 to 23, wherein the amplification reaction comprises: A first amplicon reaction performed at a first temperature; and A second amplicon reaction performed at a second temperature, wherein the first amplicon reaction is performed before the second amplicon reaction, wherein the first amplicon reaction and the second amplicon reaction are each at least about 2 minutes, optionally 5 minutes, and Wherein the second temperature is at least 2°C higher than the first temperature, optionally, the first temperature is 66°C and the second temperature is 70°C.

25. The method according to any one of claims 1 to 24, wherein: The first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence are shorter than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence; The first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence have a lower Tm than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence; and / or The first forward primer, the first reverse primer, the first signal generating oligonucleotide and / or the first nucleic acid sequence are present at a lower concentration than the second forward primer, the second reverse primer, the second signal generating oligonucleotide and / or the second nucleic acid sequence.

26. The method of any one of claims 1-25, wherein the first signal generating oligonucleotide and / or the second signal generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids.

27. The method of any one of claims 1-26, wherein the first signal generating oligonucleotide and / or the second signal generating oligonucleotide is a TaqMan detection probe oligonucleotide, a 3' minor groove binder probe oligonucleotide, a hairpin probe detection probe oligonucleotide, or a Molecular Torch detection probe oligonucleotide.

28. The method according to any one of claims 1 to 27, wherein: The label comprises a quenchable label, and optionally the quenchable label is a fluorophore; and / or The first signal generating oligonucleotide and / or the second signal generating oligonucleotide comprises a quencher.

29. The method according to any one of claims 1 to 28, wherein: The method comprises determining the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample; Determining the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample comprises determining the presence, absence and / or amount of the dsDNA and / or nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence in the sample; The presence, absence and / or amount of the first signal and the second signal respectively indicates the presence, absence and / or amount of the first nucleic acid sequence and / or the second nucleic acid sequence in the sample; The presence, absence and / or amount of the first signal and the second signal respectively indicates the presence, absence and / or amount of dsDNA and / or nucleic acid comprising the first nucleic acid sequence and / or the second nucleic acid sequence in the sample; and / or Amplifying the first nucleic acid sequence and / or the second nucleic acid sequence includes generating the first nucleic acid amplification product and / or the second nucleic acid amplification product at detectable levels within about 20 minutes, about 15 minutes, or about 10 minutes.

30. The method according to any one of claims 1 to 29, wherein: wherein the method does not comprise an intercalating dye; and / or Detecting the first nucleic acid amplification product and the second nucleic acid amplification product does not include detecting a signal of an intercalating dye.

31. The method of any one of claims 1-30, wherein the melting temperatures of the first amplification product and the second amplification product are the same, and wherein the melting temperatures of the first signal generating oligonucleotide and the second signal generating oligonucleotide are different.

32. The method of any one of claims 1-31, wherein the melting temperatures of the first signal generating oligonucleotide and the second signal generating oligonucleotide differ by at least about 2°C.

33. The method of any one of claims 1-32, wherein detecting the first nucleic acid amplification product and the second nucleic acid amplification product in the same optical channel comprises a melting curve analysis (MCA).

34. The method of any one of claims 1-33, wherein the MCA is performed at least about 1 minute after the amplifying step.

35. The method of any one of claims 1-34, wherein the MCA comprises: incubating the first nucleic acid amplification product and the second nucleic acid amplification product at a series of increasing temperatures, optionally from a starting temperature to a final temperature; and The signals of the labels of the first signal generating oligonucleotide and the second signal generating oligonucleotide are detected at the series of increasing temperatures, thereby generating a melting curve.

36. The method according to any one of claims 1 to 35, wherein: The starting temperature is at least about 50°C, optionally, the starting temperature is the optimal temperature of the enzyme having hyperthermophilic biopolymerase activity; and / or The final temperature is at least about 80°C, optionally 90°C.

37. The method according to any one of claims 1 to 36, wherein: The temperature transition from said starting temperature to said final temperature is a linear function of time, optionally said linear transition is at least 0.05°C per second; MCA includes deriving the negative derivative of the signal strength with respect to temperature (-dF / dt vs. T); and / or The signal derived from the first signal generating oligonucleotide is distinguishable in the melting curve from the signal derived from the second signal generating oligonucleotide or its negative first derivative.

38. The method according to any one of claims 1 to 37, wherein: The presence, absence and / or amount of the signal at the first melting temperature in the melting curve indicates the presence, absence and / or amount of the first amplification product; and The presence, absence and / or amount of the signal at the second melting temperature in the melting curve indicates the presence, absence and / or amount of the second amplification product, Optionally, the melting temperature corresponds to the maximum level of the negative derivative of the change in fluorescence with temperature (-dF / dT) with respect to temperature (T), and further optionally corresponds to a temperature within 1°C-4°C of said maximum level.

39. The method according to any one of claims 1 to 38, wherein: The first melting temperature corresponds to the melting temperature (Tm) of the first amplification product / first signal generating oligonucleotide duplex and / or the melting temperature (Tm) of the paired stem domain of the first signal generating oligonucleotide; and / or The second melting temperature corresponds to the melting temperature (Tm) of the second amplification product / second signal generating oligonucleotide duplex and / or the melting temperature (Tm) of the paired stem domain of the second signal generating oligonucleotide.

40. The method of any one of claims 1-39, wherein the first melting temperature differs from the second melting temperature by at least about 2°C.

41. The method of any one of claims 1-40, wherein the first signal generating oligonucleotide and / or the second 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 optionally wherein the one or more LNAs increase the difference between the first melting temperature and the second melting temperature.

42. The method of any one of claims 1-41, wherein the first signal generating oligonucleotide and / or the second signal generating oligonucleotide is configured such that the first melting temperature differs from the second melting temperature by at least about 2°C, optionally via one or more LNAs located in the loop domain.

43. The method according to any one of claims 1 to 42, wherein the method comprises: 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; 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.

44. The method of claim 43, wherein the amplification reaction is performed in an amplification reaction mixture under amplification conditions, optionally isothermal amplification conditions.

45. The method of any one of claims 1-44, wherein subjecting the quality control template and the quality control primer to an amplification reaction capable of generating 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.

46. ​​The method of any one of claims 1-45, wherein the amplification reaction comprises: Reverse transcription reaction; contacting the quality control primer with the quality control template for hybridization, and extending the quality control primer hybridized with the quality control template with an enzyme having polymerase activity, thereby generating a first quality control product; contacting the quality control primer with the first quality control product for hybridization, and extending the quality control primer hybridized to the first quality control product with an enzyme having polymerase activity, thereby generating a second quality control product; contacting the 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 having polymerase activity, thereby generating a first quality control product; and / or The first quality control product and the second quality control product are linearly amplified and / or exponentially amplified.

47. The method according to any one of claims 1 to 46, wherein the method further comprises: Providing an enzyme having polymerase activity, optionally the enzyme having polymerase activity is an enzyme having hyperthermophilic polymerase activity, optionally the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity; and / or Reverse transcriptase is provided.

48. The method of any one of claims 1-47, 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.

49. The method of any one of claims 1-48, wherein the 5' subdomain comprises the sequence of at least a portion of the quality control primer.

50. The method of any one of claims 1-49, wherein both the first quality control product and the second quality control product are capable of forming a hairpin structure.

51. The method of any one of claims 1-50, 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.

52. The method of any one of claims 1-51, 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.

53. The method of any one of claims 1-52, wherein detecting the first quality control product comprises detecting the first quality control product with the second signal generating oligonucleotide, optionally wherein the second signal generating oligonucleotide is capable of hybridizing to the first quality control product.

54. The method of any one of claims 1-53, wherein the detecting comprises contacting the first quality control product with the second signal generating oligonucleotide for hybridization.

55. The method of any one of claims 1-54, wherein the second signal generating oligonucleotide comprises a quencher, a label, or both, optionally wherein the label comprises a quenchable label, and optionally wherein the quenchable label is a fluorophore.

56. The method of any one of claims 1-55, wherein the second signal generating oligonucleotide comprises a quencher, optionally the quencher is capable of quenching the label.

57. The method of any one of claims 1-56, wherein the detecting comprises contacting the first quality control product with the second signal generating oligonucleotide for hybridization.

58. The method of any one of claims 1-57, wherein: The label is capable of generating a second signal when the second signal generating oligonucleotide hybridizes to the first quality control product; and / or The label generates a second signal when the second signal generating oligonucleotide hybridizes to the first quality control product, Optionally, the second signal is fluorescence.

59. The method of any one of claims 1-58, wherein detecting the first quality control product comprises detecting a second signal generated by the label of the second signal generating oligonucleotide, optionally, the label is a fluorophore and the second signal is fluorescence.

60. The method of any one of claims 1-59, wherein the detecting comprises detecting a second signal of the label before the amplification reaction, during the amplification reaction, after the amplification reaction, or any combination thereof.

61. The method of any one of claims 1-60, wherein the method further comprises: providing a second signal generating oligonucleotide; performing the amplification reaction on the second signal generating oligonucleotide; and The first quality control product is detected using the second signal generating oligonucleotide.

62. The method of any one of claims 1-61, wherein the quality control template is a second signal generating oligonucleotide.

63. The method of any one of claims 1-62, 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.

64. The method of any one of claims 1-63, wherein the second 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.

65. The method of any one of claims 1-64, 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.

66. The method of any one of claims 1-65, 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.

67. The method of any one of claims 1-66, 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.

68. The method of any one of claims 1-67, wherein the 3' end of the extended quality control template comprises a sequence complementary to at least a portion of the quality control primer.

69. The method of any one of claims 1-68, 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.

70. The method of any one of claims 1-69, 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.

71. The method of any one of claims 1-70, wherein the quality control template is a second signal generating oligonucleotide, wherein the second 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.

72. The method of any one of claims 1-71, wherein the second 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.

73. The method of any one of claims 1-72, 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.

74. The method of any one of claims 1-73, 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.

75. The method of any one of claims 1-74, wherein detecting the first quality control product comprises detecting a second signal generated by the first cleavage product comprising a label, optionally, the label is a fluorophore and the second signal is fluorescence.

76. The method of any one of claims 1-75, wherein the method further comprises: Provides supplemental quality control primers; and The amplification reaction is performed on the supplemented quality control primers.

77. The method of any one of claims 1-76, wherein the second 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 optionally wherein the one or more LNAs enhance the detectability of the first quality control product.

78. A method according to any one of claims 1-77, wherein the second signal generating oligonucleotide is configured such that the melting temperature (Tm) of the first quality control product / second signal generating oligonucleotide duplex is equal to or greater than the melting temperature (Tm) of the paired stem domain of the second signal generating oligonucleotide, optionally configured via one or more LNAs located in the loop domain.

79. The method of any one of claims 1-78, wherein providing the quality control primer, the quality control template and / or the second signal generating oligonucleotide comprises providing a reagent composition comprising the quality control primer, the quality control template and / or the second signal generating oligonucleotide.

80. The method of any one of claims 1-79, wherein performing an amplification reaction on the quality control primer, the quality control template and / or the second signal generating oligonucleotide comprises contacting the reagent composition with the treated sample to generate the amplification reaction mixture.

81. The method of any one of claims 1-80, wherein the method comprises determining the presence, absence and / or amount of the first quality control product.

82. The method of any one of claims 1-81, wherein the presence, absence and / or amount of the second signal is indicative of 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 second signal indicates the presence, absence and / or amount of one or more interfering components in the amplification reaction mixture.

84. The method of any one of claims 1-83, wherein the presence, absence and / or amount of the second signal indicates: (i) the integrity of the one or more amplification reagents in the amplification reaction mixture; (ii) a malfunction of an instrument in which the amplification reaction is performed; and / or (iii) sample-derived inhibition of the amplification reaction, optionally comprising matrix-derived inhibition.

85. The method of any one of claims 1-84, wherein the presence, absence and / or amount of the second signal indicates the extent to which amplification of the first target nucleic acid sequence is inhibited in the amplification reaction.

86. The method of any one of claims 1-85, 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.

87. The method of any one of claims 1-86, wherein the reagent composition is lyophilized, heat dried, and / or comprises one or more additives, wherein the one or more additives comprise: 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, cyclodextrin, 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, Optionally, contacting the reagent composition with the treated sample comprises dissolving the reagent composition in the treated sample.

88. The method of any one of claims 1-87, 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.

89. The method of any one of claims 1-88, 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; Thermally and / or enzymatically denaturing the first and second nucleic acids during the amplification; and / or The first nucleic acid and the second nucleic acid are contacted with a single-stranded DNA binding protein.

90. The method of any one of claims 1-89, wherein: The first signal generating oligonucleotide and / or the second signal generating oligonucleotide has a length of about 10 nucleotides to about 100 nucleotides; The forward primer and / or the reverse primer has a length of about 5 nucleotides to about 25 nucleotides; and / or The 5' subdomain, the 3' subdomain and / or the loop domain has a length of about 1 nucleotide to about 25 nucleotides.

91. The method of any one of claims 1-90, wherein: The first nucleic acid sequence and / or the second nucleic acid sequence comprises a length of no longer than about 20 nucleotides to no longer than about 90 nucleotides, optionally the first nucleic acid sequence and / or the second nucleic acid sequence comprises a length of about 30 nucleotides; The first forward primer, the second forward primer, the first reverse primer, the second reverse primer, the first reverse transcription primer and / or the second reverse transcription primer are about 8 to 16 bases in length; The first nucleic acid amplification product and / or the second nucleic acid amplification product is about 20 to 40 bases in length; and / or The first spacer sequence and / or the second spacer sequence respectively comprise a portion of the first nucleic acid sequence and / or the second nucleic acid sequence, and optionally the first spacer sequence and / or the second spacer sequence are 1 to 10 bases long.

92. The method of any one of claims 1-91, wherein: The isothermal amplification conditions include 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; The amplification (a) 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 (b) is performed under isothermal amplification conditions without a helicase, without a single-stranded binding protein, without a cleavage agent, and without a recombinase; The amplification is performed using a method selected from the group consisting of: archaeal polymerase amplification (APA), polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification and transcription-mediated amplification (TMA), optionally the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); 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, optionally 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, and optionally the enzyme having hyperthermophilic biopolymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1, optionally the enzyme having hyperthermophilic biopolymerase activity has low exonuclease activity or no exonuclease activity; The sample RNA is contacted with the reverse transcriptase and the enzyme having a hyperthermophilic polymerase activity simultaneously, optionally the sample RNA is contacted with the reverse transcriptase, the enzyme having a hyperthermophilic polymerase activity, the first forward primer and the second forward primer and the first reverse primer and the second reverse primer simultaneously, and optionally the sample RNA is contacted with the reverse transcriptase, the enzyme having a hyperthermophilic polymerase activity, the first forward primer and the second forward primer, the first reverse primer and the second reverse primer and the first reverse transcription primer and the second reverse transcription primer simultaneously; and / or The sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid. Optionally, the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA or a combination thereof.

93. The method of any one of claims 1-92, 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 first target nucleic acid sequence and / or the second target nucleic acid sequence is a nucleic acid sequence of a virus, a bacterium, a fungus or a protozoa, and optionally the sample nucleic acid is derived from a virus, a bacterium, a fungus or a protozoa.

94. The method of any one of claims 1-93, wherein: The virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV) or hantavirus; The bacteria include Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponemapallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes; The fungi include one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis and Trichophyton rubrum; and / or The protozoa include one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.

95. The method of any one of claims 1-94, 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.

96. The method of any one of claims 1-95, wherein the more than one target nucleic acid sequence is specific for two or more different organisms, optionally comprising one or more of: SARS-CoV-2, influenza A virus, influenza B virus, and / or influenza C virus.

97. The method of any one of claims 1-96, wherein: The amplification does not include 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 the amplification does not include loop-mediated isothermal amplification (LAMP); The amplification comprises one or more of the following: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR and IMDA, optionally the amplification does not include loop-mediated isothermal amplification (LAMP); and / or 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 the one or more cleavage 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 RNase H to the treated sample or the amplification reaction mixture.

98. The method according to any one of claims 1 to 97, wherein the sample nucleic acid is suspected of comprising a third target nucleic acid sequence, and wherein the method comprises: (c) amplifying a third target nucleic acid sequence in the amplification reaction mixture to generate a third nucleic acid amplification product; and (d) detecting the third nucleic acid amplification product using a third signal generating oligonucleotide, wherein the third signal generating oligonucleotide comprises a label, wherein the detecting comprises detecting a signal of the label of the third signal generating oligonucleotide before the amplifying, during the amplifying, after the amplifying, or any combination thereof, wherein the first signal generating oligonucleotide, the second signal generating oligonucleotide and the third signal generating oligonucleotide are detectable using the same optical channel, and wherein the melting temperatures (Tm) of the first signal generating oligonucleotide, the second signal generating oligonucleotide and the third signal generating oligonucleotide differ from each other by at least about 2° C., Optionally, the first signal generating oligonucleotide, the second signal generating oligonucleotide and the third signal generating oligonucleotide comprise the same label.

99. A kit comprising: A first forward primer and a first reverse primer as defined in any one of claims 1 to 98; A second forward primer and a second reverse primer as defined in any one of claims 1 to 98; The first signal generating oligonucleotide, the second signal generating oligonucleotide and / or the third signal generating oligonucleotide as defined in any one of claims 1 to 98; A quality control template as defined in any one of claims 1 to 98; A quality control primer as defined in any one of claims 1 to 98; A signal generating oligonucleotide as defined in any one of claims 1 to 98; and / or Supplementary quality control primers as defined in any one of claims 1-98.

100. The kit according to any one of claims 99, 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.

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