Asymmetric hairpin probes for nucleic acid detection

Abstract

Disclosed herein are methods, compositions, and kits for use in detecting a target nucleic acid sequence in a sample. The methods may include the use of a signal-generating oligonucleotide (SGO) capable of hybridizing to a nucleic acid amplification product. The SGO may include a 5' subdomain, a 3' subdomain, and a loop domain located between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be capable of forming a paired stem domain. The SGO may include one or more locked nucleic acid (LNA) nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. The SGO may include a 5' terminal domain located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 USC §119(e) of U.S. Provisional Patent Application No. 63 / 385,681, filed December 1, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Sequence Listing Reference This application is filed with an electronic Sequence Listing. The Sequence Listing is provided in file number 68EB-317363-WO, created on November 30, 2023, and is 61,673 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. The present disclosure relates generally to methods and compositions for amplifying (eg, isothermal amplification) nucleic acids. [Background technology]

[0002] Nucleic acid-based diagnostics can be useful for the rapid detection of infections, diseases, and / or genetic mutations. For example, identifying bacterial or viral nucleic acids in a sample can be useful for diagnosing certain types of infectious diseases. Other examples include identifying single nucleotide polymorphisms for disease control or forensics, and identifying genetic mutations indicative of genetically modified foods. Nucleic acid-based diagnostic assays often require the amplification of specific portions of nucleic acids in a sample. A common technique for nucleic acid amplification is polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermal cycling) to proceed through the following steps: denaturation (e.g., separating the strands of a double-stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short strands of complementary DNA sequences), and extension of the primers along their complementary targets by a polymerase. Such thermal cycling can be a time-consuming process that generally requires specialized machinery. Therefore, there is a need for a faster nucleic acid amplification method that can be performed without thermal cycling.

[0003] Archaeal polymerase amplification (APA) is an isothermal technique that uses primers with Tm comparable to the reaction temperature, typically 67-68°C, much shorter than PCR or other amplification methods to generate amplicons 25-35 bases in length. Small amplicon size can present significant challenges for real-time detection, as short APA amplicons typically have only 4-7 bases in the spacer region that are neither homologous nor complementary to the primer sequence, and their sequence length does not allow for sufficient binding with hybridization probes. Therefore, currently available detection approaches are insufficient for real-time detection of short APA amplicons. Additionally, in some embodiments, nonspecific product formation can be caused by unintended interaction of the probe with the amplification primer (subsequent extension of the amplification primer), which can result in false positives. There is a need for compositions and methods of nucleic acid detection that reduce unintended extension product formation and false positives. Summary of the Invention

[0004] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture, thereby generating a nucleic acid amplification product; and detecting the nucleic acid amplification product using a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide includes a loop domain located between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be capable of forming a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes a 5' terminal domain about 1 nt to about 6 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is not capable of hybridizing to the 3' end of the nucleic acid amplification product. In some embodiments, the signaling oligonucleotide comprises one or more locked nucleic acids (LNAs) in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0005] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes the steps of amplifying a target nucleic acid sequence in an amplification reaction mixture, thereby generating a nucleic acid amplification product, and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide includes a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a 5'-terminal domain about 1 nt to about 15 nt in length and located 5' to the 5' subdomain, and in some embodiments, the 5'-terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product.

[0006] In some embodiments, the one or more LNA nucleotides increase the melting temperature (Tm) of the signal-generating oligonucleotide by about 3°C ​​to about 20°C. In some embodiments, the signal-generating oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides. In some embodiments, the loop domain comprises one or more LNA nucleotides, and optionally, the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for nucleic acid amplification products. In some embodiments, enhancing the specificity of the signal-generating oligonucleotide for nucleic acid amplification products comprises enhanced mismatch discrimination between nucleic acid amplification products and mismatch products. In some embodiments, the mismatch products comprise non-template control products and / or non-target genotypes. In some embodiments, the terminal 3' nucleotide of the signal-generating oligonucleotide is an LNA nucleotide, and optionally, the LNA nucleotide reduces or prevents digestion of the signal-generating oligonucleotide and / or removal of a quencher associated with the 3' end of the signal-generating oligonucleotide (e.g., digestion by the exonuclease activity of a polymerase). In some embodiments, the 5' subdomain and / or the 3' subdomain comprises one or more LNA nucleotides, and optionally, the one or more LNA nucleotides enhance the stability of the paired stem domain. In some embodiments, the paired stem domain comprises at least one base pairing of opposing LNA nucleotides. In some embodiments, the nucleotide located in the 5' terminal domain cannot form an intramolecular nucleotide base pair. In some embodiments, the 5' terminal domain has less than about 5 nt, less than 4 nt, less than 3 nt, less than 2 nt, or less than 1 nt complementary to the 3' end of the nucleic acid amplification product. In some embodiments, the signal-generating nucleotide does not include a nucleotide located 3' to the 3' subdomain.

[0007] In some embodiments, the signal-generating oligonucleotide comprises a label. In some embodiments, the label comprises a quenchable label (e.g., a fluorophore). In some embodiments, the signal-generating oligonucleotide comprises a quencher. In some embodiments, the label is associated with the 3' end of the signal-generating oligonucleotide and the quencher is associated with the 5' end of the signal-generating oligonucleotide, or the label is associated with the 5' end of the signal-generating oligonucleotide and the quencher is associated with the 3' end of the signal-generating oligonucleotide. In some embodiments, when the quencher and label are in close proximity, the quencher is able to quench the signal generated by the label. In some embodiments, when the quencher and label are not in close proximity, the quencher cannot quench the signal generated by the label. In some embodiments, when the quencher and label are in close proximity, the signal generated by the label is undetectable. In some embodiments, when the quencher and label are not in close proximity, the signal generated by the label is detectable. In some embodiments, when the quencher and label are not in close proximity, the signal generated by the label is detectable. In some embodiments, the quencher and label are in close proximity when intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain forms a paired stem domain. In some embodiments, when the signal-generating oligonucleotide does not comprise a paired stem domain, the quencher and label are not in close proximity. In some embodiments, the detecting step comprises contacting the nucleic acid amplification product with the signal-generating oligonucleotide for hybridization. In some embodiments, the detecting step comprises using a real-time detection method. In some embodiments, the detecting step comprises detecting the signal of the label before, during, or after the amplification reaction, or any combination thereof. In some embodiments, the detecting step comprises detecting a signal generated by the label of the signal-generating oligonucleotide. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, the detecting step comprises detecting fluorescence emitted by the label.

[0008] In some embodiments, the amplification reaction and detecting step includes contacting the nucleic acid amplification product with a signal-generating oligonucleotide for hybridization, and extending the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity, thereby generating an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. In some embodiments, the extended nucleic acid amplification product comprises a complement of the 5'-terminal domain. In some embodiments, extension of the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with the enzyme having polymerase activity can disrupt intramolecular nucleotide base pairing between the 5' and 3' subdomains, thereby unwinding the paired stem domain. In some embodiments, the label is capable of generating a detectable signal (e.g., fluorescence) upon: (i) hybridization of the signal-generating oligonucleotide to the nucleic acid amplification product; and / or (ii) extension of the nucleic acid amplification product to generate an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. In some embodiments, the label generates a detectable signal (e.g., fluorescence) when (i) the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or (ii) the nucleic acid amplification product is extended to produce an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide.

[0009] Amplifying the target nucleic acid sequence in the amplification reaction mixture can include amplifying the target nucleic acid sequence under isothermal amplification conditions. In some embodiments, isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, e.g., about 55°C to about 75°C, about 56°C to about 68°C, or about 66°C to about 68°C. The amplifying step can be performed at the optimum temperature of the enzyme having hyperthermophilic polymerase activity. In some embodiments, the optimum temperature is about 66°C to about 68°C (e.g., a constant temperature). In some embodiments, the amplifying step is performed at a constant temperature. In some embodiments, the nucleic acid amplification product has a melting temperature within at least about 5°C of the constant temperature. In some embodiments, the melting temperature (Tm) of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex is higher than the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex (e.g., by at least about 5°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about 20°C). In some embodiments, the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex is at least about 60°C; the Tm of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex is at least about 68°C. In some embodiments, the nucleic acid amplification product is unable to form a stable duplex with the signal-generating oligonucleotide in the absence of extension of the nucleic acid amplification product.

[0010] In some embodiments, the amplification reaction comprises the steps of contacting the mismatch product with a signal-generating oligonucleotide for hybridization, and extending the mismatch product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity, thereby generating an extended mismatch product hybridized to the signal-generating oligonucleotide. In some embodiments, the extended mismatch product comprises a complement of the 5'-terminal domain. In some embodiments, the mismatch product is a non-template control product and / or a non-target genotype. In some embodiments, the Tm of the mismatch product / signal-generating oligonucleotide duplex is about 50°C; and the Tm of the extended mismatch product / signal-generating oligonucleotide duplex is at least 5°C below the constant temperature (e.g., less than about 68°C). In some embodiments, the nucleic acid amplification product and the mismatch product differ in sequence by at least about 1 nt, 2 nt, 3 nt, 4 nt, or 5 nt.

[0011] In some embodiments, the signal-generating oligonucleotide is configured such that the paired stem domain is stable at a constant temperature in the absence of a nucleic acid amplification product and the paired stem domain is capable of dissociating upon hybridization of the nucleic acid amplification product to the loop domain. In some embodiments, the signal-generating oligonucleotide is configured by modifying the length of the paired domain, the GC content of the paired domain, and / or the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. In some embodiments, the nucleic acid amplification product comprises (1) the sequence of the forward primer and its reverse complement, (2) the sequence of the reverse primer and its reverse complement, and (3) a spacer sequence flanked by the sequence of (1) the forward primer and its reverse complement and the sequence of (2) the reverse primer and its reverse complement. In some embodiments, the spacer sequence is about 4 nt to about 7 nt in length and / or has less than about 50% GC.

[0012] In some embodiments, the signal-generating oligonucleotide comprises a first region comprising at least a portion of the sequence of a reverse primer. In some embodiments, the signal-generating oligonucleotide comprises a second region comprising a sequence complementary to at least a portion of a forward primer. In some embodiments, the signal-generating oligonucleotide does not comprise a second region comprising a sequence complementary to at least a portion of a forward primer. In some embodiments, the signal-generating oligonucleotide comprises a spacer region comprising at least a portion of a spacer sequence. In some embodiments, the first region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer. In some embodiments, the second region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer. In some embodiments, the spacer region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer. In some embodiments, the first region comprises at least a portion of a 5' subdomain and / or a loop domain, the spacer region comprises at least a portion of a loop domain, and the second region comprises at least a portion of a loop domain and / or a 3' subdomain. In some embodiments, the signal-generating oligonucleotide is about 10 nt to about 100 nt in length. In some embodiments, the second region, spacer region, and / or first region are about 1 nt to about 25 nt in length. In some embodiments, the 5' subdomain, 3' subdomain, loop domain, and / or 5' terminal domain are about 1 nt to about 25 nt in length. In some embodiments, the 5' terminal domain is about 1 nt to about 6 nt in length, the loop domain is about 4 nt to about 15 nt in length, and the paired stem domain is about 3 bp to about 8 bp in length. In some embodiments, the nucleic acid amplification product is about 25 nt to about 35 nt in length. In some embodiments, the target nucleic acid sequence comprises a length of about 20 nt or less to about 90 nt or less. In some embodiments, the target nucleic acid sequence comprises a length of about 30 nt. In some embodiments, the spacer sequence comprises a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1 to 10 bases in length.In some embodiments, the spacer sequence is about 4 nt to about 7 nt in length and / or has a GC content of less than about 50%.

[0013] In some embodiments, the forward primer and / or reverse primer are: configured to have a Tm of less than about 45°C; about 5 nt to about 25 nt in length (e.g., about 10 nt to about 14 nt in length); configured to generate a nucleic acid amplification product that is about 25 nt to about 35 nt in length and has a melting temperature within at least about 5°C of the constant temperature; contain one or more phosphorothioate linkages; and / or have a GC content of about 30% to about 55%. In some embodiments, the 3' region of the forward primer and / or reverse primer does not contain a thymine base. In some embodiments, the 3' region includes the first, second, third, and / or fourth nucleotide from the 3' end. In some embodiments, the 5' region of the forward primer and / or reverse primer does not include more than 3 nt complementary to the spacer sequence, the region adjacent thereto, its complement, or any combination thereof. In some embodiments, the 5' region includes the first, second, third, and / or fourth nucleotide from the 5' end. In some embodiments, the forward primer and / or reverse primer comprises a phosphorothioate bond between the first and second nucleotides from the 3' end of the forward primer and / or reverse primer. In some embodiments, the phosphorothioate bond can reduce or prevent polymerase-mediated degradation. In some embodiments, the forward primer and / or reverse primer comprises a phosphorothioate bond between the second and third nucleotides from the 3' end of the forward primer and / or reverse primer. In some embodiments, the 3' region of the forward primer and / or reverse primer does not comprise more than two phosphorothioate bonds. In some embodiments, the 3' region comprises the first, second, third, and / or fourth nucleotides from the 3' end. In some embodiments, the forward primer and / or reverse primer comprises one or more phosphorothioate bonds in the region comprising a GC dinucleotide repeat. In some embodiments, the one or more phosphorothioate bonds can destabilize base pairing.In some embodiments, the presence of one or more LNA nucleotides in the loop domain, 5' subdomain, and / or 3' subdomain improves the sensitivity and / or specificity of detection of nucleic acid amplification products by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide does not contain an LNA nucleotide. In some embodiments, the presence of the 5' terminal domain in the signal-generating oligonucleotide improves the sensitivity and / or specificity of detection of nucleic acid amplification products by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide comprises a blunt-ended hairpin structure.

[0014] In some embodiments, the method includes determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample. In some embodiments, determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample includes determining the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the presence, absence, and / or amount of a detected signal indicates the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, the presence, absence, and / or amount of a detected signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the signal-generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids. In some embodiments, the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. The method may include contacting a sample containing biological entities with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence. The method may include contacting a reagent composition with the processed sample to produce an amplification reaction mixture, the reagent composition comprising one or more amplification reagents.

[0015] In some embodiments, the signal-generating oligonucleotide comprises one or more polymerase stoppers and / or one or more phosphorothioate linkages. In some embodiments, the first region, the second region, and / or the spacer region comprise one or more polymerase stoppers. In some embodiments, the one or more polymerase stoppers are located in the loop domain, the first region, the second region, and / or the spacer region. In some embodiments, the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. In some embodiments, the one or more polymerase stoppers comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. In some embodiments, the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemical capture abasic site, or any combination thereof. In some embodiments, the chemically trapped abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride; the abasic site comprises an apurinic base site, an apyrimidinic base site, or both; and / or the abasic site is generated by an alkylating agent or an oxidizing agent.

[0016] The one or more polymerase stoppers may comprise one or more RNA bases, 2' methoxyethyl ribose (MOE), LNA nucleotides, 2' fluoro bases, nitroindole, inosine, one or more acridines, 2-aminopurine, 2-6-diaminopurine, 5-bromo-deoxyuridine, inverted thymidine (inverted dT), inverted dideoxy-thymidine (ddT), dideoxy-cytidine (ddC), 5-methylcytidine , 5-hydroxymethylcytidine, 2'-O-methyl RNA base, unmethylated RNA base, iso-deoxycytidine (iso-dC), iso-deoxyguanosine (iso-dG), C3(OC3H6OPO3) group, photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] group, hexanediol group, spacer 9 (iSp9) [(OCH2CH2)3OPO3] group, spacer 18 (iSp18) [(OCH2CH 26 OPO3] groups, or combinations thereof.

[0017] In some embodiments, the one or more polymerase stoppers comprise one or more steric blocking groups. In some embodiments, the one or more steric blocking groups increase the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex. In some embodiments, the polymerase stopper comprises a modification incorporated between two bases of the signal-generating oligonucleotide. In some embodiments, the modification is a naphthylene-azo compound (e.g., Zen or iFQ).

[0018] In some embodiments, the modification has the structure: [ka] wherein linking groups L1 and L2, which position the modifications at internal positions of the signal-generating oligonucleotide, are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R1-R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group, electron-donating group, or point of attachment to a ligand; and X is a nitrogen atom or a carbon atom, with the proviso that when X is a carbon atom, the fourth substituent attached to that carbon atom can be hydrogen or a C1-C8 alkyl group.

[0019] In some embodiments, the modification has the structure: [ka] wherein a linking group L1 positions the modification at an internal position of the signal-generating oligonucleotide; and L2 are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R1, R2, R4, and R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing groups, or electron-donating groups; R6, R7, R9 to R 12are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron-withdrawing group, or an electron-donating group; R is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, or an electron-withdrawing group; and X is a nitrogen atom or a carbon atom, provided that when X is a carbon atom, the fourth substituent attached to the carbon atom can be hydrogen or a C-C alkyl group. In some embodiments, R is NO.

[0020] In some embodiments, the modification has the structure: [ka]

[0021] In some embodiments, when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, one or more polymerase stoppers can stop the polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, one or more polymerase stoppers can stop the polymerase extension of the forward primer of the first undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, one or more polymerase stoppers can stop the polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, one or more polymerase stoppers can stop the polymerase extension of the reverse primer of the second undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers can stop polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, the one or more polymerase stoppers can stop polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, the exogenous nucleic acid is selected from a sample nucleic acid, a primer configured to hybridize to a second target nucleic acid sequence, a primer configured to hybridize to an internal control, or any combination thereof.

[0022] In some embodiments, the sample nucleic acid comprises a nucleic acid comprising a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence comprises the following steps: amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, wherein the nucleic acid comprising the target nucleic acid sequence is contacted with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the nucleic acid is double-stranded DNA. In some embodiments, the nucleic acid is a product of a reverse transcription reaction. In some embodiments, the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid. In some embodiments, the amplifying step comprises generating a nucleic acid by a reverse transcription reaction. In some embodiments, the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. In some embodiments, the step of amplifying the target nucleic acid sequence comprises the following steps: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises the target nucleic acid sequence, and the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying comprises contacting the dsDNA with (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity, thereby producing a nucleic acid amplification product.

[0023] In some embodiments, when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a first undesired extension product. In some embodiments, the first undesired extension product can be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product. In some embodiments, one or more polymerase stoppers can stop polymerase extension of the forward primer of the first undesired duplex to generate a first stalled extension product. In some embodiments, the first stalled extension product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to generate a first undesired amplification product. In some embodiments, one or more polymerase stoppers can terminate polymerase extension of the forward primer of a first undesired duplex past one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a second undesired extension product. In some embodiments, the second undesired extension product can be amplified by an enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to form a second undesired amplification product. In some embodiments, one or more polymerase stoppers can terminate polymerase extension of the reverse primer of the second undesired duplex to generate a second extension stall product.In some embodiments, the second extension stalled product cannot be amplified by an enzyme with hyperthermophile polymerase activity in the presence of a reverse primer to generate a second undesired amplification product. In some embodiments, one or more polymerase stoppers can stop polymerase extension of the reverse primer of the second undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when an exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a third undesired extension product. In some embodiments, the third undesired extension product can be amplified by an enzyme with hyperthermophile polymerase activity in the presence of a reverse primer to form a third undesired amplification product. In some embodiments, the one or more polymerase stoppers can stop polymerase extension of a third undesired duplex exogenous nucleic acid to generate a third extension stall product. In some embodiments, the third extension stall product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to generate a third undesired amplification product. In some embodiments, the one or more polymerase stoppers can stop polymerase extension of the third undesired duplex exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

[0024] The label is capable of generating a false-positive signal when the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. In some embodiments, the label generates a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. In some embodiments, the signal and the false-positive signal are indistinguishable. In some embodiments, the generation of the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, detection of a false-positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

[0025] The presence of one or more polymerase stoppers in the signal-generating oligonucleotide can increase the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in a sample by at least about 1.1 times compared to a signal-generating oligonucleotide that does not contain one or more polymerase stoppers. In some embodiments, the generation of the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, the signal-generating oligonucleotide hybridized to the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, the nucleic acid amplification product reaches a detectable level 1, 2, 5, 10, 15, or 20 minutes before, or at least about 1, 2, 5, 10, 15, or 20 minutes before, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reaches a detectable level. In some embodiments, the signal reaches a detectable level 1, 2, 5, 10, 15, or 20 minutes before, or at least about 1, 2, 5, 10, 15, or 20 minutes before, the false positive signal reaches a detectable level. In some embodiments, the appearance of detectable levels of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product is delayed by 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes, or by at least about 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes, compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers. In some embodiments, the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach detectable levels for 5 minutes, 10 minutes, 15 minutes, or 20 minutes, or for at least about 5 minutes, 10 minutes, 15 minutes, or 20 minutes, after the amplifying step begins.In some embodiments, the occurrence of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product is reduced by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers.

[0026] In some embodiments, amplifying the target nucleic acid sequence comprises producing a detectable level of nucleic acid amplification product within 20 minutes, 15 minutes, or 10 minutes, or within about 20 minutes, 15 minutes, or 10 minutes, hi some embodiments, the detecting step is performed less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the treated sample.

[0027] In some embodiments, the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. In some embodiments, the pH of the lysis buffer is about 1.0 to about 10.0 (e.g., about 2.2). In some embodiments, the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid. In some embodiments, the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, the one or more amplification reagents comprise a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity; and / or dNTPs. In some embodiments, the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity, a forward primer, a reverse primer, and a reverse transcription primer.

[0028] The reagent composition may be lyophilized, heat-dried, and / or may include one or more additives. In some embodiments, the one or more additives include Tween 20, Triton X-100, and / or Tween 80; amino acids; sugars or sugar alcohols; and / or polymers. The sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the polymers include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. In some embodiments, contacting the reagent composition with the processed sample includes dissolving the reagent composition in the processed sample. In some embodiments, the one or more lysis reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) of the processed sample (e.g., about 0.2% (wt / vol) of the processed sample); and / or a detergent (e.g., 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 a step of using any enzymes other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include a step of using any enzymes other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein.

[0029] The target nucleic acid sequence may comprise a length of about 20 nucleotides or less to about 90 nucleotides or less (e.g., about 30 nucleotides). In some embodiments, the forward primer, reverse primer, and / or reverse transcription primer are about 8 to 16 bases in length. In some embodiments, the nucleic acid amplification product is about 20 to 40 bases in length. In some embodiments, the spacer sequence comprises a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1 to 10 bases in length. In some embodiments, the isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, e.g., about 55°C to about 75°C or about 56°C to about 67°C. In some embodiments, the amplifying step is carried out for a time period of about 5 minutes to about 60 minutes (e.g., a time period of about 15 minutes). In some embodiments, the amplifying step is carried out under isothermal amplification conditions that are helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free. In some embodiments, the amplifying step is carried out using a method selected from polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immuno-amplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). In some embodiments, the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0030] The enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO: 31 or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase and the enzyme with hyperthermophilic polymerase activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, and the forward and reverse primers. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

[0031] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the biological entity includes a virus, a bacterium, a fungus, a protozoan, a portion thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoan. 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, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus.In some embodiments, the bacteria is Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella pneumophila, Legionella pneumophila, or any of a number of other bacteria. dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes. 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 include one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp. The sample can be a biological sample or an environmental sample. In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

[0032] The amplifying step comprises multiplex amplification of two or more target nucleic acid sequences, and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from said two or more target nucleic acid sequences, optionally wherein the two or more target nucleic acid sequences are specific to two or more different organisms, and further optionally wherein the two or more different organisms comprise one or more of SARS-CoV-2, influenza A, influenza B and / or influenza C. In some embodiments, the amplifying step may include and / or exclude one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplifying step does not include LAMP. In some embodiments, the methods do not include one or more of the following: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture.

[0033] Disclosed herein are signal-generating oligonucleotides, e.g., signal-generating oligonucleotides capable of hybridizing to nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain can hybridize to nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotide comprises a 5'-terminal domain about 1 nt to about 6 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5'-terminal domain cannot hybridize to the 3' end of the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0034] The present disclosure includes signal-generating oligonucleotides, e.g., signal-generating oligonucleotides capable of hybridizing to nucleic acid amplification products. The signal-generating oligonucleotide may comprise a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain can hybridize to nucleic acid amplification products. The signal-generating oligonucleotide may comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. The signal-generating oligonucleotide may comprise a 5' terminal domain approximately 1 nt to approximately 15 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain cannot hybridize to the 3' end of the nucleic acid amplification product.

[0035] In some embodiments, the nucleic acid amplification product is generated by amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other. In some embodiments, amplifying the target nucleic acid sequence in an amplification reaction mixture comprises amplifying the target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the isothermal amplification conditions comprise a constant temperature of about 30°C to about 72°C (e.g., about 55°C to about 75°C, about 56°C to about 68°C, about 66°C to about 68°C). In some embodiments, the nucleic acid amplification product hybridized to the signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby generating an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. In some embodiments, the extended nucleic acid amplification product comprises a complement of the 5'-terminal domain. In some embodiments, the signal-generating oligonucleotide can hybridize to the mismatch product. In some embodiments, the mismatch product hybridized to the signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby generating an extended mismatch product hybridized to the signal-generating oligonucleotide. In some embodiments, the extended mismatch product comprises a complement of the 5'-terminal domain. In some embodiments, the mismatch product is a non-template control product and / or a non-target genotype. In some embodiments, the forward primer can hybridize to a sequence of a first strand of the target nucleic acid sequence, and the reverse primer can hybridize to a sequence of a second strand of the target nucleic acid sequence. In some embodiments, a nucleic acid amplification product is generated by amplifying the target nucleic acid sequence with the forward primer and the reverse primer.

[0036] Disclosed herein is a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises a signal-generating oligonucleotide disclosed herein. The kit can include a lysis buffer comprising one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence; optionally, the one or more lysis agents comprise a detergent, wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit can also include: a reagent composition comprising one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplification are: (i) a forward primer provided herein and a reverse primer provided herein, wherein the forward primer is capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or ii) A reagent composition comprising an enzyme with hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, optionally wherein the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof, optionally wherein the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO: 31, and further optionally wherein the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 is a non-limiting, illustrative schematic diagram of a traditional molecular beacon probe. [Figure 2]FIG. 1 is a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein that comprises a locked nucleobase. [Figures 3A-3D] 3A-3D are non-limiting, exemplary schematic diagrams of signal-generating oligonucleotides provided herein hybridized to a target (FIG. 3A), an extended target (FIG. 3B), an NTC (FIG. 3C), and an extended NTC (FIG. 3D). [Figure 4] FIG. 1 is a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein. [Figure 5] FIG. 1 is a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein. [Figures 6A-6B] FIG. 1 depicts non-limiting exemplary signal-generating oligonucleotides provided herein. [Figure 7] FIG. 1 is a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein. [Figure 8A-8B] 1 depicts non-limiting exemplary data related to MB characterization. Vertical lines in the graph indicate assay temperatures. [Figure 9] FIG. 1 is a non-limiting, illustrative schematic relating to the importance of the 3′ end of the primer in an APA assay. [Figures 10A-10B] Figure 10A depicts a non-limiting, exemplary schematic diagram relating to the importance of the 5' end of the primer in an APA assay. Figure 10B depicts a non-limiting, exemplary schematic diagram relating to APA assay Tm and APA product Tm. [Figures 11A-11C] FIG. 11 depicts non-limiting exemplary interactions that can cause background products: primer-dimer interactions (FIG. 11A), homodimer interactions (FIG. 11B), and primer-spacer interactions (FIG. 11C). [Figure 12] FIG. 1 is a non-limiting, exemplary schematic diagram showing the incorrect products generated (in an assay without phosphorothioate-modified primers) and the correct products generated (in an assay with phosphorothioate-modified primers). [Figures 13A-13B] 13A and 13B depict data on the performance of the old (FIG. 13A) and new (FIG. 13B) Neisseria gonorrhoeae APA assays. [Figure 14] FIG. 1 depicts data regarding the effect of primer length on APA assay performance. [Figure 15] 1 is a non-limiting exemplary diagram for APA product detection. [Figure 16] FIG. 1 depicts non-limiting exemplary probes provided herein that contain locked nucleic acid (LNA) bases. [Figure 17] 1 is a non-limiting exemplary diagram of a Flu A APA assay design. [Figure 18] FIG. 1 is a non-limiting, exemplary schematic diagram of an asymmetric hairpin probe provided herein for amplicon detection. [Figures 19A-19B] 19A and 19B depict data on the detection of Chlamydia trachomatis gDNA in APA reactions with conventional molecular beacons in the HEX (FIG. 19A) and cy5 (FIG. 19B) channels. [Figure 20] FIG. 1 depicts non-limiting exemplary conventional molecular beacons for detection of Chlamydia trachomatis gDNA in an APA reaction. [Figure 21] FIG. 1 depicts non-limiting exemplary asymmetric hairpin probes provided herein. [Figures 22A-22C] 22A-C depict data on synthetic DNA target detection in an APA reaction using an asymmetric hairpin probe (FIG. 22A), followed by melting curve analysis (FIG. 22B) and melting derivative evaluation (FIG. 22C). [Figure 23] FIG. 1 depicts data regarding a limit of detection (LOD) study using hairpin probes provided herein for Flu A virus detection. [Figures 24A-24D]Figures 24A-24B depict data on real-time detection (Figures 24A-24D) and melting curve assessment (Figures 24C-24D) of SARS-CoV-2 virus with a hairpin probe (Figures 24A, 24C) and fluorescent DNA dye Syto 61 (Figures 24B, 24D) in the reaction. [Figure 25] FIG. 1 depicts non-limiting exemplary signal-generating oligonucleotides provided herein. [Figures 26A-26B] FIG. 1 is a non-limiting, exemplary schematic diagram of an isothermal amplification reaction provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0038] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments set forth in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form part of this disclosure.

[0039] All patents, published patent applications, other publications, and GenBank sequences and other databases referenced herein are incorporated by reference in their entirety with respect to the relevant art.

[0040] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0041] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture, thereby generating a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide includes a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes a 5' terminal domain about 1 nt to about 6 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is not capable of hybridizing to the 3' end of the nucleic acid amplification product. In some embodiments, the signaling oligonucleotide comprises one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0042] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture, thereby generating a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide includes a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide includes one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a 5'-terminal domain about 1 nt to about 15 nt in length and located 5' to the 5' subdomain, and in some embodiments, the 5'-terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product.

[0043] The present disclosure includes signal-generating oligonucleotides. In some embodiments, the signal-generating oligonucleotide is capable of hybridizing to a nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to a nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises a 5' terminal domain about 1 nt to about 6 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0044] The present disclosure includes signal-generating oligonucleotides. In some embodiments, the signal-generating oligonucleotide is capable of hybridizing to a nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to a nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a 5' terminal domain about 1 nt to about 15 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain cannot hybridize to the 3' end of a nucleic acid amplification product.

[0045] Asymmetric hairpin probe In some embodiments, asymmetric hairpin probes (e.g., signal-generating oligonucleotides) for nucleic acid detection are provided. The asymmetric hairpin probes provided herein can be used for real-time, specific nucleic acid detection using APA technology. Some embodiments of the methods and compositions provided herein can be used for real-time detection of short nucleotides, such as APA amplification products. In some embodiments, asymmetric hairpin probe designs are provided that include a nucleotide sequence that forms an asymmetric hairpin structure having a double-stranded segment, a single-stranded loop, and a 5'-end overhang. At least a portion of the single-stranded loop segment and a portion of the double-stranded segment can hybridize to form a region complementary to a target nucleotide sequence, while in some embodiments, the 5'-end overhang includes a non-target sequence 1 to 6 bases in length.

[0046] In some embodiments of the methods and compositions provided herein, locked nucleic acids are used to enhance specificity and increase affinity for short amplicons for real-time detection of archaeal polymerase amplification. As provided herein, LNA nucleotides can be strategically incorporated into asymmetric hairpin probes at the following positions to fulfill one or more of the following functions: (1) at the terminal base at the 3' end to block exonuclease digestion from 9°Nm polymerase; (2) in the loop domain to provide additional specificity and increase the binding strength of the probe to the amplicon; and (3) in the stem segment to improve the stability of the hairpin structure.

[0047] In some embodiments, the hairpin probes provided herein have a fluorophore attached to the 5' end and a quencher attached to the 3' end. In an inactive state, the proximity of the fluorophore and quencher, held together by the hairpin stem segment, can significantly quench the hairpin probe. In some embodiments, without being bound by any particular theory, hybridization of the target sequence (amplicon) to the loop and stem domains and extension of the amplicon along the 5' overhang sequence overcomes the energy barrier imposed by the stem, leading to unwinding of the stem and ultimately separation of the two labels, resulting in increased fluorescence. The unique design of the asymmetric hairpin probes provided herein may be due to the increased target binding affinity resulting from the 5' overhang, strong hairpin structure, and LNA modification. In some embodiments, without being bound by any particular theory, such features enable fast kinetics and real-time detection of short amplicons, allowing sensitivity down to one order of magnitude of copy input. The use of hairpin probes without blunt-ended stems for detection in molecular diagnostic assays is unknown in the art.

[0048] The methods and compositions provided herein enable real-time detection of short amplicons.Without intending to be bound by any particular theory, hairpin probes may be significantly more specific than conventional linear probes due to the presence of stem structures, and at the same time, the enhanced binding affinity and specificity derived from LNA modification may enable the detection of short amplicons with fast kinetics and increased sensitivity and specificity compared to conventional molecular beacons. Real-time detection can be extremely difficult for short targets or amplicons. In some embodiments, without intending to be bound by any particular theory, to achieve real-time detection of amplification products, the probe must form a stable hybrid with the nucleic acid, and the length of the probe sequence should be such that the probe cleaves itself from the target at a temperature 7-10°C higher than the PCR annealing temperature or the assay temperature for isothermal amplification.

[0049] Molecular beacons are used in many applications. A typical molecular beacon has a stem 6-7 nucleotides long and a loop 15-25 nucleotides long, and uses the loop region to hybridize to its target. In some embodiments, without intending to be bound by any particular theory, a delicate balance is required for molecular beacons: the stem needs to be strong enough to maintain a stable hairpin structure at the assay temperature, and at the same time, the stem needs to be weak enough to dissociate when a complementary nucleic acid hybridizes to the loop region.

[0050] The disclosed compositions and methods provided herein, in some embodiments, can overcome the above-mentioned challenges of short amplicon detection by forming asymmetric hairpin probes with a 5' overhang, a loop, and a stem for real-time detection of APA amplicons. Several approaches to improving short amplicon detection are provided herein, which can be used independently or in combination. First, some embodiments of the disclosed compositions and methods use a 5' overhang to improve amplicon / probe stability. Extension of the target sequence on the probe can form a probe / target hybrid that is longer and more stable than the stem structure. As a result, the hairpin probe undergoes a conformational change from a hairpin shape to a more rigid double helix. Second, in some embodiments, the methods and compositions provided herein use LNA modification to improve hairpin stability and target / probe binding stability, as well as specificity for the bona fide target (thereby discriminating between mismatched products). Third, the provided methods and compositions can include a target-complementary building sequence in the stem to improve hairpin opening kinetics. Fourth, in some embodiments, the signal-generating oligonucleotides provided herein comprise one or more polymerase stoppers to reduce or prevent nonspecific product formation caused by unintended interactions of the probe with amplification primers (and subsequent extension of the amplification primers). In some embodiments, the methods and compositions provided herein can be used for real-time detection of short nucleotides, detection of long amplicons, and / or detection of small RNAs (small non-coding RNAs of 20-22 nucleotides) such as microRNAs for cancer diagnosis.

[0051] The hairpin probes provided herein can vary in size of the 5' overhang, loop, and stem. Base modifications can be placed at various positions to (1) enhance the stability of the template / probe duplex or hairpin structure, (2) increase the specificity of target recognition, and / or (3) block nonspecific off-target priming and background product interactions. The 5' overhang can be 1 to 6 bases, the loop size can be 4 to 15 bases, and the stem region can be 3 to 8 bases. Locked nucleic acid modifications can be placed in the loop and stem regions to enhance hairpin stability and enhance detectability. In some embodiments, without intending to be bound by any particular theory, the principles underlying the disclosed compositions and methods include nucleic acid thermodynamics, hybridization kinetics, and hairpin thermodynamics, and locked nucleic acids may be used to increase structural stability between hairpin probes and short amplicons.

[0052] The asymmetric hairpin probes provided herein can form a stem-and-loop structure with a 5' overhang through a portion of the 5' end and a complementary sequence on the 3' end of the probe. The loop portion and a portion of the 5' end can be complementary to the target nucleic acid. A fluorophore and a quencher can be attached to the 5' end and the 3' end. When the probe is in a stem-and-loop structure, fluorescence can be sufficiently quenched. In some embodiments, without being bound by any particular theory, hybridization of the probe with the target in the presence of a complementary sequence results in extension of the target along the 5' overhang, resulting in separation of the fluorophore from the quencher and increased fluorescence emission. The stem structure of hairpin probes can be significantly more specific than conventional probes, and at the same time, the enhanced binding affinity and specificity derived from the LNA modification can enable detection of short amplicons with greater sensitivity and specificity than conventional molecular beacons.

[0053] As provided herein, it has been found that conventional molecular beacons with blunt-ended stems cannot form stable hybrids with short target sequences and emit detectable fluorescent signals (see, e.g., the Examples). On the other hand, the non-conventional hairpin probe designs provided herein with 5'-end overhangs can be sufficiently quenched by a quencher labeled at the recessed 3' end of the stem. Unexpectedly, hairpins with fluorophore-labeled 5'-overhangs and quenchers located at the 3' end of the hairpins exhibited enhanced performance compared to conventional molecular beacons with blunt-ended stems. Furthermore, contrary to the teaching of conventional probe designs that a probe should not overlap with a primer binding site on the same strand, in some embodiments, the probe designs provided herein can incorporate one of the primer sequences into the probe to achieve sensitive and specific detection of amplified targets.

[0054] Figure 1 depicts a non-limiting, exemplary schematic diagram of a traditional molecular beacon probe. In some embodiments, a single-stranded nucleic acid sequence of 15 to 40 bases forms a hairpin (stem and loop) structure, with the stem formed by 6 to 7 GC pairs. The 5' and 3' ends may contain a fluorescent reporter and a quencher molecule. The loop sequence may be designed to be complementary to the target sequence. The target sequence can disrupt the stem structure, allowing the reporter to fluoresce. APA probe design has several challenges. The first challenge is the short amplicon size. APA targets are designed so that they do not form stable duplexes at high temperatures (e.g., 68°C). Short amplicons cannot form stable hybrids with molecular beacons (MBs) (real-time detection is not possible). The second challenge is the high assay temperature (e.g., 68°C). It can be difficult to force the probe into a "closed" conformation (stem stability) and to form stable target-probe hybrids. The third challenge is the high similarity between authentic and NTC products: unlike PCR, APA products differ from NTC by only a few bases (small spacer region).

[0055] In some embodiments, the challenge of short amplicon size is overcome herein by utilizing the ability to extend on a 9°N MB. In some embodiments, the challenge of high assay temperature is overcome herein by the use of LNA. In some embodiments, the challenge of high similarity between authentic and NTC products is overcome herein by a partial amplicon / MB complementary design - sharing only one primer plus a spacer.

[0056] Figure 2 depicts a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein that includes a locked nucleobase. Strategic placement of LNAs in the signal-generating oligonucleotides provided herein can enable real-time detection of APA amplicons. In some embodiments, the presence of LNA nucleotides can increase the Tm of the MB stem (e.g., 3-4 complementary bases, 4 of which are LNA-modified), with each LNA base increasing the Tm by 3-7°C in some embodiments. The presence of LNA nucleotides can increase specificity for the authentic target amplicon—the LNA-modified spacer region (and, in some embodiments, can also discriminate mismatch products). In some embodiments, the signal-generating oligonucleotides (e.g., MBs) provided herein have an LNA base at the 3' end to prevent removal of the quencher by a polymerase (e.g., 9°N). Figure 18 depicts a non-limiting, exemplary schematic diagram of an asymmetric hairpin probe provided herein for amplicon detection.

[0057] 3A-3D depict non-limiting, exemplary schematic diagrams of signal-generating oligonucleotides provided herein hybridized to a target (FIG. 3A), an extended target (FIG. 3B), an NTC (FIG. 3C), and an extended NTC (FIG. 3D). In some embodiments, signal-generating oligonucleotide (e.g., MB) design parameters and considerations include one or more of the following: (i) a target-probe hybridization Tm greater than 60°C; (ii) a target-extended probe Tm greater than >68°C (e.g., about 70°C); (iii) an NTC-probe hybridization Tm of about 50°C; and (iv) an NTC-extended probe Tm 5°C below the assay temperature (e.g., 68°C).

[0058] Figure 4 depicts a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein. In some embodiments, the design of a signal-generating oligonucleotide provided herein takes into account one or more of the following stem stability considerations: (i) a predominantly GC pair to provide sufficient Tm; (ii) a stronger LNA modification at the opposing base; (iii) an LNA / GC pair at the base of the loop can also be stabilized; and (iv) a stable stem is important, but not too strong to prevent robust opening by the target amplicon—a balance. In some embodiments, the signal-generating oligonucleotide disclosed herein can include a mismatch-extended MB design, where the MB contains a complementary sequence one base short of the 3' end of the target. In some embodiments, this design feature can increase discrimination of NTC products.

[0059] Figure 5 depicts a non-limiting, exemplary schematic diagram of a signal-generating oligonucleotide provided herein. In some embodiments, the decision of which strand of the amplification product (P1 / P2) to use in the design takes into account one or more of the following considerations: (i) the "cleanliness" of the primer (forward or reverse)—skewness; (ii) a strand with a GC-rich 3' end may be borrowed as part of the stem structure—favorable for burying the active end within the stem; and (iii) a maximum of two bases complementary beyond the spacer region (to the right of the MB). The design of a signal-generating oligonucleotide provided herein can include calculating parameters to identify the best location of an LNA base that may favor bona fide target detection (see, e.g., Table 1). Figures 6A-6B depict non-limiting, exemplary signal-generating oligonucleotides provided herein. In some embodiments, the structures of the signaling oligonucleotides provided herein can be designed using software (e.g., Quickfold; http: / / unafold.rna.albany.edu / ?q=DINAMelt / Quickfold, or the OligoAnalyzer™ Tool, https: / / www.idtdna.com / calc / analyzer) to determine whether the intended design is thermally favorable, taking into account assay temperature and salt concentration.

[0060] [Table 1]

[0061] APA can involve very rapid polymerase amplification. APA can involve (i) an isothermal reaction, (ii) no need for heat denaturation, and / or (iii) no need for helicases, recombinases, and / or nicking enzymes. APA can involve two simple, short amplification primers and a reaction temperature of about 68°C. In some embodiments, the amplification product is about 25-35 bases long. APA can, in some embodiments, be: (i) rapid (e.g., less than about 10 minutes, faster than currently available amplification technologies), (ii) sensitive (e.g., detection of ≦10 copies of target), and / or (iii) specific (e.g., two levels—amplification and detection). APA can, in some embodiments, involve real-time fluorescence detection using modified molecular beacons with LNAs. In some embodiments, the APA methods provided herein do not require sample purification.

[0062] In some embodiments provided herein, the 9°Nm polymerase has one or more of the following properties: (i) an extremely thermophilic archaeal polymerase (e.g., an optimum temperature for polymerization of about 70°C); (ii) a remarkable ability to extend single-stranded DNA at reaction temperatures higher than the primer annealing temperature (e.g., 10-14 mer extension at 68°C); (iii) reduced 3'-5' exonuclease activity (e.g., about 5% remaining, which is responsible for certain background products and assay design approaches); (iv) terminal deoxynucleotidyl transferase (TdT) activity (e.g., +A product); and (v) temperature-sensitive strand displacement activity (e.g., no strand displacement activity at 55°C, but some at 72°C).

[0063] Assay design for the DNA assays provided herein may rely on one or more of the following assumptions: (i) use of conserved DNA target sequences (e.g., no consideration of mismatches if conserved DNA targets are assumed to be available and / or target selection / sequence alignment is omitted); (ii) primer design does not include beacon design considerations (e.g., different from direct assay screening / primer selection and / or RNA assay design); and (iii) is based on a manual design approach.

[0064] Guidelines for the design of the APA DNA assays disclosed herein may include one or more of the following: (i) primer size may be between 10 and 14 nt (e.g., a 12-mer may be used for primer screening); (ii) spacer size may be between 4 and 7 nt; (iii) product size may be between 25 and 35 bp; and (iv) 30-55% GC in each primer, interdependently of the primer Tm and size.

[0065] Figure 9 depicts a non-limiting, exemplary schematic diagram relating to the importance of the 3' end of a primer in an APA assay. In some embodiments provided herein, the 3' end primer defines the APA assay and can be a critical factor for APA assay quality. In some embodiments, the 3' end of the primer comprises an APA clean base. In some embodiments, A, G, and / or C are APA clean bases, depending on the sequence context. In some embodiments, the primers provided herein do not have a T at the 3' end. The 3' end primer can define the assay spacer. Some embodiments of the methods and compositions provided herein can have a spacer length of 4-7 nt and <50% GC in the spacer region. Specificity can be conferred by the primer and spacer (e.g., via molecular beacon detection).

[0066] 10A-10B depict non-limiting, exemplary schematic diagrams for APA assay design. FIG. 10A depicts a non-limiting, exemplary schematic diagram relating to the importance of the 5' end of the primer in an APA assay. FIG. 10B depicts a non-limiting, exemplary schematic diagram for APA assay Tm and APA product Tm. In some embodiments provided herein, the APA assay Tm is equal to or approximately equal to the product Tm. In some embodiments, primer size is selected such that product Tm ≈ assay Tm. Primer size, Tm, and GC% can be interdependent factors. Assay conditions (e.g., monovalent salt and Mg) can affect the Tm. 2+ The Tm of the primer and the Tm of the product under these conditions can be calculated using currently available tools (e.g., IDT Oligo Analyzer). In some embodiments, the 5' terminal primer does not contain more than 3 nt complementary to the spacer sequence or the sequence adjacent to the spacer.

[0067] 11A-11C depict non-limiting exemplary interactions that can cause background products: primer-dimer interactions (FIG. 11A), homodimer interactions (FIG. 11B), and primer-spacer interactions (FIG. 11C). Non-target interactions can be the primary cause of background products. In some embodiments, the methods and compositions provided herein avoid primer-dimer interactions. For example, heterodimers with GC at the 3' end cannot produce specific amplification (FIG. 11A). For example, TGCA-3' can form strong homodimers and cannot produce specific amplification (FIG. 11B). In some embodiments, the methods and compositions provided herein avoid primer-spacer interactions. For example, a three-base interaction between a primer and a spacer can generate only background products with a truncated spacer (FIG. 11C).

[0068] In some embodiments, the primers provided herein are modified with phosphorothioates. Phosphorothioates (PS) can be added after primer screening for selected primers. In some embodiments, a single PS modification at the 3' end can prevent primer degradation by 9°Nm polymerase. The position of the modification in the primer can vary depending on the embodiment. In some embodiments, the phosphorothioate is located between the last (first) 3' end and the penultimate 3' end. Phosphorothioates can be added at positions with GC repeats to destabilize base pairing. In some embodiments, there are no more than two PS modifications at the 3' end per primer. In some embodiments, without intending to be bound by any particular theory, residual exonuclease activity in the 9°N polymerase can digest the mismatched base at the 3' end, resulting in a product with a truncated spacer. In some embodiments, the phosphorothioate modifications provided herein render the DNA resistant to nuclease degradation, and amplification can proceed to generate the correct product. Figure 12 depicts a non-limiting exemplary schematic showing the incorrect product generated (in an assay without phosphorothioate-modified primers) and the correct product generated (in an assay with phosphorothioate-modified primers).

[0069] The disclosed compositions and methods may follow one or more of the following DNA assay design rules: (i) select primers based on 3'-end properties; (ii) design product sizes with Tm approximately ≦APA assay Tm; (iii) avoid all forms of adverse interactions based on APA interaction rules; and (iv) modify promising primers with phosphorothioate modifications at the 3'-end after primer screening and selection. The rules may be interdependent on each other. Table 2 provides a comparison of assay designs for the APA assays provided herein compared to PCR.

[0070] [Table 2]

[0071] A challenge with APA product detection is that the Tm of the APA product is close to the assay temperature (e.g., about 68°C). Figure 15 depicts a non-limiting exemplary diagram for APA product detection. In some embodiments, only a partial sequence in the APA product can be used for detection: primer + spacer segment. The Tm of the product sequence that can be used for detection can be much lower than the assay temperature. For example, the Tm of the Flu A PB2.2 assay product for detection can be about 20°C lower than the optimal Tm for molecular beacon detection: 68°C + 5°C = 73°C.

[0072] In some embodiments, the aforementioned problems are solved by the signal-generating oligonucleotides provided herein, which contain LNA bases. Methods and compositions for real-time fluorescence detection using LNA probes are provided herein. Each probe can be modified with up to six LNAs. The selection of fluorophore / quencher pairs can vary depending on the embodiment, including those for Fam, Hex, Rox, and / or Cy5 channels. The probes provided herein that solve the aforementioned problems can have unconventional designs. In some embodiments, product extension on the probe can stabilize the product / probe duplex. In some embodiments, an LNA base is present at the 3' end to block exonuclease activity from 9°Nm polymerase. In some embodiments, LNA bases are used within the probes provided herein to improve Tm and / or at specific positions for mismatch discrimination. Figure 16 depicts non-limiting exemplary probes provided herein that contain LNA bases. For example, a FluA LNA probe may contain a loop, a stem, and a reverse primer, a spacer region, plus two borrowed bases (15mer) sequence that allows P1 extension along five additional bases. LNA bases can increase the Tm by 3-7°C per LNA base.

[0073] The complexity of RNA assay design is significantly increased compared to DNA assay design. Several considerations can be taken into account in RNA assay design, including consideration of mismatch sequences, consideration of RT primers, and consideration of beacon design. RNA assay primer design can include consideration of mismatches, RT primers, and molecular beacons. In some embodiments, RNA assay design considers mismatch positions and / or RT primer positions that can maximize comprehensiveness. In some embodiments, LNA bases are used in probes (e.g., beacons). In some embodiments, the interaction rules provided herein are expanded to include interaction checks of consensus sequences for all mismatch sequences. The disclosed compositions and methods may follow one or more of the following RNA assay design rules: (i) defining the 3' end of the primer based on the mismatch position that can maximize inclusivity for the mismatched strand (e.g., primer length 10-14 mer, spacer selection 4-7 nt, and / or avoiding mismatches within 3 nt at the 3' end); (ii) including beacon design in primer selection (e.g., mismatch discrimination, positioning LNA bases to avoid MB-9°N interactions); (iii) including RT primer design in primer selection (e.g., using an RT primer with the same 3' end as the forward primer, using an RT primer upstream of the forward primer); (iv) considering interactions (e.g., primer dimers, primer-target, and / or primer-spacer interactions) and avoiding nonspecific interactions; and (v) 3'-end PS modification.

[0074] Some embodiments provide signal-generating oligonucleotides containing nucleotide modifications (e.g., polymerase stoppers) to block 9° Nm interactions and nonspecific extension of reaction background products on the probe. Such modifications may include LNA, RNA, 2'-F DNA, 2' methoxyethyl ribose (MOE), and / or 2'O methyl ribose (2'OMe) modifications. In some embodiments, the signal-generating oligonucleotides provided herein contain steric blocking groups to block 9° Nm interactions and nonspecific extension of reaction background products (work in progress) on the probe. In some embodiments, the signal-generating oligonucleotides contain naphthylene-azo compounds (Zen or iFQ), which, in some embodiments, not only block polymerase extension but also increase target / probe stability (Tm). Modification groups that can be used as steric blocking moieties in the methods and compositions provided herein are disclosed in WO2012033848A1, the contents of which are incorporated herein by reference in their entirety. Disclosed herein are methods and compositions comprising modified molecular beacons (e.g., "protection probes"), which, in some embodiments, can improve assay specificity. The protection probes disclosed herein can be used in assays that isothermally amplify regions of interest within a target DNA (or cDNA) template using archaeal polymerase amplification ("APA") for real-time analyte detection. In some embodiments, the protection probes provided herein comprise a polymerase stopper (e.g., one or more 2'-O-methyl RNA bases ("2'OM")) within the molecular beacon probe to reduce nonspecific product formation (and subsequent false-positive signals). While not intending to be bound by any particular theory, in some embodiments, modifying specific bases within the molecular beacon construct can prevent undesired "read-through" of the probe molecule. Currently available methods do not utilize 2'OM bases for this purpose.Spacer modifications (e.g., C3 spacers) can be used to this same effect, although it has been shown that C3 spacers are incompatible with the molecular beacons used in the APA assays described herein. While not intending to be bound by any particular theory, in some embodiments, the compositions and methods provided herein take advantage of the inability of 9dN (DNA-dependent DNA polymerase) to read RNA templates. Specifically, it is theorized that when a 2'-O-M base (i.e., a methylated RNA base) is encountered within a given template, the enzyme's processivity is halted (e.g., the enzyme is unable to successfully "read" this position within the DNA template). 2'-O-methyl RNA can be found in small RNAs (e.g., tRNA) and is a post-transcriptional modification that is a naturally occurring modification of RNA. Oligonucleotides containing 2'-O-methyl RNA can be synthesized directly. This modification can increase the melting temperature of RNA:RNA duplexes while also causing subtle changes in RNA:DNA stability. Additionally, this modification can exhibit stability against single-stranded ribonuclease attack, typically being 5-10 times less sensitive to deoxyribonucleases than DNA. 2'OM modifications can be used in antisense oligonucleotides to improve stability and target binding affinity. In addition to the 2'OM modifications described herein, the following alternative base modifications within the context of molecular beacons used in APA have also been tested: C3 spacer modifications; abasic site modifications; and unmethylated RNA bases. Based on the resulting data, 2'OM modifications offer the greatest protection (without rendering the molecular beacon incompatible with APA). Some embodiments of the methods and compositions described herein can, in some embodiments, be used in concert with the systems, methods, compositions, and kits described in International Application No. PCT / US23 / 73576, entitled "MODIFIED MOLECULAR BEACONS FOR IMPROVED DETECTION SPECIFICITY," filed September 6, 2023, the contents of which are incorporated herein by reference in their entirety.

[0075] Unintended interactions between signal-generating oligonucleotides (e.g., molecular beacons) and forward and / or reverse primers, resulting in nonspecific product formation, can be an inherent and significant problem for some embodiments of the amplification / detection assays provided herein due to the intentional overlap of primer / probe footprints—the signal-generating oligonucleotide (e.g., molecular beacon) may include a first region comprising at least a portion of the sequence of the reverse primer and / or a second region comprising a sequence complementary to at least a portion of the forward primer. Additionally, due to the hairpin nature of the signal-generating oligonucleotides provided herein, the repetition of the two 3'-terminal nucleotides of the reverse primer in the stem-loop may result in unintended reverse primer / probe interactions. Thus, these inherent elements of some of the amplification / detection assays based on the signal-generating oligonucleotides provided herein may result in nonspecific product formation (and thereby false-positive signals). However, the methods and compositions provided herein solve these problems in the art and result in assays with reduced nonspecific product formation, reduced false-positive signals, and / or increased likelihood of accurately determining the presence, absence, and / or amount of target nucleic acid sequences in a sample. In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 5' modification (e.g., 5TEX615, FAM). In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 3' modification (e.g., 3IAbRQSp, IBFQ).

[0076] In some embodiments, a method for detecting a target nucleic acid sequence in a sample is provided. In some embodiments, the method includes the steps of: amplifying the target nucleic acid sequence in an amplification reaction mixture, thereby generating a nucleic acid amplification product; and detecting the nucleic acid amplification product using a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotide may include a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotide may include a loop domain located between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be capable of forming a paired stem domain. At least a portion of the 5' subdomain and at least a portion of the loop domain may be capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotide may include a 5' terminal domain about 1 nt to about 6 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is not capable of hybridizing to the 3' end of the nucleic acid amplification product. The signaling oligonucleotide may comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0077] In some embodiments, a method for detecting a target nucleic acid sequence in a sample is provided. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture, thereby generating a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotide includes a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotide includes a loop domain located between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be capable of forming a paired stem domain. At least a portion of the 5' subdomain and at least a portion of the loop domain may be capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotide may include one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. The signal-generating oligonucleotide may include a 5' terminal domain that is about 1 nt to about 15 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product.

[0078] In some embodiments, a signal-generating oligonucleotide is provided. The signal-generating oligonucleotide may be capable of hybridizing to a nucleic acid amplification product. The signal-generating oligonucleotide may include a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotide may include a loop domain located between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be capable of forming a paired stem domain. At least a portion of the 5' subdomain and at least a portion of the loop domain may be capable of hybridizing to a nucleic acid amplification product. The signal-generating oligonucleotide may include a 5' terminal domain having a length of about 1 nt to about 6 nt and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product. The signal-generating oligonucleotide may include one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0079] In some embodiments, a signal-generating oligonucleotide is provided. The signal-generating oligonucleotide may be capable of hybridizing to a nucleic acid amplification product. The signal-generating oligonucleotide may include a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotide may include a loop domain located between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be capable of forming a paired stem domain. At least a portion of the 5' subdomain and at least a portion of the loop domain may be capable of hybridizing to a nucleic acid amplification product. The signal-generating oligonucleotide may include one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. The signal-generating oligonucleotide may include a 5' terminal domain approximately 1 nt to approximately 15 nt in length and located 5' to the 5' subdomain. In some embodiments, the 5' terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product.

[0080] The nucleic acid amplification product can be generated by amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other. Amplifying the target nucleic acid sequence in an amplification reaction mixture can include amplifying the target nucleic acid sequence under isothermal amplification conditions. The isothermal amplification conditions can include a constant temperature of about 30°C to about 72°C (e.g., about 55°C to about 75°C, about 56°C to about 68°C, or about 66°C to about 68°C). The nucleic acid amplification product hybridized to the signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby generating an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. The extended nucleic acid amplification product can include a complement of the 5'-terminal domain. The signal-generating oligonucleotide can be hybridized to the mismatch product. The mismatch product hybridized to the signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby generating an extended mismatch product hybridized to the signal-generating oligonucleotide. The extended mismatch product may comprise the complement of the 5'-terminal domain. The mismatch product may be a non-template control product and / or a non-target genotype. The forward primer may be capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence, and the reverse primer may be capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence. The nucleic acid amplification product may be generated by amplifying the target nucleic acid sequence with the forward primer and the reverse primer.

[0081] In some embodiments, the one or more LNA nucleotides increase the melting temperature (Tm) of the signal-generating oligonucleotide by about 3°C ​​to about 20°C. The signal-generating oligonucleotide may comprise 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides. The loop domain may comprise one or more LNA nucleotides, and optionally, the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for nucleic acid amplification products. Enhancing the specificity of the signal-generating oligonucleotide for nucleic acid amplification products may include enhanced mismatch discrimination between nucleic acid amplification products and mismatch products. The mismatch products may include non-template control products and / or non-target genotypes. The terminal 3' nucleotide of the signal-generating oligonucleotide may be an LNA nucleotide, and optionally, the LNA nucleotide reduces or prevents digestion of the signal-generating oligonucleotide and / or removal of a quencher associated with the 3' end of the signal-generating oligonucleotide (e.g., digestion by the exonuclease activity of a polymerase). The 5' subdomain and / or the 3' subdomain may contain one or more LNA nucleotides, which optionally enhance the stability of the paired stem domain. The paired stem domain may contain at least one base pairing of opposing LNA nucleotides. In some embodiments, the nucleotides located in the 5' terminal domain are unable to form intramolecular nucleotide base pairs. The 5' terminal domain may have less than about 5 nt, less than 4 nt, less than 3 nt, less than 2 nt, or less than 1 nt complementary to the 3' end of the nucleic acid amplification product. In some embodiments, the signal-generating nucleotide does not include a nucleotide located 3' to the 3' subdomain.

[0082] The signal-generating oligonucleotide may comprise a label. The label may comprise a quenchable label (e.g., a fluorophore). The signal-generating oligonucleotide may comprise a quencher. The label may be associated with the 3' end of the signal-generating oligonucleotide and the quencher may be associated with the 5' end of the signal-generating oligonucleotide, or the label may be associated with the 5' end of the signal-generating oligonucleotide and the quencher may be associated with the 3' end of the signal-generating oligonucleotide. When the quencher and label are in close proximity, the quencher may be able to quench the signal generated by the label. In some embodiments, when the quencher and label are not in close proximity, the quencher cannot quench the signal generated by the label. In some embodiments, when the quencher and label are in close proximity, the signal generated by the label is undetectable. When the quencher and label are not in close proximity, the signal generated by the label may be detectable. The quencher and label may be in close proximity when intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain forms a paired stem domain. In some embodiments, when the signal-generating oligonucleotide does not comprise a paired stem domain, the quencher and label are not in close proximity. The detecting step comprises contacting the nucleic acid amplification product with the signal-generating oligonucleotide for hybridization. In some embodiments, detecting the nucleic acid amplification product may comprise using a real-time detection method. The detecting step comprises detecting the signal of the label before, during, or after the amplification reaction, or any combination thereof. In some embodiments, detecting the nucleic acid amplification product may comprise detecting a signal generated by the label of the signal-generating oligonucleotide. The label may be a fluorophore, and the signal may be fluorescence. In some embodiments, detecting the signal may comprise detecting fluorescence emitted by the label.

[0083] In some embodiments, the amplification reaction and / or detecting step includes contacting the nucleic acid amplification product with a signal-generating oligonucleotide for hybridization and extending the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity, thereby generating an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. The extended nucleic acid amplification product may include a complement of the 5'-terminal domain. Extension of the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity may be capable of disrupting intramolecular nucleotide base pairing between the 5' and 3' subdomains, thereby unwinding the paired stem domain. The label may be capable of generating a detectable signal (e.g., fluorescence) upon: (i) hybridization of the signal-generating oligonucleotide to the nucleic acid amplification product; and / or (ii) extension of the nucleic acid amplification product to generate an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. In some embodiments, the label generates a detectable signal (e.g., fluorescence) when (i) the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or (ii) the nucleic acid amplification product is extended to produce an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide. Amplifying the target nucleic acid sequence in the amplification reaction mixture can include amplifying the target nucleic acid sequence under isothermal amplification conditions. The isothermal amplification conditions can include a constant temperature of about 30°C to about 72°C. The constant temperature can be about 55°C to about 75°C, about 56°C to about 68°C, or about 66°C to about 68°C. The amplifying can be performed at an optimum temperature for the enzyme having hyperthermophilic polymerase activity. The optimum temperature can be about 66°C to about 68°C (e.g., a constant temperature). The amplifying can be performed at a constant temperature. The nucleic acid amplification product can have a melting temperature within at least about 5°C of the constant temperature.The melting temperature (Tm) of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex can be higher than the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex (e.g., by at least about 5°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, or 20°C). The Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex can be as low as or as high as about 60°C, and the Tm of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex can be as low as about 68°C. In some embodiments, the nucleic acid amplification product cannot form a stable duplex with the signal-generating oligonucleotide in the absence of extension of the nucleic acid amplification product.

[0084] In some embodiments, the amplification reaction includes the following steps: contacting the mismatch product with a signal-generating oligonucleotide for hybridization, and extending the mismatch product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity, thereby generating an extended mismatch product hybridized to the signal-generating oligonucleotide. The extended mismatch product may comprise a complement of the 5'-terminal domain. The mismatch product may be a non-template control product and / or a non-target genotype. The Tm of the mismatch product / signal-generating oligonucleotide duplex may be about 50°C, and the Tm of the extended mismatch product / signal-generating oligonucleotide duplex may be at least 5°C lower than the predetermined temperature (e.g., less than about 68°C). In some embodiments, the nucleic acid amplification product and the mismatch product differ in sequence by at least about 1 nt, 2 nt, 3 nt, 4 nt, or 5 nt.

[0085] The signal-generating oligonucleotide can be configured such that the paired stem domain is stable at the constant temperature in the absence of a nucleic acid amplification product and such that the paired stem domain is capable of dissociating upon hybridization of the nucleic acid amplification product to the loop domain. This can be achieved by modifying the length of the paired domain, the GC content of the paired domain, and / or the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. In some embodiments, the nucleic acid amplification product comprises: (1) the sequence of a forward primer and its reverse complement, (2) the sequence of a reverse primer and its reverse complement, and (3) a spacer sequence flanked by the sequence of (1) the forward primer and its reverse complement and the sequence of (2) the reverse primer and its reverse complement. The spacer sequence can be about 4 nt to about 7 nt in length and / or have a GC content of less than about 50%.

[0086] The signal-generating oligonucleotide may include a first region comprising at least a portion of the sequence of the reverse primer. The signal-generating oligonucleotide may include a second region comprising a sequence complementary to at least a portion of the forward primer. The signal-generating oligonucleotide may include a spacer region comprising at least a portion of the sequence of the spacer sequence. The first region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer. The second region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer. The spacer region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer. The first region may comprise at least a portion of the 5' subdomain and / or the loop domain, the spacer region may comprise at least a portion of the loop domain, and the second region may comprise at least a portion of the loop domain and / or the 3' subdomain. The signal-generating oligonucleotide may be about 10 nt to about 100 nt in length. The second region, spacer region, and / or first region may be about 1 nt to about 25 nt in length. The 5' subdomain, 3' subdomain, loop domain, and / or 5' terminal domain may be about 1 nt to about 25 nt in length. The 5' terminal domain may be about 1 nt to about 6 nt in length, the loop domain may be about 4 nt to about 15 nt in length, and the paired stem domain may be about 3 bp to about 8 bp in length. The nucleic acid amplification product may be about 25 nt to about 35 nt in length. The target nucleic acid sequence may comprise a length of about 20 nt or less to about 90 nt or less. The target nucleic acid sequence may comprise a length of about 30 nt. The spacer sequence may comprise a portion of the target nucleic acid sequence. The spacer sequence may be 1 to 10 bases in length. The spacer sequence may be about 4 nt to about 7 nt in length and / or may have a GC content of less than about 50%.

[0087] In some embodiments, the nucleic acid amplification product comprises: (1) the sequence of the forward primer and its reverse complement, (2) the sequence of the reverse primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement. The spacer sequence can be 1 to 10 bases in length.

[0088] The forward primer and / or reverse primer may be configured to have a Tm of less than about 45°C; be about 5 nt to about 25 nt in length (e.g., about 10 nt to about 14 nt in length); be configured to generate a nucleic acid amplification product that is about 25 nt to about 35 nt in length and has a melting temperature within at least about 5°C of the constant temperature; contain one or more phosphorothioate linkages; and / or have a GC content of about 30% to about 55%. In some embodiments, the 3' region of the forward primer and / or reverse primer does not contain a thymine base. The 3' region may include the first, second, third, and / or fourth nucleotide from the 3' end. In some embodiments, the 5' region of the forward primer and / or reverse primer does not include more than 3 nt complementary to the spacer sequence, the region adjacent thereto, its complement, or any combination thereof. The 5' region may include the first, second, third, and / or fourth nucleotide from the 5' end. The forward primer and / or reverse primer may comprise a phosphorothioate bond between the first and second nucleotides from the 3' end of the forward primer and / or reverse primer. The phosphorothioate bond may be capable of reducing or preventing polymerase-mediated degradation. The forward primer and / or reverse primer may comprise a phosphorothioate bond between the second and third nucleotides from the 3' end of the forward primer and / or reverse primer. In some embodiments, the 3' region of the forward primer and / or reverse primer does not contain more than two phosphorothioate bonds. The 3' region may include the first, second, third, and / or fourth nucleotides from the 3' end. The forward primer and / or reverse primer may comprise one or more phosphorothioate bonds in the region containing the GC dinucleotide repeat. The one or more phosphorothioate bonds may be capable of destabilizing base pairing.In some embodiments, the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain improves the sensitivity and / or specificity of detection of nucleic acid amplification products by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between these values) compared to a comparable method in which the signal-generating oligonucleotide does not contain any LNA nucleotides. In some embodiments, the presence of a 5' terminal domain in the signal-generating oligonucleotide improves the sensitivity and / or specificity of detecting nucleic acid amplification products by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or any number or range between these values) compared to a comparable method in which the signal-generating oligonucleotide comprises a blunt-ended hairpin structure.

[0089] The method may include determining the presence, absence, and / or amount of a target nucleic acid sequence in the sample. In some embodiments, determining the presence, absence, and / or amount of a target nucleic acid sequence in the sample may include determining the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the presence, absence, and / or amount of the detected signal indicates the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, the presence, absence, and / or amount of the detected signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample.

[0090] As used herein, a "polymerase stopper" is a molecule (e.g., a modified nucleotide) that can terminate or inhibit polymerization. In some embodiments, at least one of the one or more polymerase stoppers is a 2'-O-methylated nucleotide. Non-limiting examples of 2'-O-methylated nucleotides include 2'-O-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methylguanosine. The one or more polymerase stoppers may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. The signal-generating oligonucleotide may comprise one or more polymerase stoppers and / or one or more phosphorothioate linkages. The first region, second region, and / or spacer region may comprise one or more polymerase stoppers. The one or more polymerase stoppers may be located in the loop domain, the first region, the second region, and / or the spacer region. In some embodiments, the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. The one or more polymerase stoppers may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. The one or more polymerase stoppers may comprise one or more of an abasic site, a stable abasic site, a chemically captured abasic site, or any combination thereof. In some embodiments, the chemically captured abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride; the abasic site comprises an apurinic base site, an apyrimidinic base site, or both; and / or the abasic site is generated by an alkylating agent or an oxidizing agent.In some embodiments, the one or more polymerase stoppers comprise one or more RNA bases, one or more 2' methoxyethyl ribose (MOE), one or more LNA nucleotides, one or more 2' fluoro bases, one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more a plurality of 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C(OCHOPO) groups, one or more photocleavable (PC) [OCH-C(O)NHCH-CHNO-CH(CH)OPO] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCHCH)OPO] groups, one or more spacer 18 (iSp18) [(OCHCH. 26 OPO3] groups, or any combination thereof.

[0091] The one or more polymerase stoppers may comprise one or more steric blocking groups. In some embodiments, the one or more steric blocking groups increase the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex. The polymerase stopper comprises a modification incorporated between two bases of the signal-generating oligonucleotide. The modification may be a naphthylene-azo compound (e.g., Zen or iFQ). In some embodiments, the modification has the following structure:

[0092] [ka] wherein linking groups L1 and L2, which position the modification at an internal position of the signal-generating oligonucleotide, are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R1-R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group, electron-donating group, or point of attachment to a ligand; and X is a nitrogen atom or a carbon atom, with the proviso that when X is a carbon atom, the fourth substituent attached to that carbon atom can be hydrogen or a C1-C8 alkyl group. In some embodiments, the modification has the following structure:

[0093] [ka] wherein the linking groups L1 and L2, which position the modifications at internal positions of the signal-generating oligonucleotide, are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R1, R2, R4, R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing groups, or electron-donating groups; R6, R7, R9 to R 12are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron-withdrawing group, or an electron-donating group; R is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, or an electron-withdrawing group; and X is a nitrogen atom or a carbon atom, provided that when X is a carbon atom, the fourth substituent attached to the carbon atom can be hydrogen or a C1-C8 alkyl group. R can be NO2. In some embodiments, the modification has the following structure:

[0094] [ka]

[0095] When the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, the one or more polymerase stoppers may be capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. When the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, the one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. When the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers may be capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

[0096] The sample nucleic acid may include a nucleic acid comprising a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence includes the following steps: amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, the step including contacting the nucleic acid comprising the target nucleic acid sequence with i) a forward primer and a reverse primer, the forward primer being capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the reverse primer being capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. The nucleic acid may be double-stranded DNA. The nucleic acid may be 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. The amplifying step may include generating a nucleic acid by a reverse transcription reaction. The sample nucleic acid may include a sample ribonucleic acid, and the method may include contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. In some embodiments, the step of amplifying the target nucleic acid sequence comprises the following steps: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises the target nucleic acid sequence, and the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying comprises contacting the dsDNA with (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby producing a nucleic acid amplification product.

[0097] In some embodiments, when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a first undesired extension product. The first undesired extension product may be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product. One or more polymerase stoppers may terminate polymerase extension of the forward primer of the first undesired duplex to generate a first extension stall product. In some embodiments, the first extension stall product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to generate a first undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the forward primer of the first undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a second undesired extension product. The second undesired extension product may be amplified by an enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to form a second undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex to generate a second extension stall product. In some embodiments, the second extension stalled product cannot be amplified by an enzyme with hyperthermophile polymerase activity in the presence of a reverse primer to produce a second undesired amplification product.The one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when an exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity generates a third undesired extension product. The third undesired extension product may be amplified by an enzyme having hyperthermophile polymerase activity in the presence of the reverse primer to form a third undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex to generate a third extension stall product. In some embodiments, the third extension stalled product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to produce a third undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the third undesired duplex exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

[0098] The label may be capable of generating a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. In some embodiments, the label generates a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. The signal and the false-positive signal may be indistinguishable. In some embodiments, the generation of the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, detection of a false-positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

[0099] In some embodiments, the presence of one or more polymerase stoppers in the signal-generating oligonucleotide increases the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in a sample by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any two of these values) compared to a signal-generating oligonucleotide that does not contain one or more polymerase stoppers. In some embodiments, production of the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, a signal-generating oligonucleotide hybridized to the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, the nucleic acid amplification product reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes, or a number or range between any two of these values, before the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reach a detectable level. In some embodiments, the signal reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes, or a number or range between any two of these values, before the false positive signal reaches a detectable level. The appearance of detectable levels of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product may be delayed by at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes, or a number or range between any two of these values, compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers. In some embodiments, the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach detectable levels for at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the amplifying step begins.The occurrence of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product may be reduced by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any two of these values) compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers.

[0100] Amplifying the target nucleic acid sequence can include producing a detectable level of nucleic acid amplification product within about 20 minutes, about 15 minutes, or about 10 minutes. The detecting step can be performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition is contacted with the treated sample.

[0101] The lysis buffer may contain one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. The pH of the lysis buffer may be about 1.0 to about 10.0 (e.g., about 2.2). The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample nucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, the one or more amplification reagents include a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity; and / or dNTPs. In some embodiments, the one or more amplification reagents include an enzyme having reverse transcriptase activity, an enzyme having hyperthermophilic polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer. The reagent composition may be lyophilized, heat-dried, and / or include one or more additives. In some embodiments, the one or more additives include Tween 20, Triton X-100, and / or Tween 80; an amino acid; a sugar or sugar alcohol; and / or a polymer. The sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof. Contacting the reagent composition with the processed sample can include dissolving the reagent composition in the processed sample. In some embodiments, the one or more solubility reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) of the processed sample (e.g., about 0.2% (wt / vol) of the processed sample); and / or include a detergent (e.g., one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant).

[0102] In some embodiments, the method is performed in a single reaction vessel; does not include the use of any enzymes other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include the use of any enzymes other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein.

[0103] The target nucleic acid sequence can have a length of about 20 nucleotides or less to about 90 nucleotides or less (e.g., about 30 nucleotides). The forward primer, reverse primer, and / or reverse transcription primer can be about 8 to 16 bases long. The nucleic acid amplification product can be about 20 to 40 bases long. The spacer sequence can comprise a portion of the target nucleic acid sequence. The spacer sequence can be 1 to 10 bases long. Isothermal amplification conditions can include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, and optionally about 56°C to about 67°C. The amplifying step can be carried out for a period of about 5 minutes to about 60 minutes (e.g., a period of about 15 minutes). The amplifying step can be carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions. The amplifying step can be performed using PCR, LCR, LAMP, SDA, replicase-mediated amplification, immunoamplification, NASBA, 3SR, rolling circle amplification, or TMA. PCR can be real-time PCR and / or QRT-PCR.

[0104] The enzyme having hyperthermophilic polymerase activity may have an amino acid sequence that may be at least about 90% identical to the amino acid sequence of SEQ ID NO: 31 or a functional fragment thereof. The enzyme having hyperthermophilic polymerase activity may have an amino acid sequence that may be at least about 95% identical to the amino acid sequence of SEQ ID NO: 31. The enzyme having hyperthermophilic polymerase activity may be a polymerase comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the enzyme having hyperthermophilic polymerase activity has low or no exonuclease activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, and the forward and reverse primers. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

[0105] In some embodiments, the amplifying step includes and / or does not include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplifying step does not include LAMP.

[0106] In some embodiments, the methods do not include one or more of the following: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture. The term "isothermal amplification reaction" shall be given its ordinary meaning and shall include reactions in which the temperature does not change significantly during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10°C, for example, not more than 5°C or not more than 2°C, during the main enzymatic reaction step in which amplification occurs. Depending on the method of isothermal amplification of nucleic acids, different enzymes can be used for amplification. Isothermal amplification compositions and methods are described in WO2017176404, the entire contents of which are incorporated herein by reference.

[0107] In some embodiments, the methods and compositions described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer is resistant to the formation of a precipitate over a period of time under storage conditions (e.g., a storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer resists precipitation are described, for example, in International Application No. PCT / US23 / 61980, filed February 3, 2023, entitled "NON-OPAQUE LYTIC BUFFER COMPOSITION FORMULATIONS," the entire contents of which are incorporated herein by reference. Some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than acid and / or low pH conditions. Compositions, kits, and methods for nucleic acid detection in which nucleic acid strands are dissociated under low pH conditions (e.g., by contact with an acidic lysis buffer) to facilitate subsequent rapid amplification and detection are described in International Application No. PCT / US23 / 61978, filed February 3, 2023, entitled "METHOD FOR SEPARATING GEOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS," the entire contents of which are incorporated herein by reference.

[0108] In some embodiments, the methods and compositions described herein can include a lysis buffer and / or reagent composition. A lysis buffer comprising a lysis agent and a reducing agent, and a reagent composition comprising one or more protecting agents (e.g., cyclodextrin compounds) capable of sequestering the amplification agent and the lysis agent are described in WO2022198086, the entire contents of which are incorporated herein by reference. Some embodiments of the methods and compositions described herein can be used in conjunction with systems, methods, compositions, and kits for monitoring amplification reactions, as described in International Application No. PCT / US23 / 73519, filed September 6, 2023, entitled "HAIRPIN INTERNAL CONTROL FOR ISOTHERMAL NUCLEIC ACID AMPLIFICATION," the entire contents of which are incorporated herein by reference.

[0109] Some embodiments of the methods and compositions described herein may include probes that melt at a temperature different from the optimal APA reaction temperature to allow for multiplexed targets and / or internal controls. Compositions, kits, and methods for multiplexed nucleic acid detection are described in International Application No. PCT / US23 / 73521, entitled "ARCHEAL POLYMERASE AMPLIFICATION," filed September 6, 2023, the contents of which are incorporated herein by reference in their entirety.

[0110] Some embodiments of the methods and compositions described herein can, in some embodiments, be used in concert with the systems, methods, compositions, and kits for detecting pathogens described in International Application No. PCT / US23 / 73576, entitled "MODIFIED MOLECULAR BEACONS FOR IMPROVED DETECTION SPECIFICITY," filed September 6, 2023, the contents of which are incorporated herein by reference in their entirety.

[0111] Nucleic acids, subjects, samples and nucleic acid processing Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein. This term refers to nucleic acids of any composition, including DNA (e.g., complementary DNA (cDNA) and genomic DNA (gDNA)), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single- or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be found in plasmids, phages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or in vivo. It may be or be derived from other nucleic acids that can be replicated or replicated in vitro or in a host cell, cell, cell nucleus, mitochondria, or cell cytoplasm. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.The term can also include nucleotide analogs, single-stranded polynucleotides ("sense" or "antisense," "plus" or "minus" strand, "forward" or "reverse" reading frame, "forward" or "reverse" strand), and RNA or DNA equivalents, derivatives, variants, and analogs synthesized from double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain and generally includes regions preceding and following the coding region (leader and trailer) involved in transcription / translation of the gene product and regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). In RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed as the number of bases.

[0112] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid may be referred to as a target sequence, a target polynucleotide, and / or a target polynucleotide sequence, and may include double-stranded and single-stranded nucleic acid molecules. The target nucleic acid may be, for example, DNA or RNA. If the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may include a single-stranded target sequence. If the target nucleic acid is double-stranded, the target nucleic acid generally comprises a first strand and a second strand. The first strand and the second strand may be referred to as the forward strand and the reverse strand, and are generally complementary to each other. If the target nucleic acid is single-stranded, the complementary strand may be generated, for example, by polymerization and / or reverse transcription, to make the target nucleic acid double-stranded and have a first / forward strand and a second / reverse strand.

[0113] A target sequence can refer to either the sense or antisense strand of a nucleic acid sequence, a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence can also be a subsequence within a larger polynucleotide. For example, a target sequence can be a short sequence (e.g., 20-50 bases) within a nucleic acid fragment, chromosome, or plasmid targeted for amplification. In some embodiments, a target sequence can refer to a sequence within a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence can refer to the entire sequence targeted for amplification, or to a subsequence within the target nucleic acid to which the oligonucleotide binds. An amplification product can be a larger molecule containing the target sequence as well as at least one other sequence or other nucleotide. An amplification product can be approximately the same length as the target sequence, e.g., exactly the same length as the target sequence. An amplification product can comprise or consist of the target sequence.

[0114] The target sequence length and / or guanosine cytosine (GC) concentration (percentage) may depend, in part, on the temperature at which the amplification reaction is performed, which may in turn depend, in part, on the stability of the polymerase used in the reaction. Trial assays may be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, if the polymerase is stable up to 60°C-65°C, the target sequence may be, for example, 19-50 nucleotides in length, or, for example, about 40-50, 20-45, 20-40, or 20-30 nucleotides in length. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0115] Target nucleic acids may include, for example, genomic nucleic acids, plasmid nucleic acids, mitochondrial nucleic acids, cellular nucleic acids, extracellular nucleic acids, bacterial nucleic acids, and viral nucleic acids. In some embodiments, target nucleic acids may include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, genes, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA, or synthetic oligonucleotides. Genomic nucleic acids may include any nucleic acid derived from any genome, for example, animal genomes, plant genomes, insect genomes, viral genomes, and bacterial genomes (e.g., genomes present in spores). In some embodiments, genomic target nucleic acids are present within a specific genomic locus or multiple genomic loci. A genomic locus may include any or a combination of open reading frame DNA, non-transcribed DNA, intronic sequences, exonic sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequences considered to be associated with a given genomic locus.

[0116] The target sequence may comprise one or more types of repetitive elements (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as a repetitive element (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellite repeats, minisatellite repeats, etc.). For example, the target sequence may occur multiple times as a repetitive element, and one, some, or all occurrences of the target sequence within the repetitive element can be amplified (e.g., using a single pair of primers) using the methods described herein. In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as duplicates and / or paralogs.

[0117] The target nucleic acid may include a microRNA. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21-23 nucleotides in length), single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNAs and are partially complementary to them. The target nucleic acid may also include microRNA precursors, such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop RNAs that are further processed into miRNAs. The target nucleic acid may also include small interfering RNAs (siRNAs), which are short (e.g., about 20-25 nucleotides in length), at least partially double-stranded RNA molecules involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0118] Nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, isolated from a sample obtained from a subject. The subject can be any living or non-living organism, including, but not limited to, humans, non-human animals, plants, bacteria, fungi, viruses, or protists. Any human or non-human animal can be selected, including, but not limited to, mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), suidae (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), ursidae (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female, and the subject can be of any age (eg, embryo, fetus, infant, child, adult).

[0119] A sample or test sample may be any specimen isolated or obtained from a subject or a portion thereof. Non-limiting examples of specimens include fluids or tissues derived from a subject, including, but not limited to, blood or blood products (such as serum or plasma), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), serum, plasma, urine, aspirate, biopsy sample, intestinal puncture sample, cells (e.g., blood cells) or portions thereof (e.g., mitochondria, nuclei, or extracts), female reproductive tract washings, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, mammary fluid, hard tissue (e.g., liver, spleen, kidney, lung, or ovary), or the like, or combinations thereof. The term blood, as conventionally defined, includes whole blood, blood products, or any fraction of blood, such as serum, plasma, or buffy coat. Plasma refers to the fraction of whole blood obtained by centrifugation of blood that has been treated with an anticoagulant. Serum refers to the aqueous portion of the fluid that remains after a blood sample has clotted. Fluid or tissue samples are often collected according to standard protocols commonly followed by hospitals or clinics. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 milliliters) is often collected and can be stored according to standard procedures before or after preparation.

[0120] The sample can include spores, viruses, cells, nucleic acids, and / or samples containing any free nucleic acid from prokaryotes or eukaryotes. For example, the methods described herein can be used to detect nucleic acids outside of spores (e.g., no lysis is required). The sample can be isolated from any material suspected of containing the target sequence, such as from a subject as described above. In some embodiments, the target sequence is present in air, plants, soil, or other material suspected of containing biological organisms. Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from biological samples can be used, including art-known DNA preparation methods and various commercially available reagents or kits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit, or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wisconsin), and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, New Jersey), or combinations thereof. U.S. Patent No. 7,888,006 provides a DNA purification method, but does not disclose the compositions (e.g., lysis buffer, protectant) and methods provided herein.

[0121] In some embodiments, a cell lysis procedure is performed. Cell lysis may be performed before initiating the amplification reaction described herein (e.g., to release DNA and / or RNA from cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical methods (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or a combination thereof), physical methods (e.g., French press and sonication, etc.), or electrolytic lysis. For example, chemical methods generally involve disrupting cells using a lysing agent, extracting nucleic acids from the cells, followed by treatment with a chaotropic salt. In some embodiments, cell lysis involves the use of a detergent (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis involves the use of an ionic detergent (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods, such as freeze / thaw trituration and the use of a cell press, may also be useful. High-salt lysis procedures can also be used. For example, alkaline lysis procedures can be used. The latter procedure traditionally incorporates the use of a phenol-chloroform solution, although an alternative phenol-chloroform-free procedure involving three solutions can also be used. In the latter procedure, for example, one solution may contain 15 mM Tris (pH 8.0), 10 mM EDTA, and 100 μg / ml RNase A, a second solution may contain 0.2 N NaOH and 1% SDS, and a third solution may contain 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and components described herein.

[0122] Nucleic acids for performing the methods described herein can be provided without processing a sample containing the nucleic acid. For example, nucleic acids for performing the amplification methods described herein can be provided without prior nucleic acid purification. In some embodiments, target sequences are amplified directly from a sample (e.g., without any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids for performing the methods described herein are provided after processing a sample containing the nucleic acid. For example, nucleic acids can be extracted, isolated, purified, or partially purified from a sample. The term "isolated" generally refers to nucleic acids that have been removed from their original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed) and thus have been altered from their original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid" can refer to nucleic acids removed from a subject (e.g., a human subject). Isolated nucleic acids can be provided that have fewer non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. A composition containing isolated nucleic acids may contain no more than about 50%-99% of non-nucleic acid components. A composition comprising an isolated nucleic acid may be free of more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of non-nucleic acid components. The term "purified" generally refers to a nucleic acid that contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. A composition comprising a purified nucleic acid may be free of more than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of other non-nucleic acid components.

[0123] Nucleic acids can be provided for performing the methods described herein without modifying the nucleic acid, for example, by denaturing, digesting, nicking, unwinding, incorporating and / or ligating heterologous sequences, adding epigenetic modifications, adding labels (e.g., 32P, 33 P, 125 I, or 35 Examples of such labels include radioactive labels such as S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, and fluorescent dyes. Thus, in some embodiments, unmodified nucleic acids are amplified.

[0124] The methods disclosed herein for detecting target nucleic acid sequences (single-stranded or dsDNA and / or RNA) in a sample can detect target nucleic acid sequences (e.g., DNA or RNA) with high sensitivity. In some embodiments, the methods can be used to detect target DNA / RNA present in a sample containing multiple RNAs / DNAs (including a target RNA / DNA and multiple non-target RNAs / DNAs), where the target RNA / DNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 It is present in one or more copies per non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meaning and shall refer to DNA, or RNA, or a combination of DNA and RNA.

[0125] The detection threshold of a method for detecting target RNA / DNA in a sample can be, for example, 10 nM or lower. The term "detection threshold" shall be given its ordinary meaning and shall describe the minimum amount of target RNA / DNA that must be present in a sample for detection to occur. As an illustrative example, if the detection threshold is 10 nM, a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the methods of the present disclosure provide a method for detecting target RNA / DNA at a concentration of 5 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower, 0.05 nM or lower, 0.01 nM or lower, 0.005 nM or lower, 0.001 nM or lower, 0.0005 nM or lower, 0.0001 nM or lower, 0.00005 nM or lower, 0.00001 nM or lower, or 0.00001 nM. or lower, 10 pM or lower, 1 pM or lower, 500 fM or lower, 250 fM or lower, 100 fM or lower, 50 fM or lower, 500 aM (attomolar) or lower, 250 aM or lower, 100 aM or lower, 50 aM or lower, 10 aM or lower, or 1 aM or lower. In some embodiments, the disclosed compositions or methods exhibit attamolar (aM), femtomolar (fM), picomolar (pM), and / or nanomolar (nM) detection sensitivity.

[0126] A sample may contain sample nucleic acids (e.g., multiple sample nucleic acids). The term "multiple" is used herein to mean two or more. Thus, in some embodiments, a sample contains two or more (e.g., three or more, five or more, ten or more, twenty or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, or five thousand or more) sample nucleic acids (e.g., DNA / RNA). The disclosed methods can be used as highly sensitive methods for detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, a sample contains 5, 10, 20, 25, 50, 100, 500, 10, 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 seeds or 10 7 The sample may contain DNA / RNA from 50 or more species, each differing in sequence from the others. The sample may contain DNA / RNA from cells (e.g., eukaryotic, mammalian, or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0127] As used herein, the term "sample" shall be given its ordinary meaning and shall include any sample containing RNA and / or DNA (e.g., for determining whether target DNA and / or target RNA is present in a population of RNA and / or DNA). A sample may be a biological sample or an environmental sample. A sample may be derived from any source; for example, a sample may be a synthetic combination of purified DNA and / or RNA. A sample may be a cell lysate, a DNA / RNA-enriched cell lysate, or DNA / RNA isolated and / or purified from a cell lysate. A sample may be derived from a patient (e.g., for diagnostic purposes). A sample may be derived from permeabilized cells, crosslinked cells, tissue sections, or combinations thereof. A sample may be derived from tissue prepared by crosslinking followed by delipidation and adjustment to a uniform refractive index. A sample may contain a target nucleic acid (e.g., target DNA / RNA) and multiple species of non-target DNA / RNA. In some embodiments, the target DNA / RNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 Present in the sample at 1 copy per non-target DNA / RNA.

[0128] Patient-related samples include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, and samples that have been manipulated in some way after procurement (e.g., treatment with reagents); washed; or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA). Samples may include biological samples, including, but not limited to, clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, and bone marrow. Biological samples may also include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), such as samples containing RNA obtained from such cells (e.g., RNA-containing cell lysates or other cell extracts). In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof.

[0129] The source of the sample may be a diseased (or suspected) cell, fluid, tissue, or organ, or may be a normal (non-diseased) cell, fluid, tissue, or organ. In some embodiments, the source of the sample is a cell, tissue, or organ infected (or suspected) with a pathogen. For example, the source of the sample may be an individual, which may be infected or uninfected - and the sample may be any biological sample collected from an individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, stool sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., buccal swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal secretion, tears, buffy coat, mucosal sample, epithelial cell sample (e.g., epithelial cell scraping), etc.), and cultures thereof. The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens may be viruses, fungi, helminths, protozoans, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites, etc. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny headworms, and tapeworms (Cestodes). Protozoan infections include infections by Giardia spp., Trichomonas spp., African trypanosomiasis, amebic dysentery, babesiosis, balantidiosis, Chagas disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens, such as parasitic / protozoan pathogens, include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza virus, dengue fever, West Nile virus, herpes virus, yellow fever virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, and papillomavirus. Pathogenic viruses include papovaviruses (e.g., HPV, polyomavirus); hepadnaviruses; herpes viruses (e.g., HSV (e.g., HSV I, HSV II), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, and pityriasis rosea). Rosea, Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; variola virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; and Phycodnaviridae. Non-limiting examples of pathogens include Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pneumoniae, Cryptococcus neoformans, Histoplasma capsulatum, and Haemophilus influenzae.influenzae B, Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, human serum parvo-like virus, respiratory syncytial virus, measles virus, adenovirus, human T-cell leukemia virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma brucei rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria species (e.g., chicken cecal coccidia (tenella)), Onchocercavolvulus), Leishmania species (e.g., Leishmania tropica), Streptococcus pneumoniae, Pneumocystis carinii, Trichophyton rubrum, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora species, SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), herpes simplex, herpesvirus 6, herpesvirus 7, JC virus, and type A Influenza, influenza B, influenza C, rotavirus, human adenovirus, human enterovirus, hantavirus, Legionella dumophila, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsii, Ehrlichia species (e.g., Ehrlichia chaffeensis), Borrelia burgdorferi, Yersinia pestis, Chlamydia pneumoniae, Trichinella spiralis spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma species (e.g., Mycoplasma arthritidis), M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae.

[0130] amplification Methods for amplifying nucleic acids are provided herein. In some embodiments, nucleic acids are amplified using a suitable amplification process. Nucleic acid amplification typically involves enzymatic synthesis of nucleic acid amplicons (copies) containing sequences complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is performed in a single vessel, a single chamber, and / or a single volume (i.e., adjacent volumes). In some embodiments, the amplification method and the detection method (e.g., the detection methods described herein) are performed in a single vessel, a single chamber, and / or a single volume (i.e., adjacent volumes).

[0131] The terms "amplify," "amplification," "amplification reaction," or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification can also refer to an "exponential" increase in the target nucleic acid. "Amplifying" can also refer to a linear increase in the number of target nucleic acids, but is distinct from a single, single primer extension step. In some embodiments, a limited amplification reaction, also known as preamplification, can be performed. Preamplification is a method in which a limited amount of amplification occurs because a small number of cycles, e.g., 10 cycles, are performed. Preamplification allows for some amplification but stops amplification before the exponential phase, typically producing approximately 500 copies of the desired nucleotide sequence. Preamplification can be used to limit inaccuracies associated with reactant depletion in a particular amplification reaction and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a single primer extension step can be performed prior to linear or exponential amplification.

[0132] A general description of the amplification process is provided herein. For example, when a primer (e.g., an oligonucleotide described herein) and a target nucleic acid are contacted, complementary sequences anneal or hybridize to each other. The primer can anneal to the target nucleic acid at or near (e.g., adjacent, abutting, etc.) the sequence of interest. A primer annealed to a target may be referred to as a primer-target hybrid, a hybridized primer-target, or a primer-target duplex. The terms "near" or "adjacent" when referring to a nucleotide sequence of interest refer to the distance (e.g., number of bases) or region between the end of the primer and one or more nucleotides (e.g., a nucleotide sequence) of the target. Generally, adjacent refers to a range of about 1 nucleotide to about 50 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides) from the nucleotide or nucleotide sequence of interest. In some embodiments, a set of primers (e.g., a pair of primers, a forward primer and a reverse primer, a first oligonucleotide and a second oligonucleotide) anneals within about 1-20 nucleotides of a nucleotide or nucleotide sequence of interest to generate an amplification product. In some embodiments, the primers anneal within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended along the target (i.e., template strand) by a polymerase to generate a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplification product is generated. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step.

[0133] Components of an amplification reaction (e.g., one or more amplification reagents) can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, and multiple primer sets for multiplex amplification), a nucleic acid target (e.g., a target nucleic acid derived from a sample), one or more polymerases, nucleotides (e.g., dNTPs), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, a monovalent ion, and a divalent ion). The amplification reaction may further include one or more of a reverse transcriptase, a reverse transcription primer, and one or more detection agents.

[0134] Nucleic acid amplification can be performed in the presence of natural nucleotides, such as deoxyribonucleoside triphosphates (dNTPs) and / or derivatized nucleotides. Natural nucleotides generally refer to adenylate, guanylate, cytidylate, thymidylate, or uridylate. Derivatized nucleotides generally are nucleotides other than natural nucleotides. Ribonucleoside triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, or U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be referred to herein as A, G, C, T, or U, without specific reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs can be used, such as analogs containing a detectable label (e.g., a fluorescent label or a colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, such as 32 P, 33 P, 125 I, or 35The amplification can be performed in the presence of a radioactive label such as S; an enzyme label such as alkaline phosphatase; a fluorescent label such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, an antigen, a hapten, or a fluorescent dye. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., TriLink's CleanAmp™ dNTPs).

[0135] The one or more amplification reagents may include non-enzymatic and enzymatic components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions. In some embodiments, the non-enzymatic components do not include proteins (e.g., nucleic acid-binding proteins), enzymes, or proteins with enzymatic activity, such as polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. In some embodiments, the enzymatic components consist of a polymerase or a polymerase and a reverse transcriptase. Thus, such enzymatic components would exclude other proteins (e.g., nucleic acid-binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases.

[0136] In some embodiments, amplification conditions include enzymatic activity (e.g., enzymatic activity provided by a polymerase, or enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include enzymatic activity provided by enzymes other than the polymerase and / or reverse transcriptase, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. The polymerase activity and reverse transcriptase activity may be provided by separate enzymes or separate enzyme types (e.g., a polymerase and a reverse transcriptase), or may be provided by a single enzyme or enzyme type (e.g., a polymerase). Nucleic acid amplification may include non-thermal cycling PCR. In some embodiments, nucleic acid amplification includes an isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process performed at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature is maintained essentially constant during the amplification reaction. Isothermal amplification conditions generally do not include a thermal cycling (i.e., cycling between upper and lower temperature limits) component to the amplification process. When amplifying under isothermal conditions, the reaction can be maintained at an essentially constant temperature, meaning that the temperature does not need to be maintained at exactly one temperature. For example, isothermal amplification processes may experience small temperature fluctuations (e.g., ±1-5°C) due to environmental or equipment-based variables. Often, the entire reaction volume is maintained at an essentially constant temperature, and isothermal reactions, as used herein, generally do not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0137] The isothermal amplification reaction herein can be carried out at an essentially constant temperature. In some embodiments, the isothermal amplification reaction herein is carried out at a temperature of about 55°C to about 75°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C, or a temperature approximately these values, or a value or range between any two of these values. In some embodiments, a temperature element (e.g., a heat source) is maintained at an essentially constant temperature, for example, about 75°C or less, about 70°C or less, about 65°C or less, or about 60°C or less.

[0138] The amplification process herein can be carried out for a certain length of time, for example, until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product can be detected by any suitable detection process and / or detection process described herein. The amplification process can be carried out within about 20 minutes or less, or within about 10 minutes or less. For example, the amplification process can be carried out within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or within any value or range between any two of these values.

[0139] In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation during the amplification step (and / or other steps). In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that promote unwinding during the amplification step (and / or other steps). Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote unwinding can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, and proteins (e.g., enzymatic agents).

[0140] In some embodiments, the methods disclosed herein do not involve heat denaturation (e.g., heating a solution containing nucleic acids to an elevated temperature, such as 75°C, 80°C, 90°C, or 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of nucleic acids. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions provided herein do not include a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and / or a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions and methods provided herein do not include intercalating agents, alkylating agents, and / or chemicals such as formamide, glycerol, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods of the present disclosure do not include contacting nucleic acids with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding). In some embodiments, the amplification step (e.g., step (c)) does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase) and / or low pH conditions (e.g., contact with acid).

[0141] Nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-strand binding proteins), or any combination thereof. For example, nucleic acid targets can be amplified without exposure to helicases, including, but not limited to, DNA helicases and RNA helicases. Amplification conditions that do not include the use of helicases are helicase-free amplification conditions.

[0142] Nucleic acid targets can be amplified without exposure to recombinases, including, but not limited to, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX. In some embodiments, nucleic acid targets are amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY). Nucleic acid targets can be amplified without exposure to nucleic acid binding proteins (e.g., single-strand binding protein or single-stranded DNA binding protein (SSB)), such as T4 gp32. In some embodiments, nucleic acid targets are amplified without exposure to topoisomerases. Nucleic acid targets can be amplified with or without exposure to agents or conditions that destabilize nucleic acids. As used herein, the term "destabilization" shall be given its ordinary meaning and shall refer to the disruption of the overall organization and geometric orientation (e.g., double helix structure) of nucleic acid molecules by one or more of tilt, rotation, twist, slip, and flip effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, page 17). Destabilization generally does not refer to the melting or separation (e.g., denaturation) of nucleic acid strands. Nucleic acid destabilization can be achieved by exposure to agents, such as intercalating or alkylating agents and / or chemicals, such as formamide, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, the nucleic acid target is amplified without exposure to a ligase and / or an RNA replicase.

[0143] In some embodiments, nucleic acid targets can be amplified without cleavage or digestion. For example, nucleic acid targets can be amplified without prior exposure to one or more cleavage agents, resulting in an intact nucleic acid being amplified. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents during amplification. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents after amplification. Amplification conditions that do not include the use of a cleavage agent may be referred to herein as cleavage agent-free amplification conditions. The term "cleavage agent" generally refers to an agent, possibly a chemical or enzyme, that can cleave nucleic acids at one or more specific or non-specific sites. Specific cleavage agents often specifically cleave specific sites according to a specific nucleotide sequence. Cleavage agents can include endonucleases (e.g., restriction enzymes and nicking enzymes); exonucleases (DNAses, RNAses (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases); and chemical cleavage agents.

[0144] Nucleic acid targets can be amplified without the use of restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without prior exposure to restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes during amplification. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNAse, RNAse (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases. In some embodiments, nucleic acids are amplified without exonuclease treatment before, during, and / or after amplification. Amplification conditions that do not include the use of exonucleases are exonuclease-free amplification conditions. In some embodiments, the nucleic acid is amplified without DNAse and / or RNAse treatment. In some embodiments, the nucleic acid is amplified without RNAse H treatment.

[0145] Amplified nucleic acids may be referred to herein as nucleic acid amplification products or amplicons. In some embodiments, amplification products include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and the like, as well as combinations of the foregoing. Amplification products typically have a nucleotide sequence that is identical or substantially identical to the sequence of a sample nucleic acid (e.g., a target sequence) or its complement. A "substantially identical" nucleotide sequence in an amplification product will generally have a high degree of sequence identity (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity) to the nucleotide sequence being amplified or its complement, with variations being the result of poor polymerase fidelity or other variables.

[0146] The nucleic acid amplification product may include a polynucleotide that is contiguously complementary or substantially identical to a target sequence in the sample nucleic acid. Contiguously complementary generally refers to, for example, a nucleotide sequence in a first strand in which each base is paired in order (e.g., reading from 5' to 3') with a correspondingly ordered base in a second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered contiguously complementary. In other words, contiguously complementary generally refers to every contiguous base in the nucleotide sequence of the first strand being complementary to the corresponding contiguous base in the nucleotide sequence of the second strand. For example, a first strand having the sequence 5'-ATGCATGCATGC-3' (SEQ ID NO: 33) would be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 34) if every contiguous base in the first strand is complementary to every corresponding contiguous base in the second strand. However, a first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO: 35) would not be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 34) because the sequence of six adenines (six As) in the middle of the first strand would not pair with bases in the second strand. Contiguous complementary sequences are optionally about 5 to about 25 contiguous bases in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is contiguous complementary to or substantially identical to a target sequence in the sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequence (e.g., at the 5' and / or 3' end or within the product) that is not contiguous with or substantially identical to the target sequence, such as additional sequences incorporated into the amplification product by tail primers or ligation, and / or additional sequences that provide cleavage agent recognition sites (e.g., nicking enzyme recognition sites). Generally, unless the target sequence includes tandem repeats, the amplification product will not include products in the form of tandem repeats.

[0147] The nucleic acid amplification product can comprise sequences complementary to or substantially identical to one or more primers used in the amplification reaction, hi some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is contiguous to or identical to a first primer sequence and a second nucleotide sequence that is contiguous to or identical to a second primer sequence.

[0148] Nucleic acid amplification products can include spacer sequences. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is contiguous with or substantially identical to a portion of a target sequence in a sample nucleic acid and is flanked by sequences in the amplification product that are complementary to or substantially identical to one or more primers used in the amplification reaction. The spacer sequence flanked by sequences in the amplification product is generally located between a first sequence (complementary to or substantially identical to the first primer) and a second sequence (complementary to or substantially identical to the second primer). Thus, an amplification product typically includes a first sequence, followed by a spacer sequence, followed by a second sequence. The spacer sequence is generally neither complementary nor substantially identical to the sequence of the primers. The spacer sequence may be or include approximately 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists of or consists essentially of a first nucleotide sequence that is contiguously complementary to or identical to the first primer sequence, a second nucleotide sequence that is contiguously complementary to or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence that is not contiguously complementary to or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product), and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other mechanism incorporated into the amplification product. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguously complementary to or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product), and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other mechanism incorporated into the amplification product.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or another extra base (e.g., at the 5' and / or 3' end or within the product) introduced into the product due to, for example, errors or promiscuity in the amplification process.

[0149] Nucleic acid amplification products may be up to 50 bases in length, including 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number or range of bases between any two of these values. In some embodiments, nucleic acid amplification products of a given target sequence have the same or substantially the same length (e.g., within 1-10 bases). Thus, nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel) and generally do not produce multiple signals representing multiple lengths (e.g., a ladder or smear on an electrophoresis gel). In multiplex reactions, nucleic acid amplification products of different target sequences may have different lengths.

[0150] The methods and components described herein can be used for multiplex amplification, which generally refers to the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence). For example, multiplex amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample. For example, the amplifying step can include multiplex amplification of two or more target nucleic acid sequences, and the detecting step can include multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. The two or more target nucleic acid sequences can be specific to two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C). Multiplex amplification can also refer to the simultaneous or sequential amplification of one or more sequences present in multiple samples. For example, multiplex amplification can be used to amplify at least two amplifiable target sequences (e.g., the amplification reaction includes appropriate primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is configured to detect at least two target sequences, but only one of the target sequences is present in the sample being tested, so that both sequences are amplifiable, but only one sequence is amplified. In some embodiments, when two target sequences are present, the amplification reaction results in the amplification of both target sequences. A multiplex amplification reaction including appropriate primers and enzymes can result in the amplification of one, some, or all of the target sequences. In some embodiments, the amplification reaction is configured to detect two sequences using a pair of primers, one sequence being the target sequence and one sequence being a control sequence (e.g., a synthetic sequence that is amplifiable with the same primers as the target sequence but has spacer bases or a sequence different from the target). In some embodiments, the amplification reaction is configured to detect multiple sets of sequences using corresponding primer pairs, each set including a target sequence and a control sequence.

[0151] Primer Nucleic acid amplification is generally performed in the presence of one or more primers. A primer is generally characterized as an oligonucleotide comprising a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid at or near (e.g., adjacent to) a specific region of interest (i.e., target sequence). A primer can, for example, enable specific determination of the nucleotide sequence of a target nucleic acid or detection of the target nucleic acid or a characteristic thereof (e.g., the presence or absence of a sequence). A primer can be naturally occurring or synthetic. The term specific or specific generally refers to the binding or hybridization of one molecule, such as a primer for a target polynucleotide, with another molecule. That is, the term specific or specific refers to the recognition, contact, and formation of a stable complex between two molecules, compared to substantially less recognition, contact, or complex formation between either of those two molecules and other molecules. The term annealing or hybridization generally refers to the formation of a stable complex between two molecules. The terms primer, oligo, or oligonucleotide can be used interchangeably herein when referring to a primer.

[0152] Primers can be designed and synthesized using any suitable process and may be of any length suitable for hybridizing to a target sequence and carrying out the amplification processes described herein. Primers are often designed according to the sequence of the target nucleic acid. In some embodiments, primers may be about 5 to about 30 bases in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases in length. Primers may be composed of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides), or mixtures thereof. Modifications and modified bases include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite™, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker™, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6dT, Uni-Link™ amino modifier), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG), thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); fluorophores (e.g., Freedom™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO™ dyes, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy750 (NHS ester)); Iowa Black® dark quencher modifications (e.g., Iowa Black® FQ, Iowa Black® RQ); dark quencher modifications (e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl);spacers (C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate (PS) bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases);and click chemistry modifications. In some embodiments, modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS linkages, 3' phosphate groups, and spacer bases (such as C3 spacers or other spacer bases). For example, a primer may contain one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is a post-transcriptional modification of RNA commonly found in tRNA and other small RNA molecules. Primers containing 2'-O-methyl RNA bases can be directly synthesized. This modification can, for example, increase the Tm of an RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in a primer to, for example, increase stability and binding affinity with the target sequence. In some embodiments, a primer can contain one or more phosphorothioate (PS) linkages (e.g., PS linkage modifications). PS linkages can substitute a sulfur atom for a non-bridging oxygen in the phosphate backbone of the primer. This modification typically renders the internucleotide linkage resistant to nuclease degradation. PS bond can be introduced between approximately the last 3-5 nucleotides at the 5' or 3' end of the primer to inhibit exonuclease degradation, for example. In some embodiments, PS bond inclusion throughout the primer can help reduce endonuclease attack. Primers may contain, for example, a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases and, in certain cases, can be used to block extension by DNA polymerase. In some embodiments, primers contain one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated internally or at the 5' end of the primer. Multiple C3 spacers can be added to either end of the primer to introduce long hydrophilic spacer arms for attachment of, for example, fluorophores or other pendant groups.

[0153] A primer may comprise DNA bases, RNA bases, or both, and one or more of the DNA and RNA bases may be modified or unmodified. For example, a primer may be a mixture of DNA bases and RNA bases. A primer may comprise DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, a primer comprises unmodified DNA bases. In some embodiments, a primer comprises modified DNA bases. A primer may comprise RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, a primer comprises unmodified RNA bases. In some embodiments, a primer comprises modified RNA bases. In some embodiments, a primer does not comprise RNA bases. In some embodiments, a primer does not comprise DNA bases. In some embodiments, a primer does not comprise a cleavage agent recognition site (e.g., does not comprise a nicking enzyme recognition site). In some embodiments, a primer does not comprise a tail (e.g., does not comprise a tail comprising a nicking enzyme recognition site).

[0154] In some embodiments, all or a portion of the primer sequence may be complementary or substantially complementary to the target nucleic acid. Substantially complementary, in the context of sequences, generally refers to nucleotide sequences that will hybridize to each other. The stringency of hybridization conditions can be varied to allow for varying amounts of sequence mismatch. The target sequence and primer sequence may be at least 75% complementary to each other, including, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to each other. A primer that is substantially complementary to a target nucleic acid sequence is typically also substantially identical to the complement of the target nucleic acid sequence (i.e., the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid may be at least 75% identical in sequence, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each other.

[0155] In some embodiments, the primers comprise a pair of primers. A pair of primers may comprise a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid). In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, amplification of a target sequence is performed using a pair of primers, and no additional primers or oligonucleotides are included in the amplification of the target sequence (e.g., the amplification reaction components do not include additional primer pairs for a given target sequence, nested primers, bumper primers, oligonucleotides other than primers, probes, etc.). In some embodiments, the primers consist of a pair of primers. In some embodiments, the amplification reaction may include additional primer pairs for amplifying different target sequences, such as in multiplex amplification. In some embodiments, the primers consist of a pair of primers, but in some embodiments, the amplification reaction may include additional primers, oligonucleotides, or probes for a detection process that is not considered part of the amplification. In some embodiments, primers are used in sets. An amplification primer set may comprise a pair of forward and reverse primers for a given target sequence. In the case of multiplex amplification, the primers that amplify a first target sequence are considered to be a primer set, and the primers that amplify a second target sequence are considered to be a different primer set.

[0156] Nucleic acids (e.g., amplification products, sample nucleic acids, target nucleic acid sequences) described herein can comprise a first strand and a second strand that are complementary to each other. Amplification reaction components may comprise or consist of a first primer (first oligonucleotide) that is complementary to a target sequence in the first strand (e.g., sense strand, forward strand) of the sample nucleic acid, and a second primer (second oligonucleotide) that is complementary to a target sequence in the second strand (e.g., antisense strand, reverse strand) of the sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) comprises a first polynucleotide that is contiguously complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide that is contiguously complementary to the target sequence in the second strand of the sample nucleic acid. Contiguously complementary, with respect to primer-target, generally refers to a nucleotide sequence of a primer in which each base pairs sequentially with a corresponding, ordered base in the target sequence, with no gaps, additional sequence, or unpaired bases present within the sequence considered contiguously complementary. In some embodiments, the primer does not include any additional sequence (e.g., at the 5' and / or 3' end or within the primer) that is not contiguous with the target sequence, such as additional sequence present in a tail primer or loop primer, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site). In some embodiments, the amplification reaction components do not include primers that include additional sequence (i.e., sequence other than sequence that is contiguous with the target sequence), such as a tail primer, a loop primer, a step-loop structure, a primer that can form a hairpin structure, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site).

[0157] In some embodiments, a primer can contain modifications such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifier that alters the binding properties of the primer. In some embodiments, a primer can include a detectable molecule or entity (e.g., a fluorophore, a radioisotope, a colorimetric reagent, a particle, an enzyme, etc.).

[0158] polymerase Amplification reaction components (e.g., one or more amplification reagents) can include one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides extending the 3' hydroxyl end of a primer molecule, such as an amplification primer described herein, into a nucleic acid target sequence (e.g., to which the primer anneals). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that can exhibit activity at high reaction temperatures (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C). Hyperthermophilic polymerases are sometimes referred to as hyperthermophilic polymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, the polymerase can incorporate from about 1 to about 50 nucleotides in a single synthesis, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range of nucleotides between any two of these values ​​in a single synthesis.

[0159] Amplification reaction components include 9°N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA polymerase; Therminator™ II DNA polymerase; Therminator™ III DNA polymerase; Therminator™ γ DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo); T4 DNA polymerase; T7 DNA polymerase; DeepVentR™ (exo) DNA polymerase; DeepVentR™ DNA polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start DNA polymerase; Phusion™ High-Fidelity The polymerase may include one or more DNA polymerases selected from DNA polymerase; VentR® DNA polymerase; VentR® (exo) DNA polymerase; RepliPHI™ Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm™ DNA polymerase); MasterAmp™ AmpliTherm™ DNA polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase beta; and ThermoPhi DNA polymerase.

[0160] The amplification reaction components can include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermostable at high temperatures). The hyperthermophilic DNA polymerases can have a half-life of about 5-10 hours at 95°C or about 1-3 hours at 100°C. For example, the amplification reaction components can include one or more hyperthermophilic DNA polymerases from archaea (e.g., a hyperthermophilic DNA polymerase from Thermococcus or a hyperthermophilic DNA polymerase from Thermococcaceaen archaean). The amplification reaction components can include one or more hyperthermophilic DNA polymerases from the genera Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0161] In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase or a functional fragment thereof. A functional fragment generally retains one or more functions of a full-length polymerase, such as the ability to polymerize DNA (e.g., in an amplification reaction). In some cases, the functional fragment performs a function (e.g., polymerizing DNA in an amplification reaction) at a level that is at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the functional level of the full-length polymerase. The level of polymerase activity can be assessed using a detectable nucleic acid amplification method, such as those described herein. In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase comprising the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32, or a functional fragment of SEQ ID NO:31 or SEQ ID NO:32.

[0162] In some embodiments, an amplification reaction component (e.g., one or more amplification reagents) comprises a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase or a functional fragment thereof, hi some embodiments, an amplification reaction component comprises a polymerase comprising an amino acid sequence at least about 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32, or a functional fragment thereof.

[0163] The polymerase may have reverse transcriptase activity. In such embodiments, the amplification reaction can amplify an RNA target in a single step, for example, without the use of a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase activity include Bst (large fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, and Therminator™ II. The amplification reaction components can include one or more separate reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction can include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0164] In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases with no or low exonuclease activity are used during amplification. In some embodiments, the polymerase with no or low exonuclease activity comprises one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, a modified polymerase with low exonuclease activity may have 10% or less exonuclease activity compared to an unmodified polymerase, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase has no or low 5'-3' exonuclease activity and / or no or low 3'-5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand-dependent exonuclease activity and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of a polymerase include one or more amino acid substitutions at or corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:31 (e.g., D141A, E143A, E143D, and A485L).

[0165] Detection and Quantification The methods described herein can include detecting and / or quantifying the nucleic acid amplification product(s). The amplification product(s) can be detected and / or quantified, for example, by any suitable detection and / or quantification method described herein.Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, end-point), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating / binding dyes, absorbance methods (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension methods (e.g., iPLEX™), Affymetrix Molecular Inversion Probes (MIR), and the like. Probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, genetic bit The methods include, but are not limited to, GBA, multiplex minisequencing, SNaPshot, GOOD assay, microarray miniseq, arrayed primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed incorporation (TDI), colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probe, Invader assay, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips, and combinations thereof. In some embodiments, detecting the nucleic acid amplification product comprises the use of a real-time detection method (i.e., the product is detected and / or continuously monitored during the amplification process).In some embodiments, detecting nucleic acid amplification products involves the use of end-point detection methods (i.e., products are detected after the amplification process is completed or stopped). Nucleic acid detection methods can also employ the use of labeled nucleotides, either directly incorporated into the target sequence or incorporated into a probe containing a complementary sequence to the target. Such labels may be radioactive and / or fluorescent in nature and can be resolved in any of the manners discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, detection methods can be used in conjunction with measuring signal intensity and / or generating (or referencing) standard curves and / or look-up tables for quantification of nucleic acid amplification products.

[0166] Detection of nucleic acid amplification products may involve the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule whose detectable property is detectable under certain conditions, thereby enabling the molecule to function as a specific, useful signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or speed at which a molecule passes through an aperture of a known size. A molecular beacon for detecting nucleic acid molecules may be, for example, a hairpin-shaped oligonucleotide containing a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin may contain a probe sequence complementary to the target sequence, and the stem is formed by annealing complementary arm sequences located on either side of the probe sequence. The fluorophore and quenching molecule may be covalently linked to the opposite ends of each arm. Under conditions that prevent the oligonucleotide from hybridizing to its complementary target, or when the molecular beacon is free in solution, the fluorescent molecule and quenching molecule are in close proximity to each other, preventing FRET. When a molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, converting the loop structure into a stable, more rigid conformation, causing the fluorophore and quencher molecules to separate, resulting in fluorescence. Because the probe is specific, fluorescence generally occurs only with the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is identical to or complementary to a sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is neither identical to nor complementary to a sequence in the target nucleic acid (e.g., hybridizes to a tail amplification primer or a sequence added to the amplification product by ligation). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with different colored fluorophores and different target sequences, allowing for simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction).In the case of quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each cycle of amplification, and unhybridized molecular beacons are dark, so there is no need to isolate the probe-target hybrid to quantitatively determine the amount of amplification product. The signal obtained is proportional to the amount of amplification product. Detection using molecular beacons can be performed in real time or as an end-point detection method. The detection of nucleic acid amplification products may involve the use of lateral flow. The use of lateral flow typically involves the use of lateral flow devices, including, but not limited to, dipstick assays and thin-layer chromatography plates with various appropriate coatings. Various binding reagents for the sample, binding partners for the sample or conjugates containing binding partners, and signal generating systems are immobilized in the flow channels.

[0167] Detection of nucleic acid amplification products may involve the use of FRET, an energy transfer mechanism between two chromophores: a donor molecule and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emission of the donor molecule as it returns to its ground state can transfer the excitation energy to the acceptor molecule via long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored and is a function of the distance between the donor and acceptor, the overlap between the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be useful, for example, for quantifying the molecular dynamics of DNA-DNA interactions, as described for molecular beacons. To monitor the production of a specific product, a probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe-target hybridization changes the distance or orientation between the donor and acceptor, and a FRET change is observed.

[0168] Detection of nucleic acid amplification products may involve the use of fluorescence polarization (FP). FP techniques are based on the principle that when a fluorescently labeled compound is excited with linearly polarized light, it will emit fluorescence with a degree of polarization inversely proportional to its rotation rate. Thus, when a molecule, such as a fluorescently labeled tracer-nucleic acid conjugate, is excited with linearly polarized light, the fluorophore's rotation is constrained between light absorption and emission, resulting in a highly polarized emission. When a free tracer compound (i.e., not bound to a nucleic acid) is excited with linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, resulting in a more random molecular orientation and depolarized emitted light. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0169] Detection of nucleic acid amplification products may involve the use of surface capture, achieved, for example, by immobilizing specific oligonucleotides on a surface to create a biosensor with both high sensitivity and selectivity. Examples of surfaces that can be used to attach probes include gold and carbon. Detection of nucleic acid amplification products may involve the use of 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN). For example, TAQMAN probes are hydrolysis probes that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The principle of TAQMAN probes relies on 1) the 5'-3' exonuclease activity of Taq polymerase, which cleaves a dual-labeled probe upon hybridization to a complementary target sequence, and 2) fluorophore-based detection. The resulting fluorescent signal allows for quantitative measurement of the accumulation of amplification product during the exponential phase of amplification, and TAQMAN probes can significantly increase the specificity of detection.

[0170] Detecting nucleic acid amplification products can include the use of intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes exhibit enhanced fluorescence after binding to DNA or RNA). Dyes can include DNA or RNA intercalating fluorophores, including, but not limited to, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Detection of nucleic acid amplification products can also include the use of absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurements at 260 nm) to concentration. Direct measurements of nucleic acids can be converted to concentration using the Beer-Lambert law, which relates absorbance to concentration using the path length and extinction coefficient of the measurement. Detection of nucleic acid amplification products may involve the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and quantity of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample is ionized, and the resulting ions can be separated according to their mass-to-charge ratio in an electric and / or magnetic field, and the mass-to-charge ratio of the ions is measured by a detector. Mass spectrometry methods include, for example, MALDI, MALDI-TOF, and electrospray. These methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry methods (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can achieve high throughput due to fast signal acquisition from solid surfaces and automated analysis.

[0171] Detecting nucleic acid amplification products may involve the use of nucleic acid sequencing. The entire or partial sequence of the amplification product may be determined, and the determined nucleotide sequence may be referred to as a read. For example, linear amplification products may be directly analyzed without further amplification (e.g., by using single-molecule sequencing). In some embodiments, linear amplification products are subjected to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single-molecule sequencing, multiplex sequencing, solid-phase single-nucleotide sequencing, and nanopore sequencing. Detecting nucleic acid amplification products may involve the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm®).

[0172] Lysis buffer Soluble drugs As disclosed herein, the dissolving agent can include a detergent. The detergent can include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. Anionic surfactants have NH4 as a counterion. + , K. + , Na + , or Li + The cationic surfactant may contain I as a counter ion. - , Br - , or Cl - may also include:

[0173] The lysis agents provided herein may be capable of acting as denaturing agents. As used herein, "denaturing agent" or "denaturant" shall be given its ordinary meaning and include any compound or substance that will cause reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined by both the properties and concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturing agents include chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents can include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(dioleyoxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergents), 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). Organic, water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols, such as ethanol or isopropanol), or lower alkanediols (especially C2-C4 alkanediols, such as ethylene glycol) can be used as denaturing agents.The phospholipid may be a naturally occurring phospholipid such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or a synthetic phospholipid derivative or variant such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0174] Suitable surfactant levels may be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, it may be advantageous to use anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof).

[0175] Anionic surfactants useful herein include the water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl radical, and the water-soluble salts of sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant.

[0176] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having a single long alkyl chain containing about 8 to 18 carbon atoms, such as lauryltrimethylammonium chloride, cetylpyridinium chloride, benzalkonium chloride, cetyltrimethylammonium bromide, di-isobutylphenoxyethyl-dimethylbenzylammonium chloride, alkyltrimethylammonium nitrite of coconut, cetylpyridinium fluoride, etc. Certain cationic surfactants can also act as antiseptics in the compositions disclosed herein.

[0177] Suitable nonionic surfactants that can be used in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds produced by the condensation of an organic hydrophobic compound, which can be aliphatic and / or aromatic, with an alkylene oxide group (hydrophilic nature).Examples of suitable nonionic surfactants include: poloxamers; sorbitan derivatives such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of aliphatic alcohols or alkylphenols; condensation products of ethylene oxide with the reaction product of propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides; and mixtures of such substances.These substances are useful for stabilizing foam without contributing to excessive viscosity increase of consumer product compositions.

[0178] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, where the aliphatic radical may be straight or branched chain, and one of the aliphatic substituents contains about 8 to 18 carbon atoms and one contains an anionic water-solubilizing group, such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of anionic short-chain surfactants include alkyl sulfates, alkyl sulfonates, alkylbenzene sulfonates, saturated or unsaturated fatty acids, and their salts. The polar head group-containing moiety in cationic surfactants can include, for example, quaternary ammonium, pyridinium, sulfonium, and / or phosphonium groups. For example, the polar head group can include trimethylammonium. Exemplary cationic short-chain surfactants include alkyltrimethylammonium halides, alkyltrimethylammonium tosylates, and N-alkylpyridinium halides.

[0179] reducing agent The lysis buffer and / or reagent composition (e.g., dried composition) can include one or more reducing agents. A "reducing agent" can be a compound or group of compounds. As used herein, a "reducing agent," also known as a "reductant," "reducing agent," or "reducing equivalent," can refer to an element or compound that donates electrons to another chemical species. In particular, reducing agents are generally compounds that cleave disulfide bonds by reduction, thereby overcoming tertiary protein folding and quaternary protein structure (multimeric subunits) stabilized by disulfide bonds. Examples of suitable reducing agents include, but are not limited to, 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED. In some embodiments of the methods, compositions, and kits provided herein, the lysis buffer and / or reagent composition (e.g., the dried composition) does not include one or more reducing agents.

[0180] Reagent Composition The reagent compositions (e.g., dry compositions) described herein can be provided in a "dry form," or a form not suspended in a liquid medium. A "dry form" of a composition can include a dry powder, a lyophilized composition, a spray-dried composition, or a precipitated composition. A "dry form" composition can include one or more lyoprotectants, such as sugars and their corresponding sugar alcohols, e.g., sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, e.g., arginine or histidine; lyotropic salts, e.g., magnesium sulfate; polyols, e.g., propylene glycol, glycerol, poly(ethylene glycol), or polypropylene glycol; and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethylcellulose. As used herein, the terms "lyophilization," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or frozen solvent is removed by sublimation under reduced pressure. "Lyophile" refers to a lyphophilized material.

[0181] The reagent composition (e.g., a dried composition) may be frozen, lyophilized, or spray-dried. The reagent composition may be heat-dried. The reagent composition may include one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). The sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymers may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The lyophilized reagent may include poly rA, EGTA, EDTA, Tween 80, and / or Tween 20.

[0182] Frozen or lyophilized or spray-dried or heat-dried compositions, or aqueous compositions for preparing frozen or lyophilized or spray-dried compositions, may be prepared using the following: (i) non-aqueous solvents, such as ethylene glycol, glycerol, dimethyl sulfoxide, and dimethylformamide; (ii) surfactants, such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic acid, also known as poloxamer; The composition may include one or more of the following: F127 (polyoxyethylene-polyoxypropylene copolymer), poloxamine, and sodium dodecyl sulfate; (iii) dissacharides, such as trehalose, sucrose, lactose, and maltose; (iv) polymers (which may have different MW), such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, ficoll, and albumin; and (v) amino acids, such as one or more of glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and gamma-aminobutyric acid.

[0183] The reagent composition (e.g., a dried composition) can include one or more protecting agents and one or more amplification reagents. The one or more protecting agents can include a cyclodextrin compound. Cyclodextrins (CDs) can be used for complexation with soluble agents (e.g., SDS). Cyclodextrins (CDs) can be cyclic oligosaccharides resembling truncated cones with a hydrophobic interior cavity and a hydrophilic exterior surface. The most commonly used natural cyclodextrins contain 6, 7, and 8 glucose units, designated α-, β-, and γ-CD. Natural CDs can have solubility. Chemically modified CDs, such as hydroxypropyl derivatives, improve solubility in aqueous media by up to 50%. CAVASOL® is a trade name for cyclodextrin derivatives from WACKER, encompassing a variety of α-, β-, and γ-CD derivatives. β-CD can form a strong inclusion complex (stronger than α-CD and β-CD) with sodium dodecyl sulfate (SDS) in a predominantly 1:1 stoichiometry. The binding constant of β-CD to SDS is 2100 M -1 ~2500M -1 The range can be as follows:

[0184] kit In some embodiments, a kit for detecting a target nucleic acid sequence in a sample is provided. In some embodiments, the kit comprises a signal-generating oligonucleotide disclosed herein. The kit can include a lysis buffer containing one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence, and optionally, the one or more lysis agents include a detergent, and the detergent can include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. 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, the one or more components for amplification comprising: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophile polymerase activity capable of producing a nucleic acid amplification product. In some embodiments, the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer.

[0185] The kit may include at least one component that provides real-time detection activity for nucleic acid amplification products. The real-time detection activity may be provided by a molecular beacon. The real-time detection activity may be provided by a signal-generating oligonucleotide provided herein. The reagent composition (e.g., a dried composition) may include a reverse transcriptase and / or a reverse transcription primer. The molar ratio of the one or more protectants to the one or more amplification reagents can be between about 10:1 and about 1:10 (e.g., about 2:1). In some embodiments, the one or more additives include Tween 20, Triton X-100, Tween 80, a non-ionic detergent (e.g., a non-ionic surfactant), or any combination thereof. In some embodiments, the one or more protectants include a cyclodextrin compound. In some embodiments, the one or more solubility reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) (e.g., about 0.2% (wt / vol)) of the processed sample. In some embodiments, the one or more solubility agents include a detergent. The detergent can include one or more of a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and an amphoteric surfactant. In some embodiments, it may be advantageous for one or more protecting agents to sequester one or more solubility agents, thereby preventing the one or more solubility agents from denaturing one or more amplification reagents.

[0186] The kit may include, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, as described herein. If one target is being amplified, a pair of primers (forward and reverse) may be included in the kit. If multiple target sequences are being amplified, multiple primer pairs may be included in the kit. The kit may include a control polynucleotide, and if multiple target sequences are being amplified, multiple control polynucleotides may be included in the kit. An enzyme with hyperthermophile polymerase activity can have an amino acid sequence that is at least about 90% or 95% identical to the amino acid sequence of SEQ ID NO: 31, or a functional fragment thereof. For example, an enzyme with hyperthermophile polymerase activity can comprise the amino acid sequence of SEQ ID NO: 31.

[0187] The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may include (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length. The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoan.

[0188] The kit may also contain one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates, and the like, or the components may be combined in various combinations in such containers. The components of the kit may, for example, be present in one or more containers. In some embodiments, all of the components are provided in a single container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a container separate from the primers. The components may, for example, be lyophilized, heat-dried, freeze-dried, or present in a stable buffer. In some embodiments, the polymerase and / or reverse transcriptase are present in a single container in lyophilized or heat-dried form, and the primers are either lyophilized, heat-dried, freeze-dried, or present in a buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase and primers are present in a single container in lyophilized or heat-dried form.

[0189] The kit may further include, for example, dNTPs or modified nucleotides used in the reaction, vessels, cuvettes, or other containers used for the reaction, or vials of water or buffer for rehydrating lyophilized or heat-dried components. The buffer used may be, for example, suitable for both polymerase activity and primer annealing activity. The kits may also include instructions for practicing one or more of the methods described herein and / or instructions for one or more of the components described herein. The instructions and / or instructions may be in printed form or may be included in the kit insert. The kits may also include a written description of an internet location that provides such instructions or instructions. The kits may further include reagents used in detection methods, such as reagents used for FRET, lateral flow devices, dipsticks, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. [Example]

[0190] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Example 1 Molecular beacon characterization experiments In this example, beacon performance was first evaluated in MB characterization experiments. 500 nM synthetic target was combined with 50 nM MB + / - 9°N. Figure 7 depicts a non-limiting, exemplary schematic of the signal-generating oligonucleotides provided herein. Figures 8A-8B depict non-limiting, exemplary data related to MB characterization. Vertical lines in the graphs indicate assay temperatures. Differences were observed between signal conditions with and without extension, and it was found that the 9°N distortion of the MB caused an elevated baseline. Experimental Tm can be assessed by melting curve analysis.

[0191] ( Example 2 ) Comparison of Neisseria gonorrhoeae assay designs This example provides a comparison of the old Neisseria gonorrhoeae assay with clean primers containing a 6-spacer versus the new N. gonorrhoeae assay with clean primers containing a 4-spacer (Table 3). Figures 13A-13B depict data on the performance of the old (Figure 13A) and new (Figure 13B) Neisseria gonorrhoeae assays. The product size and reverse primer size are the same. Changing the forward primer from an 11-mer to a 13-mer improved assay performance, with speeds increasing by over 1 minute. Therefore, primer Tm should be considered in APA assay design.

[0192] [Table 3]

[0193] ( Example 3 ) Effect of primer length on APA assay This example examines the effect of primer length on APA assay performance. Table 4 provides various APA assay designs. IDT OligoAnalyzer settings were as follows: [Oligo], 0.5 uM; [Na+], 20 mM; [Mg ++ [dNTP], 4 mM; [dNTP], 2 mM. Figure 14 depicts data relating to the effect of primer length on APA assay performance. It was found that longer primers tend to produce weaker amplification. Weaker amplification was found for longer primers when product Tm >> assay Tm (thus, in some embodiments of the methods and compositions provided herein, the APA assay Tm is equal to or approximately equal to the product Tm). Without intending to be bound by any particular theory, this may be due to the weak strand displacement activity of 9°N.

[0194] [Table 4]

[0195] ( Example 4 ) Case Study - Flu A PB2.2 Assay Figure 17 depicts a non-limiting exemplary diagram for Flu A APA assay design. In some embodiments, primers are positioned to avoid missing mismatch variant amplification. In some embodiments, the 3' end of the primer is positioned within the first 3-4 nt relative to the minimal mismatch variant. In some embodiments, primers are positioned relative to the spacer containing minimal mismatches. In some embodiments, no more than one mismatched base in the spacer region is tolerated (for fewer than three probes total). Mismatch variants m3 and m4 may be detectable using a probe for P1. In some embodiments, a conserved sequence immediately 5' upstream for the RT primer is used (not shown).

[0196] ( Example 5 ) Detection of Chlamydia trachomatis gDNA by conventional molecular beacons in APA This example examines the performance of C. trachomatis DNA detection in APA reactions containing molecular beacons and Syto 61 fluorescent dye. Figure 20 depicts a non-limiting exemplary conventional molecular beacon for the detection of C. trachomatis gDNA in an APA reaction. The molecular beacon is labeled with HEX at the 5' end and an IBFQ quencher at the 3' end. Figures 19A-19B depict data for the detection of C. trachomatis gDNA in the HEX (Figure 19A) and cy5 (Figure 19B) channels in an APA reaction with a conventional molecular beacon. Figures 19A-19B depict data for real-time detection of four replicates of a no-target control (NTC, dotted curve) and 500 copies of Ct gDNA (solid curve) in a wet APA reaction. These results demonstrate that conventional molecular beacons are unable to detect amplification products in real time, even though fluorescent dye detection indicates strong target amplification (solid curve in the fluorescent cy5 channel).

[0197] ( Example 6 ) Detection of extended APA amplicons with asymmetric hairpin probes This example examines the performance of synthetic DNA target detection in APA reactions containing molecular beacons and Syto 61 fluorescent dye. Figure 21 depicts a non-limiting exemplary asymmetric hairpin probe provided herein, and Table 5 provides the sequence of the assay components. Figures 22A-22C depict data for synthetic DNA target detection in APA reactions using an asymmetric hairpin probe (Figure 22A), followed by melting curve analysis (Figure 22B) and melting derivative evaluation (Figure 22C). Figures 22A-22C show the results of synthetic DNA target detection using an asymmetric hairpin probe. At 68°C, the hairpin probe is unable to detect the synthetic oligo target at 150 nM (green and red curves). In the presence of 9 nM, extension of the synthetic target results in stable hybrids and an increase in fluorescent signal in real time. Melting curves show that the product does not form a stable hybrid (Tm=60°C) at the assay temperature, but extension of the target on the hairpin probe increases the Tm of the hybrid to 80°C.

[0198] [Table 5]

[0199] ( Example 7 ) Detection of Flu A virus using asymmetric hairpin probes This example examines the performance of an APA assay to detect Flu A virus, Solomon Islands strain, with a hairpin probe containing a 3-base-pair stem and a 5'-end non-target overhang (lowercase letters represent artificial bases, while uppercase letters represent the target sequence) (Table 6). Figure 21 depicts a non-limiting exemplary asymmetric hairpin probe provided herein. The assay was designed to generate a 23-base DNA product containing a 4-base spacer. Product P2 forms a 15-base hybrid with the hairpin probe. The calculated Tm (IDT Oligo analyzer) of the product under assay salt conditions is 67.6°C, and the Tm for the probe / product hybrid is 60.7°C. Figure 23 depicts data related to a limit of detection (LOD) study using the hairpin probe provided herein for Flu A virus detection. The experiment was performed using an APA "hot start" approach, in which both the sample and the lyophilized mixture were preheated to 63°C and then combined. The reaction proceeded for 10 minutes at 67° C. in a BioRad CFx thermal cycler. These results demonstrate that the hairpin probes provided herein (e.g., signaling oligonucleotides containing a 5′-terminal domain) can be used for real-time, sensitive detection of short amplicons.

[0200] [Table 6]

[0201] ( Example 8 ) SARS-CoV-2 virus detection This example examines the performance of an APA assay involving real-time detection of SARS-CoV-2 virus with both a hairpin probe and the fluorescent DNA dye Syto 61 in a reaction. The assay was designed to generate a 28-base DNA product containing a 4-base spacer. Figure 25 depicts non-limiting exemplary signal-generating oligonucleotides provided herein, and Table 7 provides the sequences of the assay components. This study was performed using an APA "hot start" approach, in which both the sample and the lyophilized mixture were preheated to 63°C, then combined and run for 10 minutes at 67°C in a CFx thermal cycler. Figures 24A-24D depict data related to real-time detection (Figures 24A-24B) and melting curve evaluation (Figures 24C-24D) of SARS-CoV-2 virus with both a hairpin probe (Figures 24A, 24C) and the fluorescent DNA dye Syto 61 (Figures 24B, 24D) in a reaction. The results showed that the 28-base assay product had a melting temperature of 67°C and did not form a stable hybrid with the hairpin probe. Extension of the product onto the hairpin probe generated a robust fluorescent signal with a melting temperature of 76°C.

[0202] [Table 7]

[0203] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0204] With respect to the use of substantially any plural and / or singular terminology herein, those of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless stated otherwise.

[0205] Those skilled in the art will understand that the terms used in this specification, generally, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Furthermore, those skilled in the art will understand that where a specific number is intended in an introduced claim recitation, such intention will be expressly recited in the claim; otherwise, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means limiting any particular claim containing such an introduced claim recitation to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Additionally, even if a particular number is explicitly recited in an introduced claim recitation, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a base recitation of "two recitations" means at least two recitations, or two or more recitations, in the absence of other modifiers).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that a person of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, a system having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that a person of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, those skilled in the art will appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either of those terms, or both terms.

[0206] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is also thereby described in terms of every individual member or subgroup of members of the Markush group.

[0207] As one of ordinary skill in the art would understand, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as being capable of dividing that same range into at least two, three, four, five, ten, etc., as fully described. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Furthermore, as one of ordinary skill in the art would understand, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the recited numerical values ​​and that can be subsequently divided into subranges as discussed above. Finally, as one of ordinary skill in the art would understand, a range includes each individual member. Thus, for example, a group containing 1 to 3 items refers to groups containing 1, 2, or 3 items. Similarly, a group containing 1 to 5 items refers to groups containing 1, 2, 3, 4, or 5 items, etc.

[0208] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not limitation, with the true scope and spirit being indicated by the following claims.

Claims

1. amplifying the target nucleic acid sequence in an amplification reaction mixture, thereby producing a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, the signal-generating oligonucleotide being capable of hybridizing to the nucleic acid amplification product; 1. A method for detecting a target nucleic acid sequence in a sample, comprising: the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain; the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain; Intramolecular nucleotide base pairing between the 5' and 3' subdomains is possible to form a paired stem domain; at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to a nucleic acid amplification product; the signal-generating oligonucleotide comprises a 5' terminal domain that is about 1 nt to about 6 nt in length and is located 5' to the 5' subdomain; and the 5'-terminal domain is unable to hybridize to the 3'-end of the nucleic acid amplification product; Optionally, the method wherein the signal-generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

2. amplifying the target nucleic acid sequence in an amplification reaction mixture, thereby producing a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, the signal-generating oligonucleotide being capable of hybridizing to the nucleic acid amplification product; 1. A method for detecting a target nucleic acid sequence in a sample, comprising: the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain; the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain; Intramolecular nucleotide base pairing between the 5' and 3' subdomains is possible to form a paired stem domain; At least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to a nucleic acid amplification product; and the signal-generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain; Optionally, the signal-generating oligonucleotide comprises a 5' terminal domain about 1 nt to about 15 nt in length and located 5' to the 5' subdomain; and The method, wherein the 5' terminal domain is incapable of hybridizing to the 3' end of a nucleic acid amplification product.

3. (a) the one or more LNA nucleotides increase the melting temperature (Tm) of the signal-generating oligonucleotide by about 3°C ​​to about 20°C; (b) the signal-generating oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides; (c) the loop domain comprises one or more LNA nucleotides, optionally wherein the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for nucleic acid amplification products, and further optionally, wherein the enhancing specificity of the signal-generating oligonucleotide for nucleic acid amplification products comprises enhanced mismatch discrimination between the nucleic acid amplification products and mismatch products, and optionally, the mismatch products comprise non-template control products and / or non-target genotypes; (d) the terminal 3' nucleotide of the signal-generating oligonucleotide is an LNA nucleotide, and optionally, said LNA nucleotide reduces or prevents digestion of the signal-generating oligonucleotide and / or removal of a quencher associated with the 3' end of the signal-generating oligonucleotide, and optionally digestion by the exonuclease activity of a polymerase; (e) the 5′ subdomain and / or the 3′ subdomain comprises one or more LNA nucleotides, optionally wherein the one or more LNA nucleotides enhance the stability of the paired stem domain, and further optionally wherein the paired stem domain comprises at least one base pairing of opposing LNA nucleotides; (f) the nucleotides located in the 5′-terminal domain are unable to form intramolecular nucleotide base pairs, and / or the 5′-terminal domain has less than about 5 nt, less than 4 nt, less than 3 nt, less than 2 nt, or less than 1 nt complementary to the 3′-end of the nucleic acid amplification product; and / or (g) the signal-generating nucleotide does not include a nucleotide located 3' to the 3' subdomain; 3. The method according to claim 1 or 2.

4. The method of any one of claims 1 to 3, wherein the signal-generating oligonucleotide comprises a label, optionally wherein the label comprises a quenchable label, and further optionally wherein the quenchable label is a fluorophore.

5. The signal-generating oligonucleotide comprises a quencher, and optionally a label associated with the 3' end of the signal-generating oligonucleotide and a quencher associated with the 5' end of the signal-generating oligonucleotide; or a label associated with the 5' end of the signal-generating oligonucleotide and a quencher associated with the 3' end of the signal-generating oligonucleotide; The method according to any one of claims 1 to 4.

6. the quencher is capable of quenching the signal generated by the label when the quencher and label are in close proximity; and / or When the quencher and label are not in close proximity, the quencher is unable to quench the signal generated by the label; The method according to any one of claims 1 to 5.

7. When the quencher and label are in close proximity, the signal generated by the label is not detectable; and / or When the quencher and label are not in close proximity, the signal generated by the label is detectable. The method according to any one of claims 1 to 6.

8. 8. The method of any one of claims 1 to 7, wherein the quencher and label are in close proximity when intramolecular nucleotide base pairing between the 5' and 3' subdomains forms a paired stem domain.

9. The method of any one of claims 1 to 8, wherein the quencher and label are not in close proximity when the signal-generating oligonucleotide does not comprise a paired stem domain.

10. the detecting step comprises contacting the nucleic acid amplification product with a signal-generating oligonucleotide for hybridization; Detecting the nucleic acid amplification product comprises using a real-time detection method; the detecting step includes detecting the signal of the label before, during, or after the amplification reaction, or any combination thereof; Detecting the nucleic acid amplification product comprises detecting a signal generated by a label of the signal-generating oligonucleotide, optionally wherein the label is a fluorophore and the signal is fluorescent; and / or detecting a signal comprises detecting fluorescence emitted by the label; The method according to any one of claims 1 to 9.

11. The amplification reaction and / or detecting step comprises: contacting the nucleic acid amplification product with a signal-generating oligonucleotide for hybridization; and extending the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity, thereby producing an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide, optionally wherein the extended nucleic acid amplification product comprises a complement of the 5'-terminal domain. The method according to any one of claims 1 to 10, comprising:

12. 12. The method of any one of claims 1 to 11, wherein extension of a nucleic acid amplification product hybridized to a signal-generating oligonucleotide with an enzyme having polymerase activity disrupts intramolecular nucleotide base pairing between the 5' and 3' subdomains, thereby allowing the paired stem domain to unwind.

13. The sign, (i) when the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or (ii) the nucleic acid amplification product is extended to produce an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide; capable of producing a detectable signal; Optionally, the signal is fluorescent. The method according to any one of claims 1 to 12.

14. (i) when the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or (ii) the nucleic acid amplification product is extended to produce an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide; the label produces a detectable signal, and optionally the signal is fluorescent; The method according to any one of claims 1 to 13.

15. amplifying the target nucleic acid sequence in the amplification reaction mixture comprises amplifying the target nucleic acid sequence under isothermal amplification conditions, optionally the isothermal amplification conditions comprise a constant temperature of about 30°C to about 72°C, further optionally about 55°C to about 75°C, optionally about 56°C to about 68°C, further optionally about 66°C to about 68°C; the amplifying step is carried out at the temperature optimum of the enzyme having hyperthermophilic polymerase activity, optionally said temperature optimum is about 66°C to about 68°C, and further optionally the amplifying step is carried out at a constant temperature; the nucleic acid amplification products have melting temperatures within at least about 5°C of a given temperature; and / or the melting temperature (Tm) of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex is higher than the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex, optionally at least about 5°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about 20°C higher; The method according to any one of claims 1 to 14.

16. the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex is at most about 60°C; and the Tm of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex is at least about 68°C; The method according to any one of claims 1 to 15.

17. The method of any one of claims 1 to 16, wherein the nucleic acid amplification product is unable to form a stable duplex with the signal-generating oligonucleotide in the absence of extension of the nucleic acid amplification product.

18. The method according to any one of claims 1 to 17, wherein the amplification reaction comprises the following steps: contacting the mismatch product with a signal-generating oligonucleotide for hybridization; and Extending the mismatch product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity, thereby producing an extended mismatch product hybridized to the signal-generating oligonucleotide, optionally wherein the extended mismatch product comprises a complement of the 5'-terminal domain, and further optionally wherein the mismatch product is a non-template control product and / or a non-target genotype.

19. the Tm of the mismatch product / signal-generating oligonucleotide duplex is about 50°C; and the Tm of the extended mismatch product / signal-generating oligonucleotide duplex is at least 5°C lower than a constant temperature, optionally less than about 68°C; The method according to any one of claims 1 to 18.

20. 20. The method of any one of claims 1 to 19, wherein the nucleic acid amplification product and the mismatch product differ in sequence by at least about 1 nt, 2 nt, 3 nt, 4 nt, or 5 nt.

21. The signal generating oligonucleotide is The paired stem domain is stable at a constant temperature in the absence of nucleic acid amplification products, and The paired stem domains are capable of dissociating when a nucleic acid amplification product hybridizes to the loop domain. Optionally, by modifying the length of the paired domain, the GC content of the paired domain, and / or the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. The method of any one of claims 1 to 20, wherein

22. The method of any one of claims 1 to 21, wherein the nucleic acid amplification product comprises: (1) the sequence of the forward primer and its reverse complement; (2) the sequence of the reverse primer and its reverse complement; and (3) A spacer sequence flanked by (1) the sequence of a forward primer and its reverse complement and (2) the sequence of a reverse primer and its reverse complement, optionally wherein the spacer sequence is about 4 nt to about 7 nt in length and / or has a GC content of less than about 50%.

23. (a) the signal-generating oligonucleotide comprises a first region comprising at least a portion of the sequence of a reverse primer, the signal-generating oligonucleotide comprises a second region comprising a sequence complementary to at least a portion of the forward primer, and / or the signal-generating oligonucleotide comprises a spacer region comprising at least a portion of the sequence of a spacer sequence; (b) the first region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer, the second region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer, and / or the spacer region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer; and / or (c) the first region comprises at least a portion of the 5' subdomain and / or the loop domain, the spacer region comprises at least a portion of the loop domain, and the second region comprises at least a portion of the loop domain and / or the 3' subdomain; The method according to any one of claims 1 to 22.

24. (a) (i) the signal-generating oligonucleotide is from about 10 nt to about 100 nt in length, (ii) the second region, the spacer region, and / or the first region is from about 1 nt to about 25 nt in length, and / or (iii) the 5' subdomain, the 3' subdomain, the loop domain, and / or the 5' terminal domain is from about 1 nt to about 25 nt in length; (b) the 5'-terminal domain is about 1 nt to about 6 nt in length, the loop domain is about 4 nt to about 15 nt in length, and the paired stem domain is about 3 bp to about 8 bp in length; (c) the nucleic acid amplification product is about 25 nt to about 35 nt in length; (d) the target nucleic acid sequence comprises a length of about 20 nt or less to about 90 nt or less, and optionally, the target nucleic acid sequence comprises a length of about 30 nt; and / or (e) the spacer sequence comprises a portion of the target nucleic acid sequence, and optionally the spacer sequence is 1-10 bases in length, and optionally the spacer sequence is about 4 nt to about 7 nt in length and / or has a GC content of less than about 50%; The method according to any one of claims 1 to 23.

25. The method according to any one of claims 1 to 24, wherein the sample nucleic acid comprises a nucleic acid comprising a target nucleic acid sequence.

26. The step of amplifying the target nucleic acid sequence comprises: The method includes a step of amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplifying step comprises amplifying a nucleic acid comprising the target nucleic acid sequence by: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and ii) Enzymes with hyperthermophilic polymerase activity 26. The method of any one of claims 1 to 25, comprising contacting a nucleic acid amplification product with

27. the nucleic acid is double-stranded DNA; the nucleic acid is a product of a reverse transcription reaction, optionally the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid, and further optionally the amplifying step comprises generating the nucleic acid by a reverse transcription reaction; and / or the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA, optionally wherein the reverse transcription primer has the same 3' end as the forward primer; The method according to any one of claims 1 to 26.

28. The step of amplifying the target nucleic acid sequence comprises: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying step comprises: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophilic polymerase activity to thereby produce a nucleic acid amplification product. The method of any one of claims 1 to 27, comprising:

29. The forward primer and / or the reverse primer are configured to have a Tm of less than about 45°C; between about 5 nt and about 25 nt in length, optionally between about 10 nt and about 14 nt in length; configured to produce nucleic acid amplification products that are about 25 nt to about 35 nt in length and have a melting temperature that is within at least about 5°C of a certain temperature; containing one or more phosphorothioate linkages; and / or having a GC content of about 30% to about 55%; 29. The method according to any one of claims 1 to 28.

30. the 3' region of the forward primer and / or the reverse primer does not contain a thymine base, and optionally the 3' region contains the first, second, third, and / or fourth nucleotide from the 3' end; the 5' region of the forward primer and / or reverse primer does not comprise more than 3 nt complementary to the spacer sequence, the region adjacent thereto, its complement, or any combination thereof, and optionally the 5' region comprises the first, second, third, and / or fourth nucleotide from the 5' end; the forward primer and / or reverse primer comprises a phosphorothioate bond between the first and second nucleotides from the 3' end of the forward primer and / or reverse primer, optionally, the phosphorothioate bond is capable of reducing or preventing polymerase-mediated degradation; the forward primer and / or reverse primer contains a phosphorothioate bond between the second and third nucleotides from the 3' end of the forward primer and / or reverse primer; the 3' region of the forward primer and / or the reverse primer does not contain more than two phosphorothioate linkages, and optionally the 3' region includes the first, second, third, and / or fourth nucleotide from the 3' end; the forward primer and / or the reverse primer comprises one or more phosphorothioate linkages in the region comprising the GC dinucleotide repeat, optionally the one or more phosphorothioate linkages are capable of destabilizing base pairing; the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain improves the sensitivity and / or specificity of detecting nucleic acid amplification products by at least about 1.1 fold compared to a comparable method in which the signal-generating oligonucleotide does not contain any LNA nucleotides; and / or the presence of the 5' terminal domain in the signal-generating oligonucleotide improves the sensitivity and / or specificity of detecting nucleic acid amplification products by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide comprises a blunt-ended hairpin structure; 30. The method according to any one of claims 1 to 29.

31. The method includes determining the presence, absence, and / or amount of a target nucleic acid sequence in the sample; determining the presence, absence, and / or amount of a target nucleic acid sequence in the sample comprises determining the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample; the presence, absence, and / or amount of the detected signal indicates the presence, absence, and / or amount of the target nucleic acid sequence in the sample; and / or the presence, absence, and / or amount of the detected signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample; The method according to any one of claims 1 to 30.

32. the signal-generating oligonucleotide comprises one or more polymerase stoppers and / or one or more phosphorothioate linkages, and optionally the first region, the second region, and / or the spacer region comprises one or more polymerase stoppers; The method according to any one of claims 1 to 31.

33. (a) one or more polymerase stoppers are located in the loop domain, the first region, the second region, and / or the spacer region, and optionally, the 5′ subdomain, the paired stem domain, and / or the 3′ subdomain do not comprise one or more polymerase stoppers; (b) one or more polymerase stoppers comprise one or more 2'-O-methyl (2'OM) RNA nucleotides; (c) the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemical capture abasic site, or any combination thereof; (d) (i) the chemically trapped abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride, (ii) the abasic site comprises an apurinic base site, an apyrimidinic base site, or both, and / or (iii) the abasic site is generated by an alkylating agent or an oxidizing agent; (e) the one or more polymerase stoppers are one or more RNA bases, one or more 2' methoxyethyl ribose (MOE), one or more locked nucleic acid (LNA) nucleotides, one or more 2' fluoro bases, one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more is one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3 (OC 3 H 6 OPO 3 ) group, one or more photocleavable (PC) [OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(CH 3 ) OPO 3 ] group, one or more hexanediol groups, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, one or more spacers 18 (iSp18) [(OCH 2 CH 26 OPO 3 ] group, or any combination thereof; (f) one or more polymerase stoppers comprise one or more steric blocking groups, optionally wherein said one or more steric blocking groups increase the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex; and / or (g) the polymerase stopper comprises a modification incorporated between two bases of the signal-generating oligonucleotide; 33. The method according to any one of claims 1 to 32.

34. The modification is (i) a naphthylene-azo compound, optionally Zen or iFQ; (ii) a molecule having the following structure: 【Chemistry 1】 wherein the linking group L positions the modification at an internal position of the signal-generating oligonucleotide. 1 and L 2 are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R 1 ~R 5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawing group, an electron donating group, or a point of attachment to a ligand; and X is a nitrogen atom or a carbon atom, with the proviso that when X is a carbon atom, the fourth substituent attached to the carbon atom is hydrogen or C 1 -C 8 (which may be an alkyl group) having (iii) a compound having the following structure: 【Chemistry 2】 wherein the linking group L positions the modification at an internal position of the signal-generating oligonucleotide. 1 and L 2 are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R 1 , R 2 , R 4 , R 5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron-withdrawing group, or an electron-donating group; R 6 , R 7 , R 9 ~R 12 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron-withdrawing group, or an electron-donating group; R 8 is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, or an electron-withdrawing group; and X is a nitrogen atom or a carbon atom, with the proviso that when X is a carbon atom, the fourth substituent attached to the carbon atom is hydrogen or C 1 -C 8 (which may be an alkyl group) and / or (iv) a molecule having the following structure: 【Transformation 3】 and Optionally, R 8 No 2 That is, 34. The method according to any one of claims 1 to 33.

35. when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide; Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

35. The method according to any one of claims 1 to 34.

36. when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, the one or more polymerase stoppers can terminate polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide; Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second, undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

36. The method according to any one of claims 1 to 35.

37. when the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers can terminate polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide; Optionally, the one or more polymerase stoppers can terminate polymerase extension of the third undesired duplex exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide; Optionally, the exogenous nucleic acid is selected from the group consisting of a sample nucleic acid, a primer configured to hybridize to a second target nucleic acid sequence, a primer configured to hybridize to an internal control, and any combination thereof.

37. The method according to any one of claims 1 to 36.

38. if the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity produces a first undesired extension product; the first undesired extension product can be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a forward primer and a reverse primer to form a first undesired amplification product; the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex to generate a first extension stall product; 38. The method of any one of claims 1 to 37, wherein the first extension stalled product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a forward primer and a reverse primer to produce a first undesired amplification product.

39. 39. The method of any one of claims 1 to 38, wherein the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

40. If the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity produces a second undesired extension product; The second undesired extension product can be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to form a second undesired amplification product; the one or more polymerase stoppers can terminate polymerase extension of the reverse primer of the second, undesired duplex to generate a second extension stall product; 40. The method of any one of claims 1 to 39, wherein the second extension stalled product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to produce a second undesired amplification product.

41. 41. The method of any one of claims 1 to 40, wherein the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second, undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

42. if the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity produces a third undesired extension product; the third undesired extension product can be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to form a third undesired amplification product; the one or more polymerase stoppers can terminate polymerase extension of a third undesired duplex of exogenous nucleic acid to generate a third extension stall product; 42. The method of any one of claims 1 to 41, wherein the third extension stalled product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to produce a third undesired amplification product.

43. 43. The method of any one of claims 1 to 42, wherein the one or more polymerase stoppers are capable of terminating polymerase extension of the third undesired duplex exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

44. the label is capable of generating a false positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product, and optionally the signal and the false positive signal are indistinguishable; and / or 44. The method of any one of claims 1 to 43, wherein the label generates a false positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product, and optionally the signal and the false positive signal are indistinguishable.

45. the generation of the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample; and / or 45. The method of any one of claims 1 to 44, wherein detection of a false positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample.

46. 46. ​​The method of any one of claims 1 to 45, wherein the presence of one or more polymerase stoppers in the signal-generating oligonucleotide increases the likelihood of accurately determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample by at least about 1.1 fold compared to a signal-generating oligonucleotide that does not include one or more polymerase stoppers.

47. the production of the first elongation stall product, the second elongation stall product, and / or the third elongation stall product does not result in a false positive signal; and / or 47. The method of any one of claims 1 to 46, wherein the signal-generating oligonucleotide hybridized to the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal.

48. the nucleic acid amplification product reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reach a detectable level; and / or 48. The method of any one of claims 1 to 47, wherein the signal reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before a false positive signal reaches a detectable level.

49. the appearance of detectable levels of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product is delayed by at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers; the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach detectable levels for at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the amplifying step begins; and / or the occurrence of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product is reduced by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers; 49. The method of any one of claims 1 to 48.

50. 50. The method of any one of claims 1 to 49, wherein the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide.

51. contacting a sample containing biological entities with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence; and contacting a reagent composition with the processed sample to produce an amplification reaction mixture, the reagent composition comprising one or more amplification reagents; 51. The method of any one of claims 1 to 50, comprising:

52. amplifying the target nucleic acid sequence comprises producing a detectable level of nucleic acid amplification product within about 20 minutes, within about 15 minutes, or within about 10 minutes; and / or the detecting step is performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the processed sample; 52. The method of any one of claims 1 to 51.

53. the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA; and / or 53. The method of any one of claims 1 to 52, wherein the pH of the lysis buffer is from about 1.0 to about 10.0, and optionally, the pH of the lysis buffer is about 2.

2.

54. 54. The method of any one of claims 1 to 53, wherein 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.

55. The one or more amplification reagents include: reverse transcriptase; an enzyme having hyperthermophilic polymerase activity, optionally having reverse transcriptase activity; Forward primer; reverse primer; a reverse transcription primer; and / or dNTP 55. The method of any one of claims 1 to 54, comprising:

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

57. The one or more lytic reagents include: about 0.001% (wt / vol) to about 1.0% (wt / vol) of the treated sample, optionally about 0.2% (wt / vol) of the 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; 57. The method of any one of claims 1 to 56, comprising:

58. carried out in a single reaction vessel; does not include a step of using any enzyme other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include a step of using any enzyme other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or 58. The method of any one of claims 1 to 57, which does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein.

59. The amplifying step comprises: carried out for about 5 minutes to about 60 minutes, optionally for about 15 minutes; and / or performed under helicase-free, single-strand binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions; 59. The method of any one of claims 1 to 58.

60. (a) the amplifying step is carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA), and optionally, the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); (b) the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 31 or a functional fragment thereof, optionally, the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO: 31, further optionally, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 31, optionally, the enzyme with hyperthermophilic polymerase activity has reduced or no exonuclease activity; and / or (c) simultaneously contacting the sample ribonucleic acid with a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity, optionally simultaneously contacting the sample ribonucleic acid with a reverse transcriptase, an enzyme having hyperthermophilic polymerase activity, a forward primer, and a reverse primer, and further optionally simultaneously contacting the sample ribonucleic acid with a reverse transcriptase, an enzyme having hyperthermophilic polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer; 60. The method of any one of claims 1 to 59.

61. the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle; The biological entity includes a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof; and / or the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a viral, bacterial, fungal, or protozoan; 61. The method of any one of claims 1 to 60.

62. 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, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, 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, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae pneumonia, S. agalactiae, and Listeria monocytogenes; the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or protozoa including one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; 62. The method of any one of claims 1 to 61.

63. the sample is a biological sample or an environmental sample; The environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof; and / or 63. The method of any one of claims 1 to 62, wherein the biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, stool, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

64. (a) the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from said two or more target nucleic acid sequences, optionally the two or more target nucleic acid sequences are specific to two or more different organisms, and further optionally the two or more different organisms comprise one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C; (b) the amplifying step does not include one or more of Archaeal Polymerase Amplification (APA), Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), Transcription-Mediated Amplification (TMA), Nicking Enzyme Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), Ramification (RAM), Circular Helicase-Dependent Amplification (cHDA), Single Primer Isothermal Amplification (SPIA), Signal-Mediated RNA Amplification Technology (SMART), Self-Sustained Sequence Replication (3SR), Genomic Exponential Amplification Reaction (GEAR), and Isothermal Multiple Displacement Amplification (IMDA), optionally wherein the amplifying step does not include LAMP; (c) the amplifying step comprises one or more of APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA, optionally, the amplifying step does not comprise LAMP; and / or (d) the method does not include one or more of: (i) diluting the processed sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the processed sample; (iv) sonicating the processed sample; (v) sonicating the amplification reaction mixture; (vi) adding an RNase inhibitor to the processed sample; (vii) adding an RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lytic agents from the processed sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the processed sample or amplification reaction mixture.

64. The method of any one of claims 1 to 63.

65. a signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product; the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain; the signal-generating oligonucleotide comprises a loop domain located between the 5' and 3' subdomains; Intramolecular nucleotide base pairing between the 5' and 3' subdomains is possible to form a paired stem domain; at least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to a nucleic acid amplification product; the signal-generating oligonucleotide comprises a 5' terminal domain that is about 1 nt to about 6 nt in length and is located 5' to the 5' subdomain; and The 5'-terminal domain is incapable of hybridizing to the 3'-end of the nucleic acid amplification product; Signal-generating oligonucleotides.

66. 66. The signaling oligonucleotide of claim 65, comprising one or more locked nucleic acid (LNA) nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

67. a signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product; the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain; the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain; Intramolecular nucleotide base pairing between the 5' and 3' subdomains is possible to form a paired stem domain; At least a portion of the 5' subdomain and at least a portion of the loop domain are capable of hybridizing to a nucleic acid amplification product; and the signal-generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain; Signal-generating oligonucleotides.

68. the signal-generating oligonucleotide comprises a 5' terminal domain that is about 1 nt to about 15 nt in length and is located 5' to the 5' subdomain; and / or The 5'-terminal domain is incapable of hybridizing to the 3'-end of the nucleic acid amplification product; 68. The signal-generating oligonucleotide of claim 67.

69. (i) the one or more LNA nucleotides increase the melting temperature (Tm) of the signal-generating oligonucleotide by about 3°C ​​to about 20°C; (ii) the signal-generating oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides; (iii) the loop domain comprises one or more LNA nucleotides, optionally wherein the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for nucleic acid amplification products, and further optionally, wherein the enhancing specificity of the signal-generating oligonucleotide for nucleic acid amplification products comprises enhanced mismatch discrimination between the nucleic acid amplification products and mismatch products, and optionally, the mismatch products comprise non-template control products and / or non-target genotypes; (iv) the terminal 3′ nucleotide of the signal-generating oligonucleotide is an LNA nucleotide, and optionally said LNA nucleotide reduces or prevents digestion of the signal-generating oligonucleotide and / or removal of a quencher associated with the 3′ end of the signal-generating oligonucleotide, and optionally digestion by the exonuclease activity of a polymerase; (v) the 5′ subdomain and / or the 3′ subdomain comprises one or more LNA nucleotides, optionally wherein said one or more LNA nucleotides enhance the stability of the paired stem domain, and further optionally wherein the paired stem domain comprises at least one base pairing of opposing LNA nucleotides; (vi) the nucleotides located in the 5′-terminal domain are unable to form intramolecular nucleotide base pairs, and / or the 5′-terminal domain has less than about 5 nt, less than 4 nt, less than 3 nt, less than 2 nt, or less than 1 nt complementary to the 3′-end of the nucleic acid amplification product; and / or (vii) the signal-generating nucleotide does not include a nucleotide located 3' to the 3' subdomain; 69. The signal-generating oligonucleotide of any one of claims 65 to 68.

70. 70. The signal-generating oligonucleotide of any one of claims 65 to 69, wherein the signal-generating oligonucleotide comprises a label, optionally wherein the label comprises a quenchable label, and further optionally wherein the quenchable label is a fluorophore.

71. the signal-generating oligonucleotide comprises a quencher; Optionally, a label associated with the 3' end of the signal-generating oligonucleotide and a quencher associated with the 5' end of the signal-generating oligonucleotide; or a label associated with the 5' end of the signal-generating oligonucleotide and a quencher associated with the 3' end of the signal-generating oligonucleotide; The signal-generating oligonucleotide according to any one of claims 65 to 70.

72. the quencher is capable of quenching the signal generated by the label when the quencher and label are in close proximity; and / or When the quencher and label are not in close proximity, the quencher is unable to quench the signal generated by the label; The signal-generating oligonucleotide according to any one of claims 65 to 71.

73. When the quencher and label are in close proximity, the signal generated by the label is not detectable; and / or When the quencher and label are not in close proximity, the signal generated by the label is detectable. The signal-generating oligonucleotide according to any one of claims 65 to 72.

74. When intramolecular nucleotide base pairing between the 5' and 3' subdomains forms a paired stem domain, the quencher and label are brought into close proximity; When the signal-generating oligonucleotide does not contain a paired stem domain, the quencher and label are not in close proximity; the nucleic acid amplification product is generated by amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to one another, optionally by amplifying the target nucleic acid sequence under isothermal amplification conditions, optionally the isothermal amplification conditions comprising 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 68°C, further optionally about 66°C to about 68°C; a nucleic acid amplification product hybridized to the signal-generating oligonucleotide is extended with an enzyme having polymerase activity, thereby producing an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide, optionally wherein the extended nucleic acid amplification product comprises the complement of the 5' terminal domain; and / or Extension of the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity disrupts the intramolecular nucleotide base pairing between the 5' and 3' subdomains, thereby allowing the paired stem domain to unwind. The signal-generating oligonucleotide according to any one of claims 65 to 73.

75. The sign, (i) when the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or (ii) extending the nucleic acid amplification product to produce an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide; capable of producing a detectable signal; Optionally, the signal is fluorescent. The signal-generating oligonucleotide according to any one of claims 65 to 74.

76. (i) when the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or (ii) the nucleic acid amplification product is extended to produce an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide; the label produces a detectable signal, and optionally the signal is fluorescent; The signal-generating oligonucleotide according to any one of claims 65 to 75.

77. 77. The signal-generating oligonucleotide of any one of claims 65 to 76, wherein the nucleic acid amplification product has a melting temperature within at least about 5°C of a certain temperature.

78. 78. The signal-generating oligonucleotide of any one of claims 65 to 77, wherein the melting temperature (Tm) of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex is higher than the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex, optionally by at least about 5°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, or about 20°C higher.

79. the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex is at most about 60°C; and the Tm of the extended nucleic acid amplification product / signal-generating oligonucleotide duplex is at least about 68°C; The signal-generating oligonucleotide of any one of claims 65 to 78.

80. 80. The signal-generating oligonucleotide of any one of claims 65 to 79, wherein the nucleic acid amplification product is unable to form a stable duplex with the signal-generating oligonucleotide in the absence of extension of the nucleic acid amplification product.

81. a signal-generating oligonucleotide is capable of hybridizing to the mismatch product; Optionally, the mismatch product hybridized to the signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby generating an extended mismatch product hybridized to the signal-generating oligonucleotide, and further optionally, the extended mismatch product comprises a complement of the 5'-terminal domain, and optionally, the mismatch product is a non-template control product and / or a non-target genotype. The signal-generating oligonucleotide according to any one of claims 65 to 80.

82. the Tm of the mismatch product / signal-generating oligonucleotide duplex is about 50°C; and the Tm of the extended mismatch product / signal-generating oligonucleotide duplex is at least 5°C below a certain temperature, optionally less than about 68°C; The signal-generating oligonucleotide according to any one of claims 65 to 81.

83. 83. The signal-generating oligonucleotide of any one of claims 65 to 82, wherein the nucleic acid amplification product and the mismatch product differ in sequence by at least about 1 nt, 2 nt, 3 nt, 4 nt, or 5 nt.

84. The signal generating oligonucleotide is The paired stem domain is stable at a constant temperature in the absence of nucleic acid amplification products, and The paired stem domains are capable of dissociating when a nucleic acid amplification product hybridizes to the loop domain. Optionally, by modifying the length of the paired domain, the GC content of the paired domain, and / or the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. The signal-generating oligonucleotide of any one of claims 65 to 83, which is formed.

85. 85. The signal-generating oligonucleotide of any one of claims 65 to 84, wherein the nucleic acid amplification product comprises: (1) the sequence of the forward primer and its reverse complement; (2) the sequence of the reverse primer and its reverse complement; and (3) A spacer sequence flanked by (1) the sequence of a forward primer and its reverse complement and (2) the sequence of a reverse primer and its reverse complement, optionally wherein the spacer sequence is about 4 nt to about 7 nt in length and / or has a GC content of less than about 50%.

86. (a) the signal-generating oligonucleotide comprises a first region comprising at least a portion of the sequence of a reverse primer, the signal-generating oligonucleotide comprises a second region comprising a sequence complementary to at least a portion of the forward primer, and / or the signal-generating oligonucleotide comprises a spacer region comprising at least a portion of the sequence of a spacer sequence; (b) the first region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer, the second region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer, and / or the spacer region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or the reverse primer; and / or (c) the first region comprises at least a portion of the 5' subdomain and / or the loop domain, the spacer region comprises at least a portion of the loop domain, and the second region comprises at least a portion of the loop domain and / or the 3' subdomain; The signal-generating oligonucleotide according to any one of claims 65 to 85.

87. (a) (i) the signal-generating oligonucleotide is from about 10 nt to about 100 nt in length, (ii) the second region, the spacer region, and / or the first region is from about 1 nt to about 25 nt in length, and / or (iii) the 5' subdomain, the 3' subdomain, the loop domain, and / or the 5' terminal domain is from about 1 nt to about 25 nt in length; (b) the 5'-terminal domain is about 1 nt to about 6 nt in length, the loop domain is about 4 nt to about 15 nt in length, and the paired stem domain is about 3 bp to about 8 bp in length; (c) the nucleic acid amplification product is about 25 nt to about 35 nt in length; (d) the target nucleic acid sequence comprises a length of about 20 nt or less to about 90 nt or less, and optionally, the target nucleic acid sequence comprises a length of about 30 nt; and / or (e) the spacer sequence comprises a portion of the target nucleic acid sequence, and optionally the spacer sequence is 1-10 bases in length, and optionally the spacer sequence is about 4 nt to about 7 nt in length and / or has a GC content of less than about 50%; The signal-generating oligonucleotide according to any one of claims 65 to 86.

88. (a) a forward primer is capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence, and a reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence, and optionally, a nucleic acid amplification product is generated by amplifying the target nucleic acid sequence with the forward primer and the reverse primer; (b) the signal-generating oligonucleotide comprises a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide; and / or (c) the signal-generating oligonucleotide comprises one or more polymerase stoppers and / or one or more phosphorothioate linkages, and optionally the first region, the second region, and / or the spacer region comprise one or more polymerase stoppers; The signal-generating oligonucleotide according to any one of claims 65 to 87.

89. (i) one or more polymerase stoppers are located in the loop domain, the first region, the second region, and / or the spacer region, and optionally, the 5′ subdomain, the paired stem domain, and / or the 3′ subdomain do not comprise one or more polymerase stoppers; (ii) one or more polymerase stoppers comprise one or more 2'-O-methyl (2'OM) RNA nucleotides; (iii) the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemical capture abasic site, or any combination thereof; (iv) (a) the chemically trapped abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride; (b) the abasic site comprises an apurinic base site, an apyrimidinic base site, or both; and / or (c) the abasic site is generated by an alkylating agent or an oxidizing agent; (v) the one or more polymerase stoppers are one or more RNA bases, one or more 2' methoxyethyl ribose (MOE), one or more locked nucleic acid (LNA) nucleotides, one or more 2' fluoro bases, one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more is one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3 (OC 3 H 6 OPO 3 ) group, one or more photocleavable (PC) [OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(CH 3 ) OPO 3 ] group, one or more hexanediol groups, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, one or more spacers 18 (iSp18) [(OCH 2 CH 26 OPO 3 ] group, or any combination thereof; (vi) the one or more polymerase stoppers comprise one or more steric blocking groups, optionally wherein the one or more steric blocking groups increase the Tm of the nucleic acid amplification product / signal-generating oligonucleotide duplex; and / or (vii) the polymerase stopper comprises a modification incorporated between two bases of the signal-generating oligonucleotide; The signal-generating oligonucleotide according to any one of claims 65 to 88.

90. The modification is (i) a naphthylene-azo compound, optionally Zen or iFQ; (ii) a molecule having the following structure: 【Chemistry 4】 wherein the linking group L positions the modification at an internal position of the signal-generating oligonucleotide. 1 and L 2 are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R 1 ~R 5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawing group, an electron donating group, or a point of attachment to a ligand; and X is a nitrogen atom or a carbon atom, with the proviso that when X is a carbon atom, the fourth substituent attached to the carbon atom is hydrogen or C 1 -C 8 (which may be an alkyl group) having (iii) a compound having the following structure: 【Transformation 5】 wherein the linking group L positions the modification at an internal position of the signal-generating oligonucleotide. 1 and L 2 are independently alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R 1 , R 2 , R 4 , R 5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron-withdrawing group, or an electron-donating group; R 6 , R 7 , R 9 ~R 12 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron-withdrawing group, or an electron-donating group; R 8 is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, or an electron-withdrawing group; and X is a nitrogen atom or a carbon atom, with the proviso that when X is a carbon atom, the fourth substituent attached to the carbon atom is hydrogen or C 1 -C 8 and / or (iv) a molecule having the following structure: 【Transformation 6】 and Optionally, R 8 But NO 2 That is, The signal-generating oligonucleotide according to any one of claims 65 to 89.

91. when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide; Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. The signal-generating oligonucleotide according to any one of claims 65 to 90.

92. when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, the one or more polymerase stoppers can stop polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide; 92. The signal-generating oligonucleotide of any one of claims 65 to 91, wherein optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second, undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

93. when the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers can stop the polymerase from extending the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide; 93. The signal-generating oligonucleotide of any one of claims 65 to 92, wherein optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of a third undesired duplex of exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

94. 1. A kit for detecting a target nucleic acid sequence in a sample, comprising: (a) a signal-generating oligonucleotide according to any one of claims 1 to 93; (b) a lysis buffer comprising one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence, optionally wherein the one or more lysis agents comprise a detergent, optionally wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; and / or (c) 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, wherein the one or more components for amplification comprise: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of a target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, optionally having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 31 or a functional fragment thereof, optionally having an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO: 31, and further optionally, the polymerase comprising the amino acid sequence of SEQ ID NO:

31. A reagent composition comprising Kit including:

95. The forward primer and / or the reverse primer are configured to have a Tm of less than about 45°C; is about 5 nt to about 25 nt in length, optionally about 10 nt to about 14 nt in length; configured to produce nucleic acid amplification products that are about 25 nt to about 35 nt in length and have a melting temperature that is within at least about 5°C of a certain temperature; containing one or more phosphorothioate linkages; and / or having a GC content of about 30% to about 55%; 95. The kit of claim 94.

96. the 3' region of the forward primer and / or the reverse primer does not contain a thymine base, and optionally the 3' region contains the first, second, third, and / or fourth nucleotide from the 3' end; the 5' region of the forward primer and / or reverse primer does not comprise more than 3 nt complementary to the spacer sequence, the region adjacent thereto, its complement, or any combination thereof, and optionally the 5' region comprises the first, second, third, and / or fourth nucleotide from the 5' end; the forward primer and / or reverse primer comprises a phosphorothioate bond between the first and second nucleotides from the 3' end of the forward primer and / or reverse primer, optionally, the phosphorothioate bond is capable of reducing or preventing polymerase-mediated degradation; the forward primer and / or reverse primer contains a phosphorothioate bond between the second and third nucleotides from the 3' end of the forward primer and / or reverse primer; the 3' region of the forward primer and / or the reverse primer does not contain more than two phosphorothioate linkages, and optionally the 3' region includes the first, second, third, and / or fourth nucleotide from the 3' end; the forward primer and / or the reverse primer comprises one or more phosphorothioate linkages in the region comprising the GC dinucleotide repeat, optionally the one or more phosphorothioate linkages are capable of destabilizing base pairing; the presence of one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain improves the sensitivity and / or specificity of detecting a nucleic acid amplification product by at least about 1.1 fold compared to a comparable method in which the signal-generating oligonucleotide does not contain any LNA nucleotides; the presence of the 5' terminal domain in the signal-generating oligonucleotide improves the sensitivity and / or specificity of detecting nucleic acid amplification products by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide comprises a blunt-ended hairpin structure; and / or The reagent composition comprises a reverse transcriptase and / or a reverse transcription primer; 96. The kit of claim 94 or 95.

97. The kit of any one of claims 94 to 96, wherein the nucleic acid amplification product is about 20 to 40 bases in length, and the nucleic acid amplification product comprises: (1) the sequence of the forward primer and its reverse complement; (2) the sequence of the reverse primer and its reverse complement; and (3) A spacer sequence flanked by (1) the sequence of a forward primer and its reverse complement and (2) the sequence of a reverse primer and its reverse complement, wherein the spacer sequence is about 1 to 10 bases in length.

98. the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle; The biological entity includes a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof; the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a viral, bacterial, fungal, or protozoan; 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, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus; the bacteria include one or more of Mycobacterium tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma spp., Legionella pneumophila, Legionella dumophila, Mycoplasma fermentans, Ehrlichia spp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, S. agalactiae, and Listeria monocytogenes; the fungus comprises 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 spp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora spp., and Eimeria spp. The kit according to any one of claims 94 to 97.

99. The reagent composition is freeze-dried and / or heat-dried and comprises one or more additives, the one or more additives being: amino acid; sugars or sugar alcohols, optionally including sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or Optionally, a polymer including polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The kit according to any one of claims 94 to 98, comprising:

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