Asymmetric hairpin probes for nucleic acid detection

Abstract

The disclosure herein includes methods, compositions, and kits for detecting a target nucleic acid sequence in a sample. The method may include generating an oligonucleotide (SGO) using a signal capable of hybridizing to a nucleic acid amplification product. The SGO may comprise a 5'subdomain, a 3 'subdomain, and a ring domain 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 comprise one or more locked nucleic acid (LNA) nucleotides in the loop domain, the 5 '-subdomain, and / or the 3'-subdomain SGO may comprise a 5 '-terminal domain located 5' of the 5 '-subdomain In some embodiments, the 5'-terminal domain is not capable of hybridizing to the 3 '-terminal of the nucleic acid amplification product.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 385,681, filed on December 1, 2022, under 35 U.S.C. § 119(e). The content of the related application is incorporated herein by reference in its entirety for all purposes.

[0003] Reference to Sequence Listing

[0004] This application is filed together with a sequence listing in electronic format. The sequence listing is provided as a file entitled 68EB - 317363 - WO, created on November 30, 2023, and is 61,673 bytes in size. The information of the sequence listing in electronic format is incorporated herein by reference in its entirety.

[0005] Background

[0006] Field

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

[0008] Description of Related Technologies

[0009] Nucleic acid - based diagnostics can be used to rapidly detect infections, diseases, and / or genetic variations. For example, the identification of bacterial or viral nucleic acids in a sample can be used to diagnose a specific type of infection. Other examples include the identification of single - nucleotide polymorphisms for disease management or forensic medicine, and the identification of genetic variations indicative of genetically modified food products. Generally, nucleic acid - based diagnostic assays require amplification of a specific portion of the nucleic acids in a sample. A commonly used technique for nucleic acid amplification is the polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermal cycling) to proceed through steps of denaturation (e.g., separation of strands in a double - stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short chains of complementary DNA sequences), and extension of the primers along the complementary target by a polymerase. Such thermal cycling can be a time - consuming process that typically requires specialized equipment. Accordingly, there is a need for faster nucleic acid amplification methods that do not require thermal cycling.

[0010] Archaeal polymerase amplification (APA) is an isothermal technique that uses primers much shorter than those of PCR or other amplification methods to generate amplicons that are 25 - 35 bases in length, with a Tm comparable to the reaction temperature, typically 67 - 68 °C. The small amplicon size can present serious challenges for real-time detection because short APA amplicons typically have only 4 - 7 bases in the spacer region that are not homologous or complementary to the primer sequence and the sequence length does not allow for sufficient binding to hybridization probes. Thus, currently available detection methods are not sufficient to detect short APA amplicons in real time. Additionally, in some embodiments, non-specific product formation can be caused by the undesired interaction of the probe with the amplification primer (followed by extension of the amplification primer), which can result in false positives. There is a need for compositions and methods for nucleic acid detection that reduce the formation of undesired extension products and false positives.

[0011] Overview

[0012] The disclosure herein includes methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: 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, wherein the signal-generating oligonucleotide is capable of hybridizing to the 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. Intramolecular base pairing between the 5' subdomain and the 3' subdomain can 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 comprises a 5' terminal domain that is about 1 nt to about 6 nt in length and is located 5' of 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 signal-generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, 5' subdomain, and / or 3' subdomain.

[0013] The disclosure herein includes methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: 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, wherein the signal-generating oligonucleotide is capable of hybridizing to the 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 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 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 having a length of from about 1 nt to about 15 nt and located 5' of 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.

[0014] In some embodiments, 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 one, two, three, four, five, six, seven, or eight LNA nucleotides. In some embodiments, the loop domain comprises one or more LNA nucleotides, optionally the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for the nucleic acid amplification product. In some embodiments, enhancing the specificity of the signal-generating oligonucleotide for the nucleic acid amplification product includes increasing the mismatch discrimination between the nucleic acid amplification product and the mismatch product. In some embodiments, the mismatch product includes a non-template control product and / or a non-target genotype. In some embodiments, the terminal 3'-nucleotide of the signal-generating oligonucleotide is an LNA nucleotide, optionally the LNA nucleotide reduces or prevents digestion of the signal-generating oligonucleotide and / or removal of the 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, optionally the one or more LNA nucleotides enhance the stability of the paired stem domain. In some embodiments, the paired stem domain comprises base pairing of at least one pair of opposing LNA nucleotides. In some embodiments, the nucleotides located in the 5'-terminal domain are not capable of intramolecular nucleobase pairing. In some embodiments, less than about 5 nt, 4 nt, 3 nt, 2 nt, or 1 nt of the 5'-terminal domain is complementary to the 3'-end of the nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide does not comprise a nucleotide located 3' of the 3'-subdomain.

[0015] In some embodiments, the signal generating oligonucleotide comprises a label. In some embodiments, the label comprises a quencheable 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 the label are in close proximity, the quencher is capable of quenching the signal generated by the label. In some embodiments, when the quencher and the label are not in close proximity, the quencher is not capable of quenching the signal generated by the label. In some embodiments, when the quencher and the label are in close proximity, the signal generated by the label is undetectable. In some embodiments, when the quencher and the label are not in close proximity, the signal generated by the label is detectable. In some embodiments, when intermolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain forms a paired stem domain, the quencher and the label are in close proximity. In some embodiments, when the signal generating oligonucleotide does not comprise a paired stem domain, the quencher and the 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, detecting the nucleic acid amplification product comprises using a real-time detection method. In some embodiments, the detecting step comprises detecting the signal of the label before, during, after the amplification reaction, or any combination thereof. In some embodiments, detecting the nucleic acid amplification product comprises detecting the signal generated by the label of the signal generating oligonucleotide. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, detecting the signal comprises detecting the fluorescence emitted by the label.

[0016] In some embodiments, the amplification reaction and / or detection step comprises: contacting a 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. In some embodiments, the extended nucleic acid amplification product comprises a complement of the 5'-terminal domain. In some embodiments, extending the nucleic acid amplification product hybridized to the signal generating oligonucleotide with an enzyme having polymerase activity is capable of disrupting the intramolecular nucleotide base pairing between the 5'-subdomain and the 3'-subdomain, thereby unwinding the paired stem domain. In some embodiments, the label is capable of generating 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. 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.

[0017] Amplifying the target nucleic acid sequence in the amplification reaction mixture may comprise amplifying the target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the isothermal amplification conditions include a constant temperature of about 30 °C to about 72 °C, such as a constant temperature of about 55 °C to about 75 °C, about 56 °C to about 68 °C, or about 66 °C to about 68 °C. The amplification can be carried out at the optimal temperature of an enzyme having hyperthermophilic polymerase activity. In some embodiments, the optimal temperature is about 66 °C to about 68 °C (e.g., a constant temperature). In some embodiments, the amplification is carried out 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., 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). In some embodiments, the Tm of the nucleic acid amplification product / signal generating oligonucleotide duplex is at least or 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. In some embodiments, in the absence of extension of the nucleic acid amplification product, the nucleic acid amplification product cannot form a stable duplex with the signal generating oligonucleotide.

[0018] In some embodiments, the amplification reaction comprises: 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 to produce 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 lower than 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.

[0019] In some embodiments, the signal-generating oligonucleotide is configured such that: the paired stem domains are stable at a constant temperature in the absence of the nucleic acid amplification product, and the paired stem domains are capable of unwinding when the nucleic acid amplification product hybridizes to the loop domain. In some embodiments, the configuration is achieved by altering the length of the paired domains, the GC content of the paired domains, and / or the presence of one or more LNA nucleotides in the loop domain, 5'-subdomain, and / or 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 (1) the sequence of the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement. In some embodiments, the spacer sequence has a length of about 4 nt to about 7 nt and / or has a GC content of less than about 50%.

[0020] In some embodiments, the signal generating oligonucleotide comprises a first region that comprises a sequence of at least a portion of a reverse primer. In some embodiments, the signal generating oligonucleotide comprises a second region that comprises 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 that comprises a sequence complementary to at least a portion of a forward primer. In some embodiments, the signal generating oligonucleotide comprises a spacer region that comprises a sequence of at least a portion of a spacer sequence. In some embodiments, the first region comprises a sequence complementary to at least 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 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 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 has a length of from about 10 nt to about 100 nt. In some embodiments, the second region, the spacer region, and / or the first region has a length of from about 1 nt to about 25 nt. In some embodiments, the 5'-subdomain, the 3'-subdomain, the loop domain, and / or the 5'-terminal domain has a length of from about 1 nt to about 25 nt. In some embodiments, the 5'-terminal domain has a length of from about 1 nt to about 6 nt, the loop domain has a length of from about 4 nt to about 15 nt, and the paired stem domain has a length of from about 3 bp to about 8 bp. In some embodiments, the nucleic acid amplification product has a length of from about 25 nt to about 35 nt. In some embodiments, the target nucleic acid sequence has a length of no more than about 20 nt to no more than about 90 nt. In some embodiments, the target nucleic acid sequence has 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 has a length of from about 4 nt to about 7 nt and / or has a GC content of less than about 50%.

[0021] In some embodiments, the forward primer and / or the reverse primer are configured to have a Tm of less than about 45°C; a length of about 5 nt to about 25 nt (e.g., a length of about 10 nt to about 14 nt); be configured to produce a nucleic acid amplification product having a length of about 25 nt to about 35 nt and a melting temperature within at least about 5°C of a constant temperature; contain one or more phosphorothioate bonds; and / or have a GC content of about 30% to about 55%. In some embodiments, the 3' region of the forward primer and / or the reverse primer does not contain thymine bases. In some embodiments, the 3' region contains the first, second, third, and / or fourth nucleotides starting from the 3' end. In some embodiments, the 5' region of the forward primer and / or the reverse primer does not contain 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 contains the first, second, third, and / or fourth nucleotides starting from the 5' end. In some embodiments, the forward primer and / or the reverse primer contain a phosphorothioate bond between the first and second nucleotides starting from the 3' end of the forward primer and / or the reverse primer. In some embodiments, the phosphorothioate bond is capable of reducing or preventing polymerase-mediated degradation. In some embodiments, the forward primer and / or the reverse primer contain a phosphorothioate bond between the second and third nucleotides starting from the 3' end of the forward primer and / or the reverse primer. In some embodiments, the 3' region of the forward primer and / or the reverse primer does not contain more than 2 phosphorothioate bonds. In some embodiments, the 3' region contains the first, second, third, and / or fourth nucleotides starting from the 3' end. In some embodiments, the forward primer and / or the reverse primer contain one or more phosphorothioate bonds in a region containing a GC dinucleotide repeat. In some embodiments, the one or more phosphorothioate bonds are capable of destabilizing base pairing. In some embodiments, the presence of one or more LNA nucleotides in the loop domain, 5' subdomain, and / or 3' subdomain increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product to at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide does not contain LNA nucleotides. In some embodiments, the presence of the 5' terminal domain in the signal-generating oligonucleotide increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product to at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide contains a blunt-ended hairpin structure.

[0022] 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 in the sample that contains the target nucleic acid sequence. 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 in the sample that contains the target nucleic acid sequence. In some embodiments, the signal-generating oligonucleotide comprises one or more phosphorothioate bonds 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 can include contacting a sample containing a biological entity with a lysis buffer to produce a processed sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entity to release the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of containing the target nucleic acid sequence. The method can include contacting a reagent composition with the processed sample to produce an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0023] In some embodiments, the signal-generating oligonucleotide comprises one or more polymerase terminators and / or one or more phosphorothioate bonds. In some embodiments, the first region, the second region, and / or the spacer region comprises one or more polymerase terminators. In some embodiments, one or more polymerase terminators 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 terminators. In some embodiments, one or more polymerase terminators comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. In some embodiments, one or more polymerase terminators include one or more of abasic sites, stable abasic sites, chemically trapped abasic sites, or any combination thereof. In some embodiments, the chemically trapped abasic site comprises an abasic site that reacts with an alkoxyamine or sodium borohydride; the abasic site comprises an apurinic site, an apyrimidinic site, or both; and / or the abasic site is generated by an alkylating agent or an oxidizing agent.

[0024] One or more polymerase terminators can comprise: one or more RNA bases, 2'-methoxyethyl ribose (MOE), LNA nucleotides, 2'-fluorobases, nitroindoles, inosine, one or more acridines, 2-aminopurine, 2,6-diaminopurine, 5-bromo-deoxyuridine, reverse thymidine (reverse dT), reverse dideoxythymidine (ddT), dideoxycytidine (ddC), 5-methylcytidine, 5-hydroxymethylcytidine, 2'-O-methyl RNA bases, unmethylated RNA bases, isodeoxycytidine (iso-dC), isodeoxyguanosine (iso-dG), C3 (OC3H6OPO3) groups, photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, hexanediol groups, spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, spacer 18 (iSp18) [(OCH2CH 26 OPO3] groups, or combinations thereof.

[0025] In some embodiments, one or more polymerase terminators 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 terminator comprises a modification between two bases incorporated into the signal generating oligonucleotide. In some embodiments, the modification is a naphthylazo compound (e.g., Zen or iFQ).

[0026] In some embodiments, the modification has the following structure:

[0027] wherein the linking groups L1 and L2 that 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 groups, electron-donating groups, or attachment points for ligands; and X is a nitrogen or carbon atom, where if X is a carbon atom, the fourth substituent attached to that carbon atom can be hydrogen or a C1-C8 alkyl group.

[0028] In some embodiments, the modification has the following structure:

[0029] Among them, the linking groups L1 and L2 that position the modification at an internal position of the signal-generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl or alkoxy group; R1, R2, R4, R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group or electron-donating group; R6, R7, R9-R 12 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group or electron-donating group; R8 is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy or electron-withdrawing group; and X is a nitrogen or carbon atom, wherein if X is a carbon atom, the fourth substituent attached to the carbon atom can be hydrogen or a C1-C8 alkyl group. In some embodiments, R8 is NO2.

[0030] In some embodiments, the modification has the following structure:

[0031]

[0032] In some embodiments, when a forward primer binds to a signal-generating oligonucleotide to form a first undesired duplex, one or more polymerase terminators are capable of terminating 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 terminators are capable of terminating the polymerase extension of the forward primer of the first undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. In some embodiments, when a reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, one or more polymerase terminators are capable of terminating 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 terminators are capable of terminating the polymerase extension of the reverse primer of the second undesired duplex beyond one or more polymerase terminators 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, one or more polymerase terminators are capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, one or more polymerase terminators are capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. In some embodiments, the exogenous nucleic acid is selected from sample nucleic acids, primers configured to hybridize to a second target nucleic acid sequence, primers configured to hybridize to an internal control, or any combination thereof.

[0033] 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: amplifying the target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplification comprises contacting the nucleic acid comprising the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic polymerase activity, thereby producing 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 from sample ribonucleic acid. In some embodiments, the amplification comprises producing the nucleic acid by a reverse transcription reaction. In some embodiments, the sample nucleic acid comprises sample ribonucleic acid, and wherein the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA. In some embodiments, amplifying the target nucleic acid sequence comprises: (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 hyperthermophilic polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises the target nucleic acid sequence, and wherein the target nucleic acid sequence comprises a first strand and a second strand complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplification comprises contacting the dsDNA with: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product.

[0034] In some embodiments, if a forward primer binds to a signal-generating oligonucleotide to form a first undesired duplex, the forward primer of the first undesired duplex is extended by an enzyme having hyperthermophilic polymerase activity towards the 5'-end of the signal-generating oligonucleotide to produce a first undesired extension product. In some embodiments, the first undesired extension product can be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a forward primer and a reverse primer to form a first undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesired duplex to produce a first stalled extension product. In some embodiments, the first stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a forward primer and a reverse primer to produce a first undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. In some embodiments, if a reverse primer binds to a signal-generating oligonucleotide to form a second undesired duplex, the reverse primer of the second undesired duplex is extended by an enzyme having hyperthermophilic polymerase activity towards the 5'-end of the signal-generating oligonucleotide to produce a second undesired extension product. In some embodiments, the second undesired extension product can be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to form a second undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesired duplex to produce a second stalled extension product. In some embodiments, the second stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to produce a second undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. In some embodiments, if a foreign nucleic acid binds to a signal-generating oligonucleotide to form a third undesired duplex, the foreign nucleic acid of the third undesired duplex is extended by an enzyme having hyperthermophilic polymerase activity towards the 5'-end of the signal-generating oligonucleotide to produce a third undesired extension product. In some embodiments, the third undesired extension product can be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to form a third undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the foreign nucleic acid of the third undesired duplex to produce a third stalled extension product. In some embodiments, the third stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to produce a third undesired amplification product.In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the exogenous nucleic acid of the third undesired duplex by more than one or more polymerase terminators of the signal-generating oligonucleotide.

[0035] The label may be 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 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 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, the detection of the false positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

[0036] Compared to a signal-generating oligonucleotide that does not contain one or more polymerase terminators, the presence of one or more polymerase terminators in a signal-generating oligonucleotide can increase the likelihood of accurately determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample to at least about 1.1-fold. In some embodiments, the generation of the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate false positive signals. In some embodiments, the signal-generating oligonucleotide that hybridizes to the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate false positive signals. In some embodiments, the nucleic acid amplification product reaches a detectable level 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 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, compared to a comparable method in which the signal-generating oligonucleotide does not contain one or more polymerase terminators, the appearance of a false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product is delayed or delayed by at least about 1, 2, 5, 10, 15, or 20 minutes. 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 a detectable level for at least about 5, 10, 15, or 20 minutes after the start of the amplification step. In some embodiments, compared to a comparable method in which the signal-generating oligonucleotide does not contain one or more polymerase terminators, the generation of the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product is reduced by at least about 1.1-fold.

[0037] In some embodiments, amplifying the target nucleic acid sequence comprises generating a nucleic acid amplification product at a detectable level within or at about: 20, 15, or 10 minutes. In some embodiments, detection is performed within less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time of contact of the reagent composition with the treated sample.

[0038] 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 from about 1.0 to about 10.0 (such as 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, one or more amplification reagents comprise: reverse transcriptase; an enzyme having hyperthermophilic DNA polymerase activity; and / or dNTPs. In some embodiments, the enzyme having hyperthermophilic DNA polymerase activity has reverse transcriptase activity; forward primer; reverse primer; reverse transcription primer.

[0039] The reagent composition can be lyophilized, heat-dried, and / or comprise one or more additives. In some embodiments, one or more additives comprise: Tween 20, Triton X-100, and / or Tween 80; amino acids; sugars or sugar alcohols; and / or polymers. The sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethyl cellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. In some embodiments, contacting the reagent composition with the treated sample comprises dissolving the reagent composition in the treated sample. In some embodiments, one or more lysis reagents comprise: from about 0.001% (w / v) to about 1.0 (w / v) of the treated sample (such as about 0.2% (w / v) of the treated sample); and / or a detergent (such as one or more of a cationic surfactant, anionic surfactant, nonionic surfactant, and zwitterionic surfactant). In some embodiments, the method: is carried out in a single reaction vessel; does not include using any enzyme other than reverse transcriptase and an enzyme having hyperthermophilic DNA polymerase activity; does not include using any enzyme other than an enzyme having hyperthermophilic DNA polymerase activity; does not include thermal denaturation and / or enzymatic denaturation of nucleic acids during the amplification step; and / or does not include contacting the nucleic acids with single-stranded DNA binding protein.

[0040] The target nucleic acid sequence can have a length of no more than about 20 nucleotides to no more than about 90 nucleotides (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, such as about 55°C to about 75°C or about 56°C to about 67°C. In some embodiments, the amplification is carried out for a period of about 5 minutes to about 60 minutes (e.g., a period of about 15 minutes). In some embodiments, the amplification is carried out under isothermal amplification conditions without helicase, without single-strand binding protein, without lysing agent, and without recombinase. In some embodiments, the amplification 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, immunoamplification, 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).

[0041] An enzyme having hyperthermophilic DNA polymerase activity can have an amino acid sequence that is at least about 90% identical 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 having hyperthermophilic DNA polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:31. In some embodiments, the enzyme having hyperthermophilic DNA polymerase activity has low exonuclease activity or no exonuclease activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with a reverse transcriptase and an enzyme having hyperthermophilic DNA polymerase activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with a reverse transcriptase, an enzyme having hyperthermophilic DNA polymerase activity, and a forward primer and a reverse primer. In some embodiments, the sample ribonucleic acid is contacted simultaneously with a reverse transcriptase, an enzyme having hyperthermophilic DNA polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer.

[0042] Biological entities can include one or more of prokaryotic cells, eukaryotic cells, virus particles, exosomes, protoplasts, and microvesicles. In some embodiments, the biological entities include viruses, bacteria, fungi, protozoa, parts thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, bacterium, fungus, or protozoan. In some embodiments, the sample nucleic acid is derived from a virus, bacterium, fungus, or protozoan. Viruses can be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus. In some embodiments, the bacteria include one or more of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes.In some embodiments, the fungi include 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., or Eimeria sp. The sample can be a biological sample or an environmental sample. In some embodiments, the environmental sample is, or obtained from, a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a wastewater sample, a brine sample, a sample exposed to atmospheric air or other gases, its culture, or any combination thereof. In some embodiments, the biological sample is, or obtained from, a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, its culture, or any combination thereof.

[0043] The amplification step may include multiplex amplification of two or more target nucleic acid sequences, and wherein the detection step includes multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally wherein the two or more target nucleic acid sequences are specific for two or more different organisms, and further optionally wherein the two or more different organisms include one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C. In some embodiments, the amplification includes and / or does not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplification does not include LAMP. In some embodiments, the method does not include one or more of the following: (i) dilution of the processed sample; (ii) dilution of the amplification reaction mixture; (iii) heat denaturation of the processed sample; (iv) sonication of the processed sample; (v) sonication of the amplification reaction mixture; (vi) addition of ribonuclease inhibitor to the processed sample; (vii) addition of ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more lysis agents from the processed sample or amplification reaction mixture; (xii) heat denaturation and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of ribonuclease H to the processed sample or amplification reaction mixture.

[0044] The disclosure herein includes signal - generating oligonucleotides, such as signal - generating oligonucleotides capable of hybridizing to nucleic acid amplification products. In some embodiments, the signal - generating oligonucleotide comprises a 5' sub - domain and a 3' sub - domain. In some embodiments, the signal - generating oligonucleotide comprises a loop domain located between the 5' sub - domain and the 3' sub - domain. In some embodiments, the intramolecular nucleotide base pairing between the 5' sub - domain and the 3' sub - domain can form a paired stem domain. In some embodiments, at least a portion of the 5' sub - domain and at least a portion of the loop domain can hybridize to the nucleic acid amplification product. In some embodiments, the signal - generating oligonucleotide comprises a 5' terminal domain having a length of from about 1 nt to about 6 nt and located at the 5' of the 5' sub - domain. 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 signal - generating oligonucleotide comprises one or more LNA nucleotides in the loop domain, 5' sub - domain, and / or 3' sub - domain.

[0045] The disclosure herein includes signal - generating oligonucleotides, such as signal - generating oligonucleotides capable of hybridizing to nucleic acid amplification products. The signal - generating oligonucleotide can comprise a 5' sub - domain and a 3' sub - domain. In some embodiments, the signal - generating oligonucleotide comprises a loop domain located between the 5' sub - domain and the 3' sub - domain. In some embodiments, the intramolecular nucleotide base pairing between the 5' sub - domain and the 3' sub - domain can form a paired stem domain. In some embodiments, at least a portion of the 5' sub - domain and at least a portion of the loop domain can hybridize to the nucleic acid amplification product. The signal - generating oligonucleotide can comprise one or more LNA nucleotides in the loop domain, 5' sub - domain, and / or 3' sub - domain. The signal - generating oligonucleotide can comprise a 5' terminal domain having a length of from about 1 nt to about 15 nt and located at the 5' of the 5' sub - domain. In some embodiments, the 5' terminal domain is not capable of hybridizing to the 3' end of the nucleic acid amplification product.

[0046] In some embodiments, a nucleic acid amplification product is produced 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 from about 30 °C to about 72 °C (e.g., from about 55 °C to about 75 °C, from about 56 °C to about 68 °C, from about 66 °C to about 68 °C). In some embodiments, a nucleic acid amplification product hybridized to a signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby producing 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 a five prime (5') terminal domain. In some embodiments, the signal-generating oligonucleotide can hybridize to a mismatch product. In some embodiments, a mismatch product hybridized to the signal-generating oligonucleotide can be extended with an enzyme having polymerase activity, thereby producing an extended mismatch product hybridized to the signal-generating oligonucleotide. In some embodiments, the extended mismatch product comprises a complement of a five prime (5') terminal domain. In some embodiments, the mismatch product is a non-template control product and / or a non-target genotype. In some embodiments, a forward primer can hybridize to the sequence of the first strand of the target nucleic acid sequence, and a reverse primer can hybridize to the sequence of the second strand of the target nucleic acid sequence. In some embodiments, a nucleic acid amplification product is produced by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0047] The disclosure herein includes 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 comprise: a lysis buffer comprising one or more lysing agents capable of lysing a biological entity to release the sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of containing the target nucleic acid sequence, optionally one or more of the lysing agents comprise a detergent, and wherein the detergent comprises one or more of a cationic surfactant, anionic surfactant, nonionic surfactant, and zwitterionic surfactant. The kit can comprise: a reagent composition comprising one or more amplification reagents, the amplification reagents comprising one or more components for amplification for amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplification comprise: (i) a forward primer provided herein and a reverse primer provided herein, wherein the forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of generating a nucleic acid amplification product, optionally the enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof, optionally the enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 31, and further optionally the enzyme having 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 DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A non-limiting exemplary schematic diagram of a conventional molecular beacon probe is depicted.

[0050] Figure 2 A non-limiting exemplary schematic diagram of a signal generating oligonucleotide comprising locked nucleic acid bases provided herein is depicted.

[0051] Figures 3A - 3D Depicts hybridization with a target ( Figure 3A ), extended target ( Figure 3B ), NTC ( Figure 3C ), and extended NTC ( Figure 3D ) of a signal generating oligonucleotide provided herein. A non-limiting exemplary schematic diagram is depicted.

[0052] Figure 4 A non-limiting exemplary schematic diagram of a signal generating oligonucleotide provided herein is depicted.

[0053] Figure 5 A non-limiting exemplary schematic diagram of a signal generating oligonucleotide provided herein is depicted.

[0054] Figures 6A - 6B Depicts non - limiting exemplary signal - generating oligonucleotides provided herein.

[0055] Figure 7 Depicts non - limiting exemplary schematic diagrams of the signal - generating oligonucleotides provided herein.

[0056] Figures 8A - 8B Depicts non - limiting exemplary data related to MB characterization. The vertical lines in the figure indicate the measured temperature.

[0057] Figure 9 Depicts non - limiting exemplary schematic diagrams related to the importance of the primer 3'-end in the APA assay.

[0058] Figures 10A - 10B Depicts non - limiting exemplary schematic diagrams related to the APA assay design. Figure 10A Depicts non - limiting exemplary schematic diagrams related to the importance of the primer 5'-end in the APA assay. Figure 10B Depicts non - limiting exemplary schematic diagrams related to the APA assay Tm and the APA product Tm.

[0059] Figures 11A - 11C Depicts non - limiting exemplary interactions that can cause background product: primer - dimer interactions ( Figure 11A ), homodimer interactions ( Figure 11B ), and primer - spacer interactions ( Figure 11C ).

[0060] Figure 12 Depicts non - limiting exemplary schematic diagrams illustrating the incorrect products produced (in assays without phosphorothioate - modified primers) and the correct products produced (in assays with phosphorothioate - modified primers).

[0061] Figures 13A - 13B Depicts data related to the performance of the old ( Figure 13A ) and new ( Figure 13B ) Neisseria gonorrhoeae APA assays.

[0062] Figure 14 Depicts data related to the effect of primer length on the performance of the APA assay.

[0063] Figure 15 Depicts non - limiting exemplary graphs related to APA product detection.

[0064] Figure 16 Depicts non - limiting exemplary probes containing locked nucleic acid (LNA) bases provided herein.

[0065] Figure 17 Depicts a non - limiting exemplary diagram related to the Flu AAPA assay design.

[0066] Figure 18 Depicts a non - limiting exemplary schematic diagram of the asymmetric hairpin probe for amplicon detection provided herein.

[0067] Figures 19A - 19B Depicts data related to the detection of Chlamydia trachomatis gDNA in the APA reaction using a conventional molecular beacon in the HEX( Figure 19A ) and cy5( Figure 19B ) channels.

[0068] Figure 20 Depicts a non - limiting exemplary conventional molecular beacon for detecting Chlamydia trachomatis gDNA in the APA reaction.

[0069] Figure 21 Depicts a non - limiting exemplary asymmetric hairpin probe provided herein.

[0070] Figures 22A - 22C Depicts data related to the detection of synthetic DNA targets in the APA reaction using an asymmetric hairpin probe( Figure 22A ), followed by melting curve analysis( Figure 22B ) and melting derivative evaluation( Figure 22C ).

[0071] Figure 23 Depicts data related to the limit of detection (LOD) study using the hairpin probe provided herein for Flu A virus detection.

[0072] Figures 24A - 24D Depicts data related to the real - time detection( Figure 24A 、 Figure 24C ) and melting curve evaluation( Figure 24B 、 Figure 24D ) of SARS - CoV - 2 virus using both a hairpin probe( Figures 24A - 24B ) and a fluorescent DNA dye Syto61( Figures 24C - 24D ) in the reaction.

[0073] Figure 25 Depicts a non - limiting exemplary signal - generating oligonucleotide provided herein.

[0074] Figures 26A - 26B Shows a non - limiting exemplary schematic diagram of the isothermal amplification reaction provided herein.

[0075] Detailed description

[0076] In the following detailed description, reference is made to the accompanying drawings which form a part hereof. In the drawings, like reference numerals generally identify like components unless the context otherwise indicates. The illustrative embodiments described in the detailed description, the drawings, and the 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 drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and form a part of the present disclosure.

[0077] All patents, published patent applications, other publications, and sequences from GenBank, as well as other databases related to the relevant art, are hereby incorporated by reference in their entirety.

[0078] 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 pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

[0079] The disclosure herein includes methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: 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, wherein the signal generating oligonucleotide is capable of hybridizing to the 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, intermolecular 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 comprises a 5' terminal domain having a length of from about 1 nt to about 6 nt and located 5' of 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 signal generating oligonucleotide comprises one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0080] The disclosure herein includes methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: 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, wherein the signal generating oligonucleotide is capable of hybridizing to the 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, intermolecular 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 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 having a length of from about 1 nt to about 15 nt and located 5' of 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.

[0081] The disclosure herein includes signal-generating oligonucleotides. In some embodiments, the signal-generating oligonucleotides are capable of hybridizing to nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotides comprise a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotides comprise a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intermolecular 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 nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotides comprise a 5' terminal domain having a length of from about 1 nt to about 6 nt and located 5' of 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 signal-generating oligonucleotides comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0082] The disclosure herein includes signal-generating oligonucleotides. In some embodiments, the signal-generating oligonucleotides are capable of hybridizing to nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotides comprise a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotides comprise a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intermolecular 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 nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotides comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain. In some embodiments, the signal-generating oligonucleotides comprise a 5' terminal domain having a length of from about 1 nt to about 15 nt and located 5' of 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.

[0083] Asymmetric hairpin probe

[0084] 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 employed for real-time, specific nucleic acid detection in APA technology. Some embodiments of the methods and compositions provided herein can be employed for real-time detection of short nucleotides, such as APA amplification products. In some embodiments, an asymmetric hairpin probe design is provided that comprises a nucleotide sequence forming an asymmetric hairpin structure having a double-stranded segment, a single-stranded loop, and a 5'-terminal overhang. At least a portion of the single-stranded loop segment and a portion of the double-stranded segment can form a region complementary to the target nucleotide sequence to be hybridized, and in some embodiments, the 5'-terminal overhang comprises a non-target sequence that is 1 to 6 bases in length.

[0085] In some embodiments of the methods and compositions provided herein, locked nucleic acids are used to enhance specificity and increase the affinity for short amplicons for real-time detection of archaeal polymerase amplification. As provided herein, LNA nucleotides can be strategically incorporated into the asymmetric hairpin probes to achieve one or more of the following functions: (1) at the terminal base at the 3'-end to block exonuclease digestion from the 9°Nm polymerase; (2) in the loop segment to provide additional specificity and increase the binding strength of the amplicon to the probe; and (3) in the stem segment to increase the stability of the hairpin structure.

[0086] In some embodiments, a fluorophore is attached to the 5'-end of the hairpin probe provided herein, and a quencher is attached to the 3'-end. In the non-activated state, the hairpin probe can be significantly quenched because the fluorophore and the quencher are held together by the hairpin stem segment. In some embodiments, and 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 overcome the energy barrier imposed by the stem, resulting in stem unwinding and ultimately separation of the two labels, thereby generating increased fluorescence. The unique design of the asymmetric hairpin probes provided herein can lie in their 5'-overhangs, strong hairpin structures, and increased binding affinity for targets from LNA modification. In some embodiments, and without being bound by any particular theory, such features enable rapid kinetics and real-time detection of short amplicons with a sensitivity as low as single-digit copy input. Detection using hairpin probes without blunt-ended stems in molecular diagnostic assays is unknown in the art.

[0087] The methods and compositions provided herein enable real-time detection of short amplicons. Without being bound by any particular theory, due to the presence of the stem structure, the hairpin probe can be significantly more specific than conventional linear probes, and compared to conventional molecular beacons, the enhanced binding affinity and specificity from LNA modification can detect short amplicons with rapid kinetics and increased sensitivity and specificity.

[0088] For short targets or amplicons, real-time detection can be extremely difficult. In some embodiments, and without being bound by any particular theory, in order to achieve real-time detection of an amplification product, the probe needs to form a stable hybrid with the nucleic acid, and the length of the probe sequence should be such that it dissociates from the target at a temperature 7-10 °C higher than the PCR annealing temperature or the assay temperature of isothermal amplification.

[0089] Molecular beacons can be used in many applications. A typical molecular beacon has a stem that is 6-7 nucleotides in length and a loop that is 15-25 nucleotides in length, and uses the loop region to hybridize to its target. In some embodiments, and without being bound by any particular theory, molecular beacons require a delicate balance: the stem needs to be strong enough to maintain a stable hairpin structure at the assay temperature and at the same time weak enough to dissociate when the complementary nucleic acid hybridizes to the loop region.

[0090] In some embodiments, the disclosed compositions and methods herein can overcome the above challenges for short amplicon detection by configuring asymmetric hairpin probes having a 5' overhang, loop, and stem for real-time detection of APA amplicons. Multiple methods for improving short amplicon detection are provided herein, which can be employed alone 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 onto the probe can form a probe / target hybrid that is longer and more stable than the stem structure. Thus, the hairpin probe is undergoing a conformational change from a hairpin shape to a more rigid double helix. Second, in some embodiments, the methods and compositions provided herein employ LNA modifications to improve hairpin stability and target / probe binding stability, as well as specificity for the true target and discrimination against mismatched products. Third, the methods and compositions provided can include constructing a sequence complementary to the target in the stem to improve the kinetics of hairpin opening. Fourth, in some embodiments, the signal-generating oligonucleotides provided herein contain one or more polymerase terminators to reduce or prevent non-specific product formation caused by unintended interactions of the probe with the amplification primer (followed by extension of the amplification primer). In some embodiments, the method compositions provided herein can be employed for real-time detection assays of short nucleotides, for detection of large amplicons, and / or for detection of small RNAs such as microRNAs (small non-coding RNAs of 20-22 nucleotides) for cancer diagnosis.

[0091] The hairpin probes provided herein can vary with respect to the size of the 5' overhang, loop, and stem. Base modifications can be placed at various positions for (1) enhancing the stability of the template / probe duplex or hairpin structure, (2) increasing the specificity of target recognition, and / or (3) blocking non-specific off-target priming and background product interactions. The 5' terminal 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 detectability.

[0092] In some embodiments, and without being bound by any particular theory, the underlying principles of the disclosed compositions and methods relate to the thermodynamics of nucleic acids, the hybridization kinetics and thermodynamics of hairpins, and locked nucleic acids can be employed to increase the structural stability between hairpin probes and short amplicons.

[0093] The asymmetric hairpin probes provided herein can form a stem-loop structure with a 5' overhang by complementary sequences on a portion of the 5' end and 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' and 3' ends. When the probe is in the stem-loop structure, fluorescence can be sufficiently quenched. In some embodiments, and without being bound by any particular theory, in the presence of a complementary sequence, hybridization of the target and the probe results in the extension of the target along the 5' terminal overhang, and thus the separation of the fluorophore from the quencher, increasing fluorescence emission. Due to the presence of the stem structure, the hairpin probe can be significantly more specific than conventional probes, and the enhanced binding affinity and specificity derived from LNA modifications can enable the detection of short amplicons with superior sensitivity and specificity compared to conventional molecular beacons.

[0094] As provided herein, it has been found that conventional molecular beacons with blunt-ended stems cannot form stable hybrids with short target sequences and release detectable fluorescence signals (see, for example, the Examples). On the other hand, the probe design of the unconventional hairpin probes with 5' terminal overhangs provided herein can be sufficiently quenched by a quencher labeled at the recessed 3' end of the stem. Unexpectedly, compared to conventional molecular beacons with blunt-ended stems, the hairpin with a 5' overhang labeled with a fluorophore and a quencher placed at the 3' end of the hairpin results in enhanced performance. Additionally, contrary to the teachings of conventional probe designs, i.e., the probe should not overlap with the primer binding site on the same strand, in some embodiments, the probe design provided herein incorporates one of the primer sequences in the probe and enables sensitive and specific detection of the amplified target.

[0095] Figure 1Depicts a non - limiting exemplary schematic diagram of a traditional molecular beacon probe. In some embodiments, a single - stranded nucleic acid sequence of 15 - 40 bases forms a hairpin (stem and loop) structure, and the stem is formed by 6 - 7 GC pairs. The 5' and 3' ends may contain a fluorescent reporter and a quencher molecule. The loop sequence can be designed to be complementary to the target sequence. The target sequence can disrupt the stem structure and allow the reporter to fluoresce.

[0096] There are several challenges in the design of APA probes. The first challenge is the short amplicon size. The APA target is designed not to form a stable double - strand at high temperature (e.g., 68°C). The short amplicon may not be able to form a stable hybrid with the molecular beacon (MB) - unable to detect in real - time. The second challenge is the high assay temperature (e.g., 68°C). This may make it difficult for the probe to be in a "tight" conformation (stem stability), and it may be difficult to form a stable target - probe hybrid. The third challenge is the high similarity between the true product and the NTC product: unlike PCR, the APA product and the NTC differ by only a few bases (small spacer).

[0097] In some embodiments, the challenge of short amplicon size is addressed herein by leveraging the ability of 9°N to extend on the MB. In some embodiments, the challenge of high assay temperature is addressed by employing LNA. In some embodiments, the challenge of high similarity between the true product and the NTC product is addressed by partial amplicon / MB complementary design (sharing only one primer + spacer).

[0098] Figure 2 Depicts a non - limiting exemplary schematic diagram of a signal - generating oligonucleotide containing locked nucleic acid bases provided herein. Strategically placing LNA in the signal - generating oligonucleotides provided herein enables 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, where 4 LNAs are modified), and in some embodiments, each LNA base can increase by 3 - 7°C. The presence of LNA nucleotides can increase the specificity for the true target amplicon - LNA - modified spacer (and in some embodiments, can also discriminate against mismatched products). In some embodiments, the signal - generating oligonucleotides provided herein (e.g., MB) have LNA bases at the 3' end to prevent the polymerase (e.g., 9°N) from removing the quencher. Figure 18 Depicts a non - limiting exemplary schematic diagram of an asymmetric hairpin probe for amplicon detection provided herein.

[0099] Figures 3A - 3D Depicts the target ( Figure 3A ), extended target ( Figure 3B ), NTC ( Figure 3C ), and extended NTC ( Figure 3D)Non-limiting exemplary schematic diagrams of the signal-generating oligonucleotides provided herein for hybridization. In some embodiments, the design parameters and considerations for the signal-generating oligonucleotides (e.g., MB) include one or more of the following: (i) a target-probe hybridization Tm greater than 60 °C; (ii) a Tm for target probe extension greater than >68 °C (e.g., ~70 °C); (iii) an NTC-probe hybridization Tm of ~50 °C; and (iv) a Tm for NTC probe extension: 5 °C lower than the assay temperature (e.g., 68 °C).

[0100] Figure 4 A non-limiting exemplary schematic diagram of the signal-generating oligonucleotides provided herein is depicted. In some embodiments, the design of the signal-generating oligonucleotides provided herein takes into account one or more of the following stem stability considerations: (i) predominantly G-C pairs to provide sufficient Tm; (ii) stronger LNA modifications on opposing bases; (iii) LNA modifications / GC pairs at the bases of the loop can also be stabilizing; and (iv) a stable stem is important, but not so strong as to prevent robust opening of the target amplicon - a balance. In some embodiments, the signal-generating oligonucleotides disclosed herein can include a mismatched extension MB design, where the MB contains a complementary sequence that is one base shorter than the 3' end of the target. In some examples, this design feature can increase discrimination against NTC products.

[0101] Figure 5 A non-limiting exemplary schematic diagram of the signal-generating oligonucleotides provided herein is depicted. 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) - skewing; (ii) the strand with a GC-rich 3' end can be borrowed as part of the stem structure - facilitating burying the active end within the stem; and (iii) at most 2 bases complementary beyond the spacer region (to the right of the MB). The design of the signal-generating oligonucleotides provided herein can include computational parameters to identify the optimal positions of LNA bases to facilitate true target detection (see, for example, Table 1). Figures 6A - 6B A non-limiting exemplary signal-generating oligonucleotide provided herein is depicted. In some embodiments, software (e.g., Quickfold; http: / / unafold.rna.albany.edu / ?q=DINAMelt / Quickfold, or the OligoAnalyzer TM tool, https: / / www.idtdna.com / calc / analyzer) is used to design the structure of the signal-generating oligonucleotides provided herein to check whether the expected design is thermally favorable and can take into account the assay temperature and salt concentration.

[0102] Table 1: Signal - Generating Oligonucleotide Design

[0103]

[0104] APA can include very rapid polymerase amplification. APA can include (i) isothermal reactions, (ii) not requiring thermal denaturation, and / or (iii) not requiring helicase, recombinase, and / or nicking enzyme. APA can include two simple, short amplification primers and a reaction temperature of about 68 °C. In some embodiments, the amplification product is about 25-35 bases in length. In some embodiments, APA can be: (i) rapid (e.g., less than about 10 minutes, faster than currently available amplification techniques), (ii) sensitive (e.g., ≤10 copy target detection), and / or (iii) specific (e.g., two levels - amplification and detection). In some embodiments, APA can include real-time fluorescence detection with a modified molecular beacon having LNA. In some embodiments, the APA methods provided herein do not require sample purification.

[0105] In some embodiments provided herein, the 9°Nm polymerase has one or more of the following characteristics: (i) an extremely thermophilic archaeal polymerase (e.g., the optimal temperature for polymerization is ~70 °C); (ii) a significant ability to extend single-stranded DNA at a reaction temperature above the primer annealing temperature (e.g., extend 10-14mer at 68 °C); (iii) reduced 3'-5' exonuclease activity (e.g., remaining ~5%, resulting in certain background products and assay design methods); (iv) terminal deoxynucleotidyl transferase (TdT) activity (e.g., +A products); and (v) temperature-sensitive strand displacement activity (e.g., no strand displacement activity at 55 °C, but some strand displacement activity at 72 °C).

[0106] The assay design of the DNA assays provided herein can rely on one or more of the following assumptions: (i) using a conserved DNA target sequence (e.g., assuming a conserved DNA target is available and / or omitting target selection / sequence alignment without considering mismatches); (ii) primer design does not include beacon design considerations (e.g., direct assay screening / primer selection and / or different from RNA assay design); and (iii) based on manual design methods.

[0107] The design guidelines for the APA DNA assays disclosed herein can include one or more of the following: (i) the primer size can be between 10 nt and 14 nt (e.g., 12mer can be used for primer screening); (ii) the spacer size can be between 4-7 nt; (iii) the product size can be between 25 bp and 35 bp; and (iv) 30%-55% GC in each primer, which is interdependent with the Tm and size of the primer.

[0108] Figure 9 Depicts a non - limiting exemplary schematic related to the importance of the 3' end of primers in the APA assay. In some embodiments provided herein, the 3' end primer defines the APA assay and can be an important factor in the quality of the APA assay. In some embodiments, the 3' end of the primer contains APA - clean bases. In some embodiments, A, G, and / or C are APA - clean bases, with sequence - context dependence. 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 provided by the primer and the spacer (e.g., detected via molecular beacon).

[0109] Figures 10A - 10B Depicts a non - limiting exemplary schematic related to APA assay design. Figure 10A Depicts a non - limiting exemplary schematic related to the importance of the 5' end of primers in the APA assay. Figure 10B Depicts a non - limiting exemplary schematic related to 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, the primer size is selected such that the product Tm≈assay Tm. Primer size, Tm, and GC% can be interdependent factors. Primer and product Tm can be calculated using currently available tools (e.g., IDT Oligo Analyzer) under assay conditions (e.g., monovalent salts and Mg 2+ )). In some embodiments, the 5' end primer does not have more than 3 nt that are complementary to or adjacent to the spacer region sequence.

[0110] Figures 11A - 11C Depicts non - limiting exemplary interactions that can lead to background products: primer - dimer interactions ( Figure 11A ), homodimer interactions ( Figure 11B ), and primer - spacer interactions ( Figure 11C ). Non - target - specific interactions can be the main cause of background products. In some embodiments, the methods and compositions provided herein avoid primer - dimer interactions. For example, a heterodimer with a GC at the 3' end may not produce specific amplification ( Figure 11A ). For example, TGCA - 3' can form a strong homodimer and may not produce specific amplification ( Figure 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 only produce background products with truncated spacers ( Figure 11C )。

[0111] In some embodiments, the primers provided herein are modified with phosphorothioates. The phosphorothioates (PS) can be added after primer screening of the selected primers. In some embodiments, a single PS modification at the 3'-end can prevent the primer from being degraded 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 (1st) and the penultimate (2nd) 3'-end bases. In some embodiments, the phosphorothioate is located between the 2nd and 3rd 3'-end bases. Phosphorothioates can be added at positions containing repeated GCs to destabilize base pairing. In some embodiments, each primer has no more than 2 PS modifications at the 3'-end.

[0112] In some embodiments, and without being bound by any particular theory, the residual exonuclease activity in 9°N polymerase can digest mismatched bases at the 3'-end, producing products with truncated spacers. In some embodiments, the phosphorothioate modification provided herein renders the DNA resistant to nuclease degradation, and amplification can continue to produce the correct products. Figure 12 A non-limiting exemplary schematic diagram depicting the incorrect products produced (in an assay without phosphorothioate-modified primers) and the correct products produced (in an assay with phosphorothioate-modified primers) is shown.

[0113] The compositions and methods disclosed herein can follow one or more of the following DNA assay design rules: (i) select primers based on 3'-end characteristics; (ii) design the product size such that its Tm is generally ≤ the Tm of the APA assay; (iii) avoid all forms of harmful interactions based on the APA interaction rules; and (iv) after primer screening and selection, modify the promising primers with phosphorothioates at the 3'-end. The rules can be interdependent. Table 2 provides a comparison of the assay design of the APA assay provided herein with that of PCR.

[0114] Table 2: Assay Design - APA Compared to PCR

[0115] Standard PCR APA Primer Length 17 - 28bp 10 - 14bp Primer GC Content 50％-60％ 40％-55％ Primer Tm 55-70℃ <45℃ Amplicon (Product) Tm >80℃ ≈ Assay Tm (68°C) Amplicon Length 100 - 300bp 25 - 35bp 3’ - Clamp Characteristic (3’ - Terminal GC%) 3’ - Terminal is CG or GC No GC Clamp, but Rule #1, #3 The 3’ - Terminal of the Primer Should Not Be Complementary Yes Yes Avoid Hairpins Yes No (Due to Short Primer Length) Avoid Primer - Dimer Interaction Yes Yes Degenerate Bases at Specific Positions Yes Not Required

[0116] A challenge in APA product detection is that the Tm of APA products is close to the assay temperature (e.g., ~68 °C). Figure 15Depicts a non - limiting exemplary diagram related to the detection of APA products. In some embodiments, only a partial sequence in the APA product can be used for detection: the 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 APB2.2 assay product for detection can be ~20 °C lower than the optimal Tm for molecular beacon detection: 68 °C + 5 °C = 73 °C.

[0117] In some embodiments, the foregoing challenges are addressed by the signal - generating oligonucleotides containing LNA bases provided herein. Methods and compositions for real - time fluorescence detection using LNA probes are provided herein. Each probe can be modified with up to 6 LNAs. The choice of fluorophore / quencher pair can vary depending on the embodiment and includes those for the Fam, Hex, Rox, and / or Cy5 channels. The probes provided herein for addressing the foregoing problems can have an unconventional design. In some embodiments, the product extension on the probe can stabilize the product / probe duplex. In some embodiments, LNA bases are present at the 3' end to block the exonuclease activity from the 9°Nm polymerase. In some embodiments, LNA bases are employed in the probes provided herein to increase the Tm and / or LNA bases are employed at specific positions for mismatch discrimination. Figure 16 Depicts a non - limiting exemplary probe containing LNA bases provided herein. For example, the FluALNA probe can contain the reverse primer, the sequence of the spacer region, plus a two - base borrowing form (15mer) that allows P1 to extend along the loop, the stem, and 5 added bases. Each LNA base can increase the Tm by 3 - 7 °C.

[0118] The complexity of RNA assay design is increased many-fold compared to DNA assay design. Many considerations can be taken into account in RNA assay design, including mismatch sequence considerations, RT primer considerations, and beacon design considerations. RNA assay primer design can include mismatch, RT primer, and molecular beacon considerations. In some embodiments, RNA assay design takes into account mismatch positions to maximize inclusivity and / or RT primer positions. In some embodiments, LNA bases are used for probes (e.g., beacons). In some embodiments, the interaction rules provided herein are extended to include an interaction check of the consensus sequence relative to all mismatch sequences. The disclosed compositions and methods can follow one or more of the following RNA assay design rules: (i) define the 3’ end of the primer based on the mismatch position to maximize inclusivity of the mismatch strand (e.g., primer length 10-14mer, spacer selection 4-7nt, and / or avoid mismatches within 3nt of the 3’ end); (ii) include beacon design in primer selection (e.g., position LNA bases to avoid mismatch discrimination, MB-9°N interaction); (iii) include RT primer design in primer selection (e.g., use an RT primer having the same 3’ end as the forward primer, use an RT primer upstream of the forward primer); (iv) consider interactions (e.g., primer-dimer, primer-target, and / or primer-spacer interactions) and avoid non-specific interactions; and (v) 3’ end PS modification.

[0119] Some embodiments provide signal - generating oligonucleotides that contain nucleotide modifications (e.g., polymerase terminators) to block 9°Nm extension on the probe and non - specific extension of reaction background products. Such modifications can include LNA, RNA, 2′ - FDNA, 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 non - specific extension of reaction background products on the probe (work in progress). In some embodiments, the signal - generating oligonucleotides contain naphthyl azo compounds (Zen or iFQ), which in some embodiments not only block polymerase extension but also increase target / probe stability (Tm). Modifying groups that can be used as steric blocking moieties in the methods and compositions provided herein are disclosed in WO2012033848A1, the content of which is incorporated herein by reference in its entirety. The disclosure herein includes methods and compositions that contain modified molecular beacons (e.g., “protected probes”), which in some embodiments can improve assay specificity. The protected probes disclosed herein can be used in assays that employ archaeal polymerase amplification (“APA”) to isothermally amplify regions of interest within a target DNA (or cDNA) template for real - time analyte detection purposes. In some embodiments, the protected probes provided herein contain polymerase terminators (e.g., one or more 2′ - O′ - methyl RNA bases (“2′OM”)) within the molecular beacon probe to reduce non - specific product formation (and subsequent false - positive signals). Without being bound by any particular theory, in some embodiments, modifying specific bases within the modified molecular beacon construct can prevent unwanted “read - through” of the probe molecule. Currently available methods have not specifically used 2′OM bases for this purpose. Spacer modifications (such as C3 spacers) can be used for the same effect, but C3 spacers have been shown to be incompatible with the molecular beacons employed in the APA assays described herein. Without being bound by any particular theory, in some embodiments, the compositions and methods provided herein utilize the ability of 9dN (DNA - dependent DNA polymerase) to not read an RNA template. In particular, when a 2′OM base (i.e., methylated RNA base) is encountered within a given template, enzymatic processive synthesis is theoretically blocked (e.g., the enzyme cannot successfully “read” this position within the DNA template). 2′ - O - methyl RNA can be present in small RNAs (e.g., tRNA) and is a post - transcriptional modification, a natural 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 only causing a modest change in RNA:DNA stability. Additionally, this modification can show stability to single - strand ribonuclease attack and is generally 5 to 10 times less sensitive to DNase than DNA.2'-O-methyl (2’OM) modifications can be used in antisense oligonucleotides to improve stability and binding affinity to the target. In addition to the 2’OM modifications described herein, the following alternative base modifications were tested in the context of molecular beacons for APA: C3 spacer modifications; abasic site modifications; and unmethylated RNA bases. Based on the data generated, the 2’OM modification provided the greatest protection (without rendering the molecular beacon incompatible with APA). In some embodiments, some embodiments of the methods and compositions described herein can be used in concert with the systems, methods, compositions, and kits described in International Application No. PCT / US23 / 73521, filed September 6, 2023, entitled "MODIFIED MOLECULAR BEACONS FOR IMPROVED DETECTION SPECIFICITY", the content of which is incorporated herein by reference in its entirety.

[0120] Due to the expected overlap of the primer / probe footprints (the signal-generating oligonucleotide (e.g., molecular beacon) can include a first region that includes at least a portion of the sequence that includes the reverse primer and / or a second region that includes a sequence that is complementary to at least a portion of the forward primer), non-desired interactions between the signal-generating oligonucleotide (e.g., molecular beacon) and the forward and / or reverse primers result in non-specific product formation, which can be a unique and significant challenge for some embodiments of the amplification / detection assays provided herein. 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 can result in non-desired reverse primer / probe interactions. Thus, these inherent elements of some of the signal-generating oligonucleotide-based amplification / detection assays provided herein can result in non-specific product formation (and thus false-positive signals). However, the methods and compositions provided herein address these problems in the art and result in assays with reduced non-specific product formation, reduced false-positive signals, and / or an increased likelihood of accurately determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample. In some embodiments, the probes (e.g., molecular beacons) provided herein include a 5' modification (e.g., 5TEX615, FAM). In some embodiments, the probes (e.g., molecular beacons) provided herein include a 3' modification (e.g., 3IAbRQSp, IBFQ).

[0121] In some embodiments, methods are provided for detecting a target nucleic acid sequence in a sample. In some embodiments, the method comprises: 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 with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotide may comprise a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotide may comprise 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 comprise a 5' terminal domain having a length of from about 1 nt to about 6 nt and located 5' of 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 signal-generating oligonucleotide may comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0122] In some embodiments, methods are provided for detecting a target nucleic acid sequence in a sample. In some embodiments, the method comprises: 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 with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotide may comprise a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotide may comprise 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 comprise a 5' terminal domain having a length of from about 1 nt to about 15 nt and located 5' of 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 signal-generating oligonucleotide may comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0123] In some embodiments, signal-generating oligonucleotides are provided. The signal-generating oligonucleotides can be capable of hybridizing to a nucleic acid amplification product. The signal-generating oligonucleotides can comprise a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotides can comprise a loop domain positioned between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can 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 can be capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotides can comprise a 5' terminal domain having a length of from about 1 nt to about 6 nt and positioned 5' of 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 signal-generating oligonucleotides can comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0124] In some embodiments, signal-generating oligonucleotides are provided. The signal-generating oligonucleotides can be capable of hybridizing to a nucleic acid amplification product. The signal-generating oligonucleotides can comprise a 5' subdomain and a 3' subdomain. The signal-generating oligonucleotides can comprise a loop domain positioned between the 5' subdomain and the 3' subdomain. Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can 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 can be capable of hybridizing to the nucleic acid amplification product. The signal-generating oligonucleotides can comprise a 5' terminal domain having a length of from about 1 nt to about 15 nt and positioned 5' of 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 signal-generating oligonucleotides can comprise one or more LNA nucleotides in the loop domain, the 5' subdomain, and / or the 3' subdomain.

[0125] Nucleic acid amplification products can be produced 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, about 66°C to about 68°C). The nucleic acid amplification product hybridized to the signal generating oligonucleotide can be capable of being extended by an enzyme having polymerase activity, thereby producing an extended nucleic acid amplification product hybridized to the signal generating oligonucleotide. The extended nucleic acid amplification product can comprise a complement of the 5' terminal domain. The signal generating oligonucleotide can be capable of hybridizing to a mismatch product. The mismatch product hybridized to the signal generating oligonucleotide can be capable of being extended by an enzyme having polymerase activity, thereby producing an extended mismatch product hybridized to the signal generating oligonucleotide. The extended mismatch product can comprise a complement of the 5' terminal domain. The mismatch product can be a non-template control product and / or a non-target genotype. The forward primer can be capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer can be capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence. The nucleic acid amplification product can be produced by amplifying the target nucleic acid sequence with the forward primer and the reverse primer.

[0126] In some embodiments, 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 can comprise one, two, three, four, five, six, seven, or eight LNA nucleotides. The loop domain can comprise one or more LNA nucleotides, optionally the one or more LNA nucleotides enhancing the specificity and / or affinity of the signal generating oligonucleotide for the nucleic acid amplification product. Enhancing the specificity of the signal generating oligonucleotide for the nucleic acid amplification product can include increasing the mismatch discrimination between the nucleic acid amplification product and the mismatch product. The mismatch product can include a non-template control product and / or a non-target genotype. The terminal 3' nucleotide of the signal generating oligonucleotide can be an LNA nucleotide, optionally the LNA nucleotide reducing or preventing 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 can comprise one or more LNA nucleotides, optionally the one or more LNA nucleotides enhancing the stability of the paired stem domain. The paired stem domain can comprise base pairing of at least one pair of opposing LNA nucleotides. In some embodiments, the nucleotides located in the 5' terminal domain are not capable of intramolecular nucleobase pairing. The 5' terminal domain can have fewer than about 5 nt,

[0127] The signal generating oligonucleotide can comprise a label. The label can include a quencheable label (e.g., a fluorophore). The signal generating oligonucleotide can comprise a quencher. The label can be associated with the 3'-end of the signal generating oligonucleotide and the quencher can be associated with the 5'-end of the signal generating oligonucleotide, or the label can be associated with the 5'-end of the signal generating oligonucleotide and the quencher can be associated with the 3'-end of the signal generating oligonucleotide. When the quencher and the label are in close proximity, the quencher can be capable of quenching the signal generated by the label. In some embodiments, when the quencher and the label are not in close proximity, the quencher is not capable of quenching the signal generated by the label. In some embodiments, when the quencher and the label are in close proximity, the signal generated by the label is undetectable. When the quencher and the label are not in close proximity, the signal generated by the label can be detectable. When intermolecular base pairing between the 5'-subdomain and the 3'-subdomain forms a paired stem domain, the quencher and the label can be in close proximity. In some embodiments, when the signal generating oligonucleotide does not comprise a paired stem domain, the quencher and the label are not in close proximity. The detecting step can comprise contacting the nucleic acid amplification product with the signal generating oligonucleotide for hybridization. In some embodiments, detecting the nucleic acid amplification product can comprise using a real-time detection method. The detecting step can comprise detecting the signal of the label before, during, after the amplification reaction, or any combination thereof. In some embodiments, detecting the nucleic acid amplification product can comprise detecting the signal generated by the label of the signal generating oligonucleotide. The label can be a fluorophore and the signal can be fluorescence. In some embodiments, detecting the signal can comprise detecting the fluorescence emitted by the label.

[0128] In some embodiments, the amplification reaction and / or detection step comprises: contacting a 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 comprise a complement of the 5'-terminal domain. Extending the nucleic acid amplification product hybridized to the signal generating oligonucleotide with an enzyme having polymerase activity may be capable of disrupting the intramolecular nucleotide base pairing between the 5'-subdomain and the 3'-subdomain, thereby unwinding the paired stem domain. The label may be capable of generating 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 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 generate an extended nucleic acid amplification product hybridized to the signal generating oligonucleotide. Amplifying the target nucleic acid sequence in the amplification reaction mixture may comprise amplifying the target nucleic acid sequence under isothermal amplification conditions. The isothermal amplification conditions may comprise a constant temperature of about 30°C to about 72°C. The constant temperature may be about 55°C to about 75°C, about 56°C to about 68°C, or about 66°C to about 68°C. The amplification may be carried out at the optimal temperature of an enzyme having hyperthermophilic polymerase activity. The optimal temperature may be about 66°C to about 68°C (e.g., the constant temperature). The amplification may be carried out at a constant temperature. The nucleic acid amplification product may 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 may be higher than the Tm of the nucleic acid amplification product / signal generating oligonucleotide duplex (e.g., 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 Tm of the nucleic acid amplification product / signal generating oligonucleotide duplex may be at least or at most about 60°C; and the Tm of the extended nucleic acid amplification product / signal generating oligonucleotide duplex may be at least about 68°C. In some embodiments, in the absence of extension of the nucleic acid amplification product, the nucleic acid amplification product cannot form a stable duplex with the signal generating oligonucleotide.

[0129] In some embodiments, the amplification reaction comprises: 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 to produce 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 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.

[0130] The signal-generating oligonucleotide may be configured such that: the paired stem domain is stable at a constant temperature in the absence of the nucleic acid amplification product, and the paired stem domain is capable of unwinding when the nucleic acid amplification product hybridizes to the loop domain. The configuration may be achieved by varying 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, 5'-subdomain, and / or 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 (1) the sequence of the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement. The length of the spacer sequence may be from about 4 nt to about 7 nt and / or may have a GC content of less than about 50%.

[0131] The signal generating oligonucleotide can comprise a first region that comprises a sequence of at least a portion of a reverse primer. The signal generating oligonucleotide can comprise a second region that comprises a sequence complementary to at least a portion of a forward primer. The signal generating oligonucleotide can comprise a spacer region that comprises a sequence of at least a portion of a spacer sequence. The first region can comprise a sequence complementary to at least two 3'-terminal nucleotides of the forward primer and / or the reverse primer. The second region can comprise a sequence complementary to at least two 3'-terminal nucleotides of the forward primer and / or the reverse primer. The spacer region can comprise a sequence complementary to at least two 3'-terminal nucleotides of the forward primer and / or the reverse primer. The first region can comprise at least a portion of a 5'-subdomain and / or a loop domain, the spacer region can comprise at least a portion of a loop domain, and the second region can comprise at least a portion of a loop domain and / or a 3'-subdomain. The length of the signal generating oligonucleotide can be from about 10 nt to about 100 nt. The length of the second region, the spacer region, and / or the first region can be from about 1 nt to about 25 nt. The length of the 5'-subdomain, the 3'-subdomain, the loop domain, and / or the 5'-terminal domain can be from about 1 nt to about 25 nt. The length of the 5'-terminal domain can be from about 1 nt to about 6 nt, the length of the loop domain can be from about 4 nt to about 15 nt, and the length of the paired stem domain can be from about 3 bp to about 8 bp. The length of the nucleic acid amplification product can be from about 25 nt to about 35 nt. The target nucleic acid sequence can have a length no greater than about 20 nt to no greater than about 90 nt. The target nucleic acid sequence can have a length of about 30 nt. The spacer sequence can comprise a portion of the target nucleic acid sequence. The spacer sequence can be 1 to 10 bases in length. The length of the spacer sequence can be from about 4 nt to about 7 nt and / or can have a GC content of less than about 50%.

[0132] 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.

[0133] Forward primers and / or reverse primers can be configured to have a Tm of less than about 45°C; a length of about 5 nt to about 25 nt (e.g., a length of about 10 nt to about 14 nt); be configured to produce a nucleic acid amplification product having a length of about 25 nt to about 35 nt and a melting temperature within at least about 5°C of a constant temperature; contain one or more phosphorothioate bonds; 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 thymine bases. The 3' region may contain the first, second, third, and / or fourth nucleotides starting from the 3' end. In some embodiments, the 5' region of the forward primer and / or reverse primer does not contain more than 3 nt complementary to a spacer sequence, the region adjacent thereto, its complement, or any combination thereof. The 5' region may contain the first, second, third, and / or fourth nucleotides starting from the 5' end. The forward primer and / or reverse primer may contain a phosphorothioate bond between the first and second nucleotides starting 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 contain a phosphorothioate bond between the second and third nucleotides starting 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 2 phosphorothioate bonds. The 3' region may contain the first, second, third, and / or fourth nucleotides starting from the 3' end. The forward primer and / or reverse primer may contain one or more phosphorothioate bonds in a region containing a 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, 5' subdomain, and / or 3' subdomain increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product to at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or a number or range between any of these values) compared to a comparable method in which the signal-generating oligonucleotide does not contain LNA nucleotides. In some embodiments, the presence of the 5' terminal domain in the signal-generating oligonucleotide increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product to at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or a number or range between any of these values) compared to a comparable method in which the signal-generating oligonucleotide contains a blunt-ended hairpin structure.

[0134] The method can include 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 can include determining the presence, absence, and / or amount of dsDNA and / or nucleic acid containing 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 containing the target nucleic acid sequence in the sample.

[0135] As used herein, a "polymerase terminator" is a molecule (e.g., a modified nucleotide) capable of terminating or inhibiting polymerization. In some embodiments, at least one of the one or more polymerase terminators 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 terminators can comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. The signal generating oligonucleotide can comprise one or more polymerase terminators and / or one or more phosphorothioate linkages. The first region, the second region, and / or the spacer region can comprise one or more polymerase terminators. The one or more polymerase terminators can 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 terminators. The one or more polymerase terminators can comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. The one or more polymerase terminators can comprise an abasic site, a stable abasic site, a chemically trapped 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 site, an apyrimidinic 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 terminators comprise: one or more RNA bases, one or more 2'-methoxyethyl riboses (MOE), one or more LNA nucleotides, one or more 2'-fluorobases, 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-deoxyuridine, one or more inverted thymidines (inverted dT), one or more inverted dideoxythymidines (ddT), one or more dideoxycytidines (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 isodeoxycytidines (iso-dC), one or more isodeoxyguanosines (iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH 26 OPO3] groups, or any combination thereof.

[0136] One or more polymerase terminators can include 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 terminator can contain a modification incorporated between two bases of the signal generating oligonucleotide. The modification can be a naphthyl azo compound (e.g., Zen or iFQ). In some embodiments, the modification has the following structure:

[0137]

[0138] wherein the linking groups L1 and L2 that position the modification at an internal position of the signal generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy group; 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 an attachment point for a ligand; and X is a nitrogen or carbon atom, wherein if X is a carbon atom, the fourth substituent attached to the carbon atom can be hydrogen or a C1-C8 alkyl group. In some embodiments, the modification has the following structure:

[0139]

[0140] wherein the linking groups L1 and L2 that position the modification at an internal position of the signal generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy group; R1, R2, R4, R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group, or electron-donating group; R6, R7, R9-R 12 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group, or electron-donating group; R8 is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, or electron-withdrawing group; and X is a nitrogen or carbon atom, wherein if X is a carbon atom, the fourth substituent attached to the carbon atom can be hydrogen or a C1-C8 alkyl group. R8 can be NO2. In some embodiments, the modification has the following structure:

[0141]

[0142] When a forward primer binds to a signal-generating oligonucleotide to form a first undesired duplex, one or more polymerase terminators may be capable of terminating the polymerase extension of the forward primer of the first undesired duplex toward the 5'-end of the signal-generating oligonucleotide. One or more polymerase terminators may be capable of terminating the polymerase extension of the forward primer of the first undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. When a reverse primer binds to a signal-generating oligonucleotide to form a second undesired duplex, one or more polymerase terminators may be capable of terminating the polymerase extension of the reverse primer of the second undesired duplex toward the 5'-end of the signal-generating oligonucleotide. One or more polymerase terminators may be capable of terminating the polymerase extension of the reverse primer of the second undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. When an exogenous nucleic acid binds to a signal-generating oligonucleotide to form a third undesired duplex, one or more polymerase terminators may be capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5'-end of the signal-generating oligonucleotide. One or more polymerase terminators may be capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide.

[0143] The sample nucleic acid can comprise a nucleic acid containing a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence comprises: amplifying the target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplification comprises contacting the nucleic acid containing the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product. The nucleic acid can be double-stranded DNA. The nucleic acid can be a product of a reverse transcription reaction. The nucleic acid can be a product of a reverse transcription reaction from sample ribonucleic acid. The amplification can comprise producing the nucleic acid by a reverse transcription reaction. The sample nucleic acid can comprise sample ribonucleic acid, and the method can comprise contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA. In some embodiments, amplifying the target nucleic acid sequence comprises: (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 hyperthermophilic polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA contains the target nucleic acid sequence, and wherein the target nucleic acid sequence comprises a first strand and a second strand complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplification comprises contacting the dsDNA with: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product.

[0144] In some embodiments, if a forward primer binds to a signal-generating oligonucleotide to form a first undesired duplex, the forward primer of the first undesired duplex is extended by an enzyme having hyperthermophilic polymerase activity toward the 5'-end of the signal-generating oligonucleotide to produce a first undesired extension product. The first undesired extension product may be capable of being amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a forward primer and a reverse primer to form a first undesired amplification product. One or more polymerase terminators may be capable of terminating the polymerase extension of the forward primer of the first undesired duplex to produce a first stalled extension product. In some embodiments, the first stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a forward primer and a reverse primer to produce a first undesired amplification product. One or more polymerase terminators may be capable of terminating the polymerase extension of the forward primer of the first undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. In some embodiments, if a reverse primer binds to a signal-generating oligonucleotide to form a second undesired duplex, the reverse primer of the second undesired duplex is extended by an enzyme having hyperthermophilic polymerase activity toward the 5'-end of the signal-generating oligonucleotide to produce a second undesired extension product. The second undesired extension product may be capable of being amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to form a second undesired amplification product. One or more polymerase terminators may be capable of terminating the polymerase extension of the reverse primer of the second undesired duplex to produce a second stalled extension product. In some embodiments, the second stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to produce a second undesired amplification product. One or more polymerase terminators may be capable of terminating the polymerase extension of the reverse primer of the second undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide. In some embodiments, if a foreign nucleic acid binds to a signal-generating oligonucleotide to form a third undesired duplex, the foreign nucleic acid of the third undesired duplex is extended by an enzyme having hyperthermophilic polymerase activity toward the 5'-end of the signal-generating oligonucleotide to produce a third undesired extension product. The third undesired extension product may be capable of being amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to form a third undesired amplification product. One or more polymerase terminators may be capable of terminating the polymerase extension of the foreign nucleic acid of the third undesired duplex to produce a third stalled extension product. In some embodiments, the third stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of a reverse primer to produce a third undesired amplification product. One or more polymerase terminators may be capable of terminating the polymerase extension of the foreign nucleic acid of the third undesired duplex beyond one or more polymerase terminators of the signal-generating oligonucleotide.

[0145] The label may be capable of generating a false positive signal when the signal - generating oligonucleotide hybridizes to the first unwanted amplification product, the second unwanted amplification product, and / or the third unwanted amplification product. In some embodiments, when the signal - generating oligonucleotide hybridizes to the first unwanted amplification product, the second unwanted amplification product, and / or the third unwanted amplification product, the label generates a false positive signal. The signal and the false positive signal may be indistinguishable. In some embodiments, the generation of the first unwanted amplification product, the second unwanted amplification product, and / or the third unwanted 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, the detection of the false positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

[0146] In some embodiments, the presence of one or more polymerase terminators in a 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 (e.g., 1.1 - fold, 1.3 - fold, 1.5 - fold, 1.7 - fold, 1.9 - fold, 2 - fold, 3 - fold, 4 - fold, 5 - fold, 6 - fold, 7 - fold, 8 - fold, 9 - fold, 10 - fold, or a number or range between any of these values) compared to a signal - generating oligonucleotide that does not contain one or more polymerase terminators. In some embodiments, the production of the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate false - positive signals. In some embodiments, a signal - generating oligonucleotide that hybridizes to the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate false - positive signals. 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 of these values, 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 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 of these values, before a false - positive signal reaches a detectable level. The appearance of false - positive signals, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product can 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 of these values, compared to a comparable method in which the signal - generating oligonucleotide does not contain one or more polymerase terminators. In some embodiments, false - positive signals, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach a detectable level within at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the start of the amplification step. The production of false - positive signals, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product can be reduced by at least about 1.1 - fold (e.g., 1.1 - fold, 1.3 - fold, 1.5 - fold, 1.7 - fold, 1.9 - fold, 2 - fold, 3 - fold, 4 - fold, 5 - fold, 6 - fold, 7 - fold, 8 - fold, 9 - fold, 10 - fold, or a number or range between any of these values) compared to a comparable method in which the signal - generating oligonucleotide does not contain one or more polymerase terminators.

[0147] Amplifying a target nucleic acid sequence can include generating a nucleic acid amplification product at a detectable level in about 20 minutes, about 15 minutes, or about 10 minutes. Detection is performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes starting from the time when the reagent composition contacts the treated sample.

[0148] The lysis buffer can contain one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. The pH of the lysis buffer can be from about 1.0 to about 10.0 (e.g., about 2.2). The sample nucleic acid can include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample nucleic acid can include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, one or more amplification reagents include: reverse transcriptase; an enzyme having hyperthermophilic DNA polymerase activity; and / or dNTPs. In some embodiments, the enzyme having hyperthermophilic DNA polymerase activity has reverse transcriptase activity; forward primer; reverse primer; reverse transcription primer. The reagent composition can be lyophilized, heat-dried, and / or contain one or more additives. In some embodiments, one or more additives include: Tween 20, Triton X-100, and / or Tween 80; amino acids; sugars or sugar alcohols; and / or polymers. 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, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. Contacting the reagent composition with the treated sample can include dissolving the reagent composition in the treated sample. In some embodiments, one or more lysis reagents contain: from about 0.001% (w / v) to about 1.0 (w / v) of the treated sample (e.g., about 0.2% (w / v) of the treated sample); and / or a detergent (e.g., one or more of a cationic surfactant, anionic surfactant, nonionic surfactant, and zwitterionic surfactant).

[0149] In some embodiments, the method: is performed in a single reaction vessel; does not include using any enzyme other than reverse transcriptase and an enzyme having hyperthermophilic DNA polymerase activity; does not include using any enzyme other than an enzyme having hyperthermophilic DNA polymerase activity; does not include thermal denaturation and / or enzymatic denaturation of the nucleic acid during the amplification step; and / or does not include contacting the nucleic acid with single-stranded DNA binding protein.

[0150] The target nucleic acid sequence can have a length of no more than about 20 nucleotides to no more than about 90 nucleotides (e.g., about 30 nucleotides). The forward primer, reverse primer, and / or reverse transcription primer can be about 8 to 16 bases in length. The nucleic acid amplification product can be about 20 to 40 bases in length. The spacer sequence can include a portion of the target nucleic acid sequence. The spacer sequence can be 1 to 10 bases in length. The isothermal amplification conditions can include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, optionally about 56°C to about 67°C. The amplification can be carried out for a period of about 5 minutes to about 60 minutes (e.g., a period of about 15 minutes). The amplification can be carried out under isothermal amplification conditions without helicase, without single-stranded binding protein, without lysing agent, and without recombinase. The amplification can be carried out using PCR, LCR, LAMP, SDA, replicase-mediated amplification, immunoamplification, NASBA, 3SR, rolling circle amplification, or TMA. The PCR can be real-time PCR and / or QRT-PCR.

[0151] The enzyme having hyperthermophilic DNA polymerase activity has an amino acid sequence that can 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 DNA polymerase activity has an amino acid sequence that can be at least about 95% identical to the amino acid sequence of SEQ ID NO: 31. The enzyme having hyperthermophilic DNA polymerase activity can be a polymerase comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the enzyme having hyperthermophilic DNA polymerase activity has low exonuclease activity or no exonuclease activity. The sample ribonucleic acid can be contacted simultaneously with a reverse transcriptase and an enzyme having hyperthermophilic DNA polymerase activity. The sample ribonucleic acid can be contacted simultaneously with a reverse transcriptase, an enzyme having hyperthermophilic DNA polymerase activity, a forward primer, and a reverse primer. The sample ribonucleic acid can be contacted simultaneously with a reverse transcriptase, an enzyme having hyperthermophilic DNA polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer.

[0152] In some embodiments, the amplification includes and / or does not include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplification does not include LAMP.

[0153] In some embodiments, the method does not include one or more of the following: (i) dilution of the processed sample; (ii) dilution of the amplification reaction mixture; (iii) heat denaturation of the processed sample; (iv) sonication of the processed sample; (v) sonication of the amplification reaction mixture; (vi) addition of ribonuclease inhibitor to the processed sample; (vii) addition of ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of sample nucleic acid; (x) purification of nucleic acid amplification products; (xi) removal of one or more lysis agents from the processed sample or the amplification reaction mixture; (xii) heat denaturation and / or enzymatic denaturation of sample nucleic acid before and / or during amplification; and (xiii) addition of ribonuclease H to the processed sample or the amplification reaction mixture.

[0154] The term "isothermal amplification reaction" shall be given its ordinary meaning and shall also include reactions in which there is no significant change in temperature during the reaction. In some embodiments, during the main enzymatic reaction step in which amplification occurs, the temperature deviation of the isothermal amplification reaction does not exceed 10 °C, such as not exceeding 5 °C or not exceeding 2 °C. According to the methods of nucleic acid isothermal amplification, different enzymes can be used for amplification. Isothermal amplification compositions and methods are described in WO2017176404, the content of which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the methods and components described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer resists the formation of precipitation 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, entitled "NON-OPAQUELYTIC BUFFER COMPOSITION FORMULATIONS" and filed on February 3, 2023, the content of which is incorporated herein by reference in its entirety.

[0156] Except for acid and / or low pH conditions, some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote melting). Compositions, kits, and methods for nucleic acid detection in which nucleic acid strands dissociate under low pH conditions (e.g., via 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 GENOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS", the content of which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the methods and compositions described herein can include a lysis buffer and / or a reagent composition. A lysis buffer containing a lytic agent and a reducing agent, and a reagent composition containing an amplification agent and one or more protective agents (e.g., cyclodextrin compounds) capable of chelating the lytic agent are described in WO2022198086, the content of which is incorporated herein by reference in its entirety.

[0158] Some embodiments of the methods and compositions described herein can be used in conjunction with the systems, methods, compositions, and kits for monitoring amplification reactions described in International Application No. PCT / US23 / 73519, filed September 6, 2023, entitled "HAIRPIN INTERNAL CONTROL FOR ISOTHERMAL NUCLEIC ACID AMPLIFICATION", the content of which is incorporated herein by reference in its entirety.

[0159] Some embodiments of the methods and compositions described herein can include probes that melt at a temperature different from the optimal APA reaction temperature to enable multiplexing of targets and / or one or more internal controls. Compositions, kits, and methods for multiplex nucleic acid detection are described in International Application No. PCT / US23 / 73521, filed September 6, 2023, entitled "ARCHEAL POLYMERASE AMPLIFICATION", the content of which is incorporated herein by reference in its entirety.

[0160] In some embodiments, some embodiments of the methods and compositions described herein can be used in conjunction with the systems, methods, compositions, and kits for detecting pathogens described in International Application No. PCT / US23 / 73521, entitled "MODIFIED MOLECULAR BEACONS FOR IMPROVED DETECTION SPECIFICITY", filed on September 6, 2023, the content of which is incorporated herein by reference in its entirety.

[0161] Nucleic Acids, Subjects, Samples, and Nucleic Acid Processing

[0162] The present disclosure provides methods and compositions for amplifying nucleic acids. The terms “nucleic acid” and “nucleic acid molecule” are used interchangeably herein. These terms refer to any composed nucleic acid, such as DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), etc.), RNA (e.g., messenger RNA (mRNA), short interfering RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-natural backbones, etc.), RNA / DNA hybrids, and polyamide nucleic acids (PNA)), all of which can be in single-stranded or double-stranded form and, unless otherwise restricted, can include known analogs of natural nucleotides that are capable of functioning in a manner similar to the naturally occurring nucleotides. The nucleic acid can be or can be from a plasmid, phage, autonomously replicating sequence (ARS), centromere, artificial chromosome, chromosome, or other nucleic acid capable of replicating or being replicated in vitro or in a host cell, cell, nucleus, mitochondrion, or cytoplasm of a cell. Unless specifically defined, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to the naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, interspecies homologs, single nucleotide polymorphisms (SNP), and complementary sequences, as well as the explicitly indicated sequence. In particular, degenerate codon substitutions can be achieved by generating a sequence in which one or more selected (or all) codons have the third position substituted with a mixture of bases and / or deoxyinosine residues. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene. As equivalents, the term can also include derivatives, variants, and analogs of RNA or DNA synthesized from nucleotide analogs, single-stranded (“sense” or “antisense,” “plus” or “minus” strand, “forward” reading frame or “reverse” reading frame, “forward” strand or “reverse” strand) and double-stranded polynucleotides. The term “gene” means a DNA segment involved in producing a polypeptide chain; and typically includes regions (leader and trailer) before and after the coding region involved in transcription / translation and regulation of transcription / translation of the gene product, as well as intervening sequences (introns) between individual coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of a nucleic acid (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). For RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed in terms of the number of bases.

[0163] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid may be referred to as a target sequence, target polynucleotide, and / or target polynucleotide sequence, and may include double-stranded and single-stranded nucleic acid molecules. The target nucleic acid may be, for example, DNA or RNA. When the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may contain a single-stranded target sequence. When the target nucleic acid is double-stranded, the target nucleic acid generally includes a first strand and a second strand. The first strand and the second strand may be referred to as the sense strand and the antisense strand, and are generally complementary to each other. When the target nucleic acid is single-stranded, a complementary strand may be produced, for example, by polymerization and / or reverse transcription, rendering the target nucleic acid double-stranded and having a first / sense strand and a second / antisense strand.

[0164] The target sequence may refer to the sense or antisense strand of a nucleic acid sequence, and may also refer to a sequence present on the target nucleic acid, amplified copy, or amplification product of the original target sequence. The target sequence may be a subsequence within a larger polynucleotide. For example, the target sequence may be a short sequence (e.g., 20 to 50 bases) within a nucleic acid fragment, chromosome, plasmid that is targeted for amplification. In some embodiments, the target sequence may refer to the sequence within the target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, the target sequence may refer to the entire sequence that is targeted for amplification, or may refer to the subsequence within the target nucleic acid to which the oligonucleotide binds. The amplification product may be a larger molecule that includes the target sequence and at least one other sequence or other nucleotides. The amplification product may be approximately the same length as the target sequence, e.g., exactly the same length as the target sequence. The amplification product may include or consist of the target sequence.

[0165] The length and / or guanine-cytosine (GC) concentration (percentage) of the target sequence may depend in part on the temperature at which the amplification reaction is run, and this temperature may depend in part on the stability of the polymerase used in the reaction. Sample assays may be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, when the polymerase is stable up to 60°C to 65°C, the length of the target sequence may be, for example, from 19 to 50 nucleotides, or the length may be, for example, from about 40 to 50, 20 to 45, 20 to 40, or 20 to 30 nucleotides. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0166] Target nucleic acids can 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, the target nucleic acid can 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 can include any nucleic acid from any genome, such as, for example, animal, plant, insect, viral, and bacterial genomes (e.g., genomes present in spores). In some embodiments, the genomic target nucleic acid is within a specific genomic locus or more than one genomic locus. Genomic loci can include any one or combination of open reading frame DNA, non-transcribed DNA, intron sequences, exon sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequence deemed to be associated with a given genomic locus.

[0167] The target sequence can contain one or more repeat elements (e.g., multiple repeat sequences, inverted repeat sequences, 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, chromosome, genome, plasmid) as a repeat element (e.g., multiple repeat sequences, inverted repeat sequences, palindromic sequences, tandem repeats, microsatellite repeats, minisatellite repeats, etc.). For example, the target sequence can occur multiple times as a repeat element, and one, some, or all occurrences of the target sequence within the repeat element can be amplified using the methods described herein (e.g., using a single pair of primers). In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, chromosome, genome, plasmid) as a repeat and / or an intragenic homolog.

[0168] The target nucleic acid can include microRNAs. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21 to 23 nucleotides in length) and single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNA and are partially complementary to messenger RNA. The target nucleic acid can include microRNA precursors, such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop structure RNAs that are further processed into miRNAs. The target nucleic acid can include short interfering RNAs (siRNAs), which are short (e.g., about 20 to 25 nucleotides in length) and at least partially double-stranded RNA molecules involved in RNA interference (e.g., downregulation of viral replication or gene expression).

[0169] The nucleic acids used in the methods described herein can be obtained from any suitable biological sample or specimen, e.g., isolated from a specimen 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), swine (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), bears (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female, and the subject can be of any age (e.g., embryo, fetus, infant, child, adult).

[0170] A sample or test sample can be any specimen isolated or obtained from a subject or a part thereof. Non-limiting examples of specimens include fluids or tissues from a subject, including but not limited to blood or blood products (e.g., serum, plasma, etc.), cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluids (e.g., bronchoalveolar, gastric, peritoneal, catheter, ear, arthroscopic), serum, plasma, urine, aspirate, biopsy specimens, paracentesis specimens, cells (e.g., blood cells) or parts thereof (e.g., mitochondria, nuclei, extracts, etc.), washings from the female genital tract, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph fluid, bile, tears, sweat, breast milk, mammary fluid, hard tissues (e.g., liver, spleen, kidney, lung, or ovary), etc. or combinations thereof. The term blood includes whole blood, blood products, or any fraction of blood, such as serum, plasma, buffy coat, or analogous fractions as conventionally defined. Plasma refers to the fraction of whole blood produced by centrifuging blood treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after a blood sample has coagulated. Fluid or tissue specimens are typically collected according to standard protocols commonly followed by a hospital or clinic. For blood, an appropriate volume of peripheral blood (e.g., between 3 - 40 mL) is typically collected and can be stored according to standard procedures either before or after preparation.

[0171] A sample can include a sample containing spores, viruses, cells, nucleic acids from prokaryotes or eukaryotes, and / or any free nucleic acids. For example, the methods described herein can be used to detect nucleic acids outside of spores (e.g., without the need for lysis). A sample can be isolated from any material suspected of containing a target sequence, such as from a subject as described above. In some embodiments, the target sequence is present in air, plants, soil, or other materials suspected of containing organisms.

[0172] Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method can be used to isolate, extract, and / or purify nucleic acids from biological samples, including DNA preparation methods in the art, as well as various commercially available reagents or kits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Micro Kit, or QiaAmp DNA Blood Micro Kit (Qiagen, Hilden, Germany), GenomicPrep TM Blood DNA Isolation Kit (Promega, Madison, Wis.), GFX TM Genomic Blood DNA Purification Kit (Amersham, Piscataway, N.J.), etc. or combinations thereof. U.S. Patent No. 7,888,006 provides a DNA purification method and does not disclose the compositions (e.g., lysis buffers, protectants) and methods provided herein.

[0173] In some embodiments, a cell lysis procedure is performed. Cell lysis can be carried out prior to initiating the amplification reactions 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 carried out by chemical (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or combinations thereof), physical (e.g., French press, sonication, etc.), or electroporation lysis methods. For example, chemical methods typically use lysis agents to disrupt cells and extract nucleic acids from cells, followed by treatment with chaotropic salts. In some embodiments, cell lysis includes the use of detergents (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis includes the use of ionic detergents (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods such as grinding after freeze / thaw, use of a cell press, etc. can also be useful. A high-salt lysis procedure can also be used. For example, an alkaline lysis procedure can be used. The latter procedure traditionally involves the use of a phenol-chloroform solution, and an alternative phenol-chloroform-free procedure involving three solutions can be used. In the latter procedure, one solution can contain 15 mM Tris, pH 8.0; 10 mM EDTA, and 100 μg / ml RNase; a second solution can contain 0.2 N NaOH and 1% SDS; and a third solution can contain, for example, 3 M KOAc, pH 5.5. In some embodiments, cell lysis buffers are used in conjunction with the methods and components described herein.

[0174] Nucleic acids can be provided for use in the methods described herein without processing a sample containing the nucleic acids. For example, nucleic acids can be provided for use in the amplification methods described herein without prior nucleic acid purification. In some embodiments, target sequences are amplified directly from a sample (e.g., without performing any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids are provided for use in the methods described herein after processing a sample containing the nucleic acids. For example, nucleic acids can be extracted, isolated, purified, or partially purified from a sample. The term “isolated” generally refers to a nucleic acid that has been removed from its original environment (e.g., the natural environment if it is naturally occurring or the host cell if it is exogenously expressed) and thus altered from its original environment by human intervention (e.g., “by the hand of man”). An “isolated nucleic acid” can refer to a nucleic acid removed from a subject (e.g., a human subject). An isolated nucleic acid can provide a lower amount of non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than is present in the source sample. A composition comprising an isolated nucleic acid can be free of from about 50% to greater than 99% of non-nucleic acid components. A composition comprising an isolated nucleic acid can be free of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of non-nucleic acid components. The term “purified” generally refers to a nucleic acid provided that contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present prior to the nucleic acid purification procedure. A composition comprising a purified nucleic acid can be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of other non-nucleic acid components.

[0175] Nucleic acids can be provided for use in the methods described herein without modifying the nucleic acids. Modifications can include, for example, denaturation, digestion, nicking, unwinding, incorporation and / or ligation of heterologous sequences, addition of epigenetic modifications, addition of labels (e.g., radioactive labels such as 32 P, 33 P, 125 I, or 35 S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxin, antigens, haptens, fluorescent dyes), etc. Thus, in some embodiments, unmodified nucleic acids are amplified.

[0176] The methods for detecting target nucleic acid sequences (single-stranded or dsDNA and / or RNA) in a sample disclosed herein can detect target nucleic acid sequences (e.g., DNA or RNA) with a high degree of sensitivity. In some embodiments, the method can be used to detect target DNA / RNA present in a sample containing more than one RNA / DNA (including the target RNA / DNA and more than one non-target RNA / DNA), where the target RNA / DNA is present at one or more copies per 10, 20, 25, 50, 100, 500, 10 3 ones, 5×10 3 ones, 10 4 ones, 5×10 4 ones, 10 5 ones, 5×10 5 ones, 10 6 ones or 10 7 ones of non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meaning and shall also refer to DNA, or RNA, or a combination of DNA and RNA.

[0177] For the method of detecting target RNA / DNA in a sample, the detection threshold can be, for example, 10 nM or less. The term "detection threshold" shall be given its ordinary meaning and shall also describe the minimum amount of target RNA / DNA that must be present in the sample for detection to occur. As an illustrative example, when the detection threshold is 10 nM, then a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the disclosed method has a detection threshold of 5 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, 0.005 nM or less, 0.001 nM or less, 0.0005 nM or less, 0.0001 nM or less, 0.00005 nM or less, 0.00001 nM or less, 10 pM or less, 1 pM or less, 500 fM or less, 250 fM or less, 100 fM or less, 50 fM or less, 500 aM (attomolar) or less, 250 aM or less, 100 aM or less, 50 aM or less, 10 aM or less or 1 aM or less. In some embodiments, the disclosed compositions or methods exhibit attomolar (aM), femtomolar (fM), picomolar (pM) and / or nanomolar (nM) detection sensitivity.

[0178] The sample can comprise sample nucleic acids (e.g., more than one sample nucleic acid). The term "more than one" as used herein is meant to refer to two or more. Thus, in some embodiments, the sample comprises two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more) sample nucleic acids (e.g., DNA / RNA). The disclosed methods can be used as a very sensitive way to detect target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, the sample comprises 5, 10, 20, 25, 50, 100, 500, 10 3 species, 5×10 3 species, 10 4 species, 5×10 4 species, 10 5 species, 5×10 5 species, 10 6 species, or 10 7 species, 50 species or more of DNA / RNA. The sample can comprise DNA / RNA from cells (e.g., eukaryotic cells, mammalian cells, or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0179] As used herein, the term "sample" should be given its ordinary meaning and should include any sample containing RNA and / or DNA (e.g., to determine whether target DNA and / or target RNA is present in a population of RNA and / or DNA). The sample can be a biological sample or an environmental sample. The sample can be derived from any source. For example, the sample can be a synthetic combination of purified DNA and / or RNA; the sample can be a cell lysate, a DNA / RNA-rich cell lysate, or DNA / RNA isolated and / or purified from a cell lysate. The sample can be from a patient (e.g., for diagnostic purposes). The sample can be from permeabilized cells, cross-linked cells, tissue sections, or a combination thereof. The sample can be from tissue prepared by cross-linking followed by defatting and conditioning to form a uniform refractive index. The sample can comprise target nucleic acids (e.g., target DNA / RNA) and more than one non-target DNA / RNA. In some embodiments, the target DNA / RNA is present at 10, 20, 25, 50, 100, 500, 10 3 per, 5×10 3 per, 10 4 per, 5×10 4 per, 10 5 per, 5×10 5individuals, 10 6 individuals or 10 7 copies of non-target DNA / RNA are present in the sample.

[0180] Samples regarding a patient include biological source blood and other liquid samples, solid tissue samples such as biopsy samples or tissue cultures or cells derived therefrom and their progeny, and samples that are manipulated in any way (such as by treatment with reagents), washed, or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA) after their acquisition. Samples can include or be the following: biological samples, including but not limited to clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), and also include tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, etc. Biological samples can include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), e.g., samples containing RNA obtained from such cells (e.g., cell lysates containing RNA or other cell extracts). In some embodiments, environmental samples are the following, or obtained from the following: food samples, beverage samples, paper surfaces, fabric surfaces, metal surfaces, wood surfaces, plastic surfaces, soil samples, fresh water samples, wastewater samples, brine samples, samples exposed to atmospheric air or other gases, their cultures, or any combination thereof.

[0181] The source of the sample can be (or be suspected to be) diseased cells, fluids, tissues, or organs; or normal (non-diseased) cells, fluids, tissues, or organs. In some embodiments, the source of the sample is (or is suspected to be) a pathogen-infected cell, tissue, or organ. For example, the source of the sample can be an individual who may or may not be infected, and the sample can be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, fecal sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., oral swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal mucus, tears, buffy coat, mucosal sample, epithelial cell sample (e.g., epithelial cell scrape), etc.) and its culture. The sample can be a cell-free liquid sample or a liquid sample containing cells. Pathogens can be viruses, fungi, worms, protozoa, malaria parasites, Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites, etc. "Worms" include roundworms, heartworms, and phytophagous nematodes (Nematoda), flukes (Trematoda), acanthocephalans, and tapeworms (Cestoda). Protozoan infections include Giardia spp., Trichomonas spp. infections, African trypanosomiasis, amebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens such as parasitic / protozoan pathogens include, but are not limited to: Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include, for example, immunodeficiency viruses (e.g., HIV); influenza viruses; dengue fever; West Nile virus; herpes viruses; yellow fever virus; hepatitis C virus; hepatitis A virus; hepatitis B virus; papillomavirus, etc. Pathogenic viruses can include DNA viruses, such as: Papovaviridae (e.g., HPV, polyomavirus); Hepadnaviridae; Herpesviridae (e.g., HSV (e.g., HSV I, HSV II), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, pityriasisRosea), Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., Aviadenovirus, Atadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, goatpox virus, pseudocowpox, bovine papular stomatitis virus; Tanapox virus, Yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parvovirus 4G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like. Non-limiting examples of pathogens include Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Borrelia burgdorferi, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, human serum parvovirus-like virus, respiratory syncytial virus, measles virus, adenovirus, human T-cell leukemia virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, papillomavirus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovisbovis), Eimeria species (e.g., Eimeria tenella), Onchocerca volvulus, 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-lymphotropic Virus type 1 (HTLV-1), Herpes simplex virus, Herpesvirus 6, Herpesvirus 7, JC virus, Influenza A virus, Influenza B virus, Influenza C virus, Rotavirus, Human adenovirus, Human enterovirus, Hantavirus, Legionella dumoffii, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsii, Ehrlichia species (e.g., Ehrlichia chaffeensis), Borrelia burgdorferi, Yersinia pestis, Chlamydia pneumoniae, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma species (e.g., Mycoplasma arthritidis), Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini, Acholeplasma laidlawiilaidlawii), Mycoplasma salivarium, and Mycoplasma pneumoniae.

[0182] Amplification

[0183] The present disclosure provides methods for amplifying nucleic acids. In some embodiments, nucleic acids are amplified using a suitable amplification method. Nucleic acid amplification generally involves the enzymatic synthesis of nucleic acid amplicons (copies) that contain a sequence complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is performed in a single container, single chamber, and / or single volume (i.e., a continuous volume). In some embodiments, the amplification method and the detection method (e.g., the detection methods described herein) are performed in a single container, single chamber, and / or single volume (i.e., a continuous volume).

[0184] The terms "amplify", "amplification", "amplification reaction", or "amplifying" refer to any in vitro process for doubling the copy number of a target nucleic acid. Amplification sometimes refers to an "exponential" increase in the target nucleic acid. "Amplification" can also refer to a linear increase in the quantity of the target nucleic acid, but is distinct from a single, one-step primer extension. In some embodiments, a limited amplification reaction, also known as pre-amplification, can be performed. Pre-amplification is a method in which a limited amount of amplification occurs due to a small number of cycles being performed, e.g., 10 cycles. Pre-amplification can allow some amplification, but stops amplification before the exponential phase and generally produces approximately 500 copies of the desired nucleotide sequence. The use of pre-amplification can limit inaccuracies associated with depleted reactants in certain amplification reactions and can also reduce amplification bias due to the nucleotide sequence or species abundance of the target. In some embodiments, a single primer extension can be performed as a prelude to linear or exponential amplification.

[0185] This document presents a general description of the amplification process. Primers (e.g., the oligonucleotides described herein) are contacted with the target nucleic acid, and for example, complementary sequences anneal or hybridize to each other. The primers can anneal to the target nucleic acid at or near the sequence of interest (e.g., adjacent, contiguous, etc.). The primer that anneals to the target can be referred to as a primer-target hybrid, hybridized primer-target, or primer-target duplex. When referring to a nucleotide sequence of interest, the terms near or adjacent refer to the distance (e.g., number of bases) or region between the primer terminus and one or more nucleotides (e.g., nucleotide sequence) of the target. Generally, adjacent is in the range of about 1 nucleotide to about 50 nucleotides from the nucleotide or nucleotide sequence of interest (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides). In some embodiments, a set of primers (e.g., a pair of primers, forward and reverse primers, a first oligonucleotide and a second oligonucleotide) anneal within about 1 to 20 nucleotides of the nucleotide or nucleotide sequence of interest and produce an amplification product. In some embodiments, the primer anneals within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended by a polymerase along the target (i.e., the template strand) to produce a complementary strand. For example, several cycles of primer annealing and extension can be carried out until a detectable amount of amplification product is produced. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step.

[0186] The components of the amplification reaction (e.g., one or more amplification reagents) can include, for example, one or more primers (e.g., a single primer, primer pair, primer set, oligonucleotide, multiple primer sets for multiplex amplification, etc.), a nucleic acid target (e.g., the target nucleic acid from a sample), one or more polymerases, nucleotides (e.g., dNTPs, etc.), and a suitable buffer (e.g., a buffer containing a detergent, reducing agent, monovalent ions, and divalent ions). The amplification reaction can also include one or more of the following: reverse transcriptase, reverse transcription primer, and one or more detection agents.

[0187] Nucleic acid amplification can be carried out in the presence of natural nucleotides, such as deoxyribonucleoside triphosphates (dNTPs) and / or derived nucleotides. Natural nucleotides generally refer to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid. Derived nucleotides are generally nucleotides different from natural nucleotides. Ribonucleoside triphosphates are referred to as NTP or rNTP, where N can be A, G, C, U. Deoxynucleoside triphosphate substrates are referred to as dNTP, where N can be A, G, C, T, or U. Monomeric nucleotide subunits can be represented 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 or colorimetric label). For example, nucleic acid amplification can be carried out in the presence of labeled dNTPs, such as radiolabels, such as 32 P, 33 P, 125 I, or 35 S; enzyme labels, such as alkaline phosphatase; fluorescent labels, such as fluorescein isothiocyanate (FITC); or other labels, such as biotin, avidin, digoxigenin, antigen, hapten, or fluorescent dyes. In some embodiments, nucleic acid amplification can be carried out in the presence of modified dNTPs, such as, for example, heat-activated dNTPs (e.g., CleanAmp TM dNTP from TriLink).

[0188] One or more amplification reagents can include non-enzyme components and enzyme components. Non-enzyme components can include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions. In some embodiments, the non-enzyme 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 endonucleases, nicking enzymes, recombinases, etc. In some embodiments, the enzyme component consists of a polymerase or consists of a polymerase and a reverse transcriptase. Thus, such an enzyme component will exclude other proteins (e.g., nucleic acid-binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nicking enzymes, recombinases, etc.

[0189] In some embodiments, the amplification conditions include enzymatic activity (e.g., the enzymatic activity provided by a polymerase or the enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include the enzymatic activity provided by an enzyme other than a polymerase and / or a reverse transcriptase, such as helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nicking enzyme, recombinase, etc. The polymerase activity and the reverse transcriptase activity can be provided by separate enzymes or separate enzyme types (e.g., a polymerase and a reverse transcriptase), or by a single enzyme or enzyme type (e.g., a polymerase).

[0190] The amplification of nucleic acids can include non-thermal cycling types of PCR. In some embodiments, the amplification of nucleic acids includes an isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process carried out at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature remains substantially constant during the amplification reaction process. Isothermal amplification conditions generally do not include the thermal cycling (i.e., cycling between a higher temperature and a lower temperature) component in the amplification process. When amplifying under isothermal conditions, the reaction can be maintained at a substantially constant temperature, which means that the temperature may not be precisely maintained at one temperature. For example, during an isothermal amplification process, small temperature fluctuations (e.g., ±1°C to 5°C) may occur due to, for example, environmental or device-based variables. Generally, the entire reaction volume is maintained at a substantially constant temperature, and the isothermal reactions herein generally do not include amplification conditions that rely on temperature gradients generated within the reaction vessel and / or convection-based temperature cycling.

[0191] The isothermal amplification reactions herein can be carried out at a substantially constant temperature. In some embodiments, the isothermal amplification reactions herein are carried out at a temperature from about 55°C to about 75°C, such as at or below the following temperatures or at or below about the following temperatures: 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, or about 75°C, or any quantity or range between any two of these values. In some embodiments, a temperature element (e.g., a heat source) is maintained at a substantially constant temperature, such as at or below about 75°C, at or below about 70°C, at or below about 65°C, or at or below about 60°C.

[0192] The amplification process of the present disclosure can be carried out for a certain length of time, such as until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product can be detected by any suitable detection process and / or the detection processes described herein. The amplification process can be carried out for a length of time of about 20 minutes or less, or about 10 minutes or less. For example, the amplification process can be carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or any amount or range between any two of these values.

[0193] In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that denature nucleic acids. In some embodiments, during the amplification step (and / or other steps), the nucleic acid target can be amplified without exposure to agents or conditions that promote strand separation. In some embodiments, during the amplification step (and / or other steps), the nucleic acid target can be amplified without exposure to agents or conditions that promote melting. Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote melting can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, proteins (e.g., enzymatic agents), and the like.

[0194] In some embodiments, the methods disclosed herein do not include thermal denaturation of nucleic acids (e.g., heating a solution containing nucleic acids to a high temperature, such as, for example, a temperature above 75 °C, 80 °C, 90 °C, or 95 °C or higher) or protein-based (e.g., enzymatic) denaturation. Protein-based (e.g., enzymatic) denaturation can include contacting the nucleic acid with one or more of a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nicking enzyme, recombinase, RNA replicase, and nucleic acid binding protein (e.g., single-stranded binding protein). In some embodiments, the compositions provided herein do not contain a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nicking enzyme, recombinase, RNA replicase, and / or nucleic acid binding protein (e.g., 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 disclosed methods do not include contacting the nucleic acid with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include an agent and / or condition that denatures the nucleic acid (e.g., promotes strand separation and / or promotes melting). In some embodiments, except for a polymerase (e.g., a hyperthermophilic polymerase), the amplification step (e.g., step (c)) does not include an agent and / or condition that denatures the nucleic acid (e.g., promotes strand separation and / or promotes melting). In some embodiments, except for a polymerase (e.g., a hyperthermophilic polymerase) and / or low pH conditions (e.g., contacting with an acid), the methods and compositions provided herein do not include an agent and / or condition that denatures the nucleic acid (e.g., promotes strand separation and / or promotes melting).

[0195] A nucleic acid target can be amplified without exposure to an agent or condition that promotes strand separation and / or melting, such as a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nicking enzyme, recombinase, RNA replicase, nucleic acid binding protein (e.g., single-stranded binding protein), or any combination thereof. For example, a nucleic acid target can be amplified without exposure to a helicase, including but not limited to DNA helicases and RNA helicases. Amplification conditions that do not include the use of a helicase are helicase-free amplification conditions.

[0196] 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, 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-stranded binding proteins or single-stranded DNA binding proteins (SSB)), such as T4 gp32. In some embodiments, nucleic acid targets are amplified without exposure to topoisomerases. Nucleic acid targets can be amplified in the presence or absence of agents or conditions that destabilize nucleic acids. As used herein, the term "destabilize" shall be given its ordinary meaning and shall also refer to disruption of the overall organization and geometric orientation of a nucleic acid molecule (e.g., a double helix structure) by one or more of the effects of tilt, roll, twist, slide, and flip (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, page 17). Destabilization generally does not refer to the unwinding or separation of nucleic acid strands (e.g., denaturation). Nucleic acid destabilization can be achieved, for example, by exposure to agents such as intercalating agents or alkylating agents and / or chemicals such as formamide, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, nucleic acid targets are amplified without exposure to ligases and / or RNA replicases.

[0197] In some embodiments, nucleic acid targets can be amplified without lysis or digestion. For example, nucleic acid targets can be amplified without prior exposure to one or more cleavage agents, and intact nucleic acids are 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 cleavage agents are referred to herein as cleavage agent-free amplification conditions. The term "cleavage agent" generally refers to an agent that can cleave nucleic acids at one or more specific or non-specific sites, sometimes a chemical or an enzyme. Specific cleavage agents typically cleave specifically according to a specific nucleotide sequence at a specific site. Cleavage agents can include endonucleases (e.g., restriction endonucleases, nicking enzymes, etc.); exonucleases (DNases, RNases (e.g., RNase H), 5' to 3' exonucleases (e.g., exonuclease II), 3' to 5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3' to 5' exonucleases); and chemical cleavage agents.

[0198] Nucleic acid targets can be amplified without the use of restriction endonucleases and / or nicking enzymes. In some embodiments, nucleic acids are amplified without prior exposure to restriction endonucleases and / or nicking enzymes. In some embodiments, nucleic acids are amplified without exposure to restriction endonucleases and / or nicking enzymes during amplification. In some embodiments, nucleic acids are amplified without exposure to restriction endonucleases and / or nicking enzymes after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNases, RNases (e.g., RNase H), 5' to 3' exonucleases (e.g., exonuclease II), 3' to 5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3' to 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, nucleic acids are amplified without DNase treatment and / or RNase treatment. In some embodiments, nucleic acids are amplified without RNase H treatment.

[0199] The amplified nucleic acid can be referred to herein as a nucleic acid amplification product or amplicon. In some embodiments, the amplification product comprises naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, etc., and combinations of the foregoing. The amplification product typically has the same or substantially the same nucleotide sequence as the sequence in the sample nucleic acid (e.g., the target sequence) or its complement. The "substantially the same" nucleotide sequence in the amplification product will generally have a high degree of sequence identity with the nucleotide sequence being amplified or its complement (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), and the variations are sometimes the result of polymerase infidelity or other variables.

[0200] The nucleic acid amplification product can comprise a polynucleotide that is continuously complementary or substantially identical to a target sequence in the sample nucleic acid. Continuous complementarity generally refers to the nucleotide sequence in the first strand, e.g., where each ordered base (e.g., reading 5' to 3') pairs with the corresponding ordered base in the second strand and there are no gaps, additional sequences, or unpaired bases in the sequence considered to be continuously complementary. In other words, continuous complementarity generally refers to all consecutive bases in the nucleotide sequence of the first strand being complementary to the corresponding consecutive bases 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 continuously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO:34), where all consecutive bases in the first strand are complementary to all corresponding consecutive bases in the second strand. However, a first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO:35) would not be considered continuously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO:34) because the six adenine (6 A) sequence in the middle of the first strand does not pair with the bases in the second strand. Continuously complementary sequences are sometimes about 5 to about 25 consecutive bases in length, e.g., lengths of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 consecutive bases, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is continuously complementary or substantially identical to a target sequence in the sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not contain any additional sequences (e.g., at the 5' and / or 3' termini, or within the product) that are not continuously complementary or substantially identical to the target sequence, e.g., additional sequences incorporated into the amplification product by a tailing primer or ligation, and / or additional sequences that provide a cleavage agent recognition site (e.g., a nicking enzyme recognition site). Generally, unless the target sequence contains tandem repeats, the amplification product does not contain the product in the form of tandem repeats.

[0201] The nucleic acid amplification product can comprise a sequence that is complementary or substantially identical to one or more primers used in the amplification reaction. In some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is continuously complementary or identical to the first primer sequence, and a second nucleotide sequence that is continuously complementary or identical to the second primer sequence.

[0202] The nucleic acid amplification product can contain a spacer sequence. As described herein, a spacer sequence in an amplification product is a sequence (1 or more bases) that is continuously complementary or substantially identical to a portion of a target sequence in a sample nucleic acid, and is flanked by a sequence in the amplification product that is complementary or substantially identical to one or more primers used in the amplification reaction. A spacer sequence flanked by sequences in the amplification product is typically located between a first sequence (complementary or substantially identical to a first primer) and a second sequence (complementary or substantially identical to a second primer). Thus, the amplification product typically includes a first sequence, followed by a spacer sequence, followed by a second sequence. The spacer sequence is typically not complementary or substantially identical to the sequence in the primer. The spacer sequence can be or can contain from about 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists of or consists essentially of a first nucleotide sequence that is continuously complementary or identical to a first primer sequence, a second nucleotide sequence that is continuously complementary or identical to a second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not contain any additional sequence (e.g., at the 5' and / or 3' end; or within the product) that is not continuously complementary or identical to the first primer sequence and the second primer sequence and that is not part of the spacer sequence, e.g., an additional sequence incorporated into the amplification product by tailing or circularizing primers, ligation, or other mechanisms. In some embodiments, the nucleic acid amplification product generally does not contain an additional sequence (e.g., at the 5' and / or 3' end; or within the product) that is not continuously complementary or identical to the first primer sequence and the second primer sequence and that is not part of the spacer sequence, e.g., an additional sequence incorporated into the amplification product by tailing or circularizing primers, ligation, or other mechanisms. However, in such embodiments, the nucleic acid amplification product can contain some mismatched (i.e., non-complementary) bases or one or more additional bases (e.g., at the 5' and / or 3' end; or within the product) introduced into the product, for example, by errors or contamination during the amplification process.

[0203] The length of the nucleic acid amplification product can be up to 50 bases, including 10, 15, 20, 25, 30, 35, 40, 45, 50 bases, or a number or range of bases between any two of these values. In some embodiments, the nucleic acid amplification products of a given target sequence have the same length or substantially the same length (e.g., within 1 to 10 bases). Thus, the nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoretic gel) and generally do not produce multiple signals indicative of multiple lengths (e.g., a ladder or smear on an electrophoretic gel). For multiplex reactions, the nucleic acid amplification products of different target sequences can have different lengths.

[0204] The methods and compositions described herein can be used for multiplex amplification, which generally refers to the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence). For example, multiplex amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample. For example, the amplification step can include the multiplex amplification of two or more target nucleic acid sequences, and the detection step can include the 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 for two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A virus, influenza B virus, and / or influenza C virus). Multiplex amplification can also refer to the amplification of one or more sequences present in multiple samples simultaneously or in a stepwise manner. For example, multiplex amplification can be used to amplify at least two target sequences that can be amplified (e.g., the amplification reaction includes suitable primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is prepared to detect at least two target sequences, but only one target sequence is present in the sample being tested, such that although both sequences can be amplified, only one sequence is amplified. In some embodiments, when two target sequences are present, the amplification reaction results in the amplification of both target sequences. The multiplex amplification reaction can result in the amplification of one, some, or all of the target sequences, including the appropriate primers and enzymes. In some embodiments, the amplification reaction is prepared to detect two sequences with a pair of primers, where one sequence is a target sequence and one sequence is a control sequence (e.g., a synthetic sequence that can be amplified by the same primers as the target sequence and has spacer bases or a sequence different from the target). In some embodiments, the amplification reaction is prepared to detect multiple sets of sequences with corresponding primer pairs, where each set of sequences includes a target sequence and a control sequence.

[0205] Primer

[0206] Nucleic acid amplification is generally carried out in the presence of one or more primers. Primers are generally characterized as oligonucleotides that include a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid at or near a specific region of interest (i.e., the target sequence). For example, a primer can allow for the specific determination of a target nucleic acid nucleotide sequence or the detection of a target nucleic acid or its characteristics (e.g., the presence or absence of a sequence). Primers can be naturally occurring or synthetic. The term specific, or specificity, generally refers to the binding or hybridization of one molecule to another molecule, such as a primer to a target polynucleotide. That is, specific or specificity refers to the recognition, contact, and formation of a stable complex between two molecules, as compared to much less recognition, contact, or complex formation of either of these two molecules with other molecules. The terms annealing or hybridization generally refer to the formation of a stable complex between two molecules. When referring to primers, the terms primer, oligomer, or oligonucleotide can be used interchangeably herein.

[0207] Primers can be designed and synthesized using suitable methods and can have any length suitable for hybridizing with the target sequence and performing the amplification processes described herein. Primers are typically designed based on sequences in the target nucleic acid. The length of the primers in some embodiments can be from about 5 to about 30 bases, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases in length. Primers can contain naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides) or mixtures thereof. Modified and modified bases can include, for example, phosphorylation (e.g., 3'-phosphorylation, 5'-phosphorylation); attachment of chemical or linker modifications (e.g., Acrydite TM , adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesterol-TEG, I-Linker TM , amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link TM amino 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 C3 S-S, dithiol, thiol modifier C6 S-S)); fluorophores (e.g., Freedom TM Dye, Alexa Dye, LI-COR ATTO TM Dye, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas -X (NHS ester), 640 (NHS ester), Dy 750 (NHS ester)), Iowa dark quencher modification (e.g., Iowa FQ, Iowa RQ), dark quencher modification (e.g., Black Hole -1, Black Hole -2, Dabcyl); Spacers (C3 Spacer, PC Spacer, hexanediol, Spacer 9, Spacer 18, 1’,2’-dideoxyribose (dSpacer)); Modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo dU, deoxyuridine, reverse dT, reverse dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinosine, Super Super Locked nucleic acids (LNA’s), 5-nitroindole, 2’-O-methyl RNA bases, hydroxymethyl dC, unlocked nucleic acids (UNA) (e.g., UNA-A, UNA-U, UNA-C, UNA-G), iso-dC, iso-dG, fluoro-C, fluoro-U, fluoro-A, fluoro-G); Thioester phosphate (PS) bond modifications (e.g., thiophosphorylated DNA bases, thiophosphorylated RNA bases, thiophosphorylated 2’-O-methyl bases, thiophosphorylated LNA bases); and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleic acid bases, O-methyl RNA bases, PS linkages, 3’ phosphate groups, spacer bases (such as C3 spacer bases or other spacer bases). For example, a primer can comprise one or more O-methyl RNA bases (e.g., 2’-O-methyl RNA bases). 2’-O-methyl RNA is typically a post-transcriptional modification of RNA found in tRNA and other small RNAs. Primers containing 2’-O-methyl RNA bases can be synthesized directly. For example, such a modification can increase the Tm of an RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and deoxyribonucleases. 2’-O-methyl RNA bases can be included in a primer, for example, to increase stability and binding affinity to a target sequence. In some embodiments, a primer can comprise one or more thioester phosphate (PS) linkages (e.g., PS bond modifications). The PS bond replaces the non-bridging oxygen in the primer phosphate backbone with a sulfur atom. This modification generally renders the internucleotide linkage resistant to nuclease degradation. For example, a PS bond can be introduced between approximately the last 3 to 5 nucleotides at the 5’-end or 3’-end of a primer to inhibit exonuclease degradation. In some embodiments, PS bonds included throughout the primer can help reduce endonuclease attack. A primer can, for example, comprise a 3’ phosphate group. In certain cases, 3’ phosphorylation can inhibit degradation by certain 3’-exonucleases and can be used to block DNA polymerase extension. In some embodiments, a primer comprises one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated into the interior or 5’-end of a primer. For example, multiple C3 spacers can be added to either end of a primer to introduce long hydrophilic spacer arms for attaching fluorophores or other pendant groups.

[0208] Primers can comprise DNA bases, RNA bases, or both, where one or more of the DNA bases and RNA bases are modified or unmodified. For example, a primer can be a mixture of DNA bases and RNA bases. A primer can consist of DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, the primer consists of unmodified DNA bases. In some embodiments, the primer consists of modified DNA bases. A primer can consist of RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, the primer consists of unmodified RNA bases. In some embodiments, the primer consists of modified RNA bases. In some embodiments, the primer does not contain RNA bases. In some embodiments, the primer does not contain DNA bases. In some embodiments, the primer does not contain a cleavage agent recognition site (e.g., does not contain a nickase recognition site). In some embodiments, the primer does not contain a tail (e.g., does not contain a tail of a nickase recognition site).

[0209] In some embodiments, all or part of the primer sequence can be complementary or substantially complementary to the target nucleic acid. Substantial complementarity of sequences generally refers to nucleotide sequences that will hybridize to each other. The stringency of the hybridization conditions can be varied to tolerate different amounts of sequence mismatch. The target sequence and the primer sequence can be, for example, at least 75% complementary to each other, including 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to each other. A primer that is substantially complementary to a target nucleic acid sequence is generally 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 can be at least 75% identical in sequence, such as 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.

[0210] In some embodiments, the primer comprises a pair of primers. A pair of primers can include a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of the target nucleic acid). In some embodiments, the primer consists of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, a pair of primers is used for amplification of the target sequence, and no additional primers or oligonucleotides are included in the amplification of the target sequence (e.g., the amplification reaction components do not contain an additional primer pair for a given target sequence, do not contain nested primers, do not contain bumper primers, do not contain oligonucleotides other than primers, do not contain probes, etc.). In some embodiments, the primer consists of a pair of primers. In some embodiments, the amplification reaction can contain additional primer pairs for amplifying different target sequences, such as in multiplex amplification. In some embodiments, the primer consists of a pair of primers. However, in some embodiments, the amplification reaction can contain additional primers, oligonucleotides, or probes for a detection process that are not considered part of the amplification. In some embodiments, primers are used in groups. An amplification primer set can include a pair of forward and reverse primers for a given target sequence. For multiplex amplification, the primers that amplify the first target sequence are considered a primer set, and the primers that amplify the second target sequence are considered a different primer set.

[0211] The nucleic acids described herein (e.g., amplification products, sample nucleic acids, target nucleic acid sequences) can comprise a first strand and a second strand that are complementary to each other. The amplification reaction components can 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 continuously 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 continuously complementary to the target sequence in the second strand of the sample nucleic acid. Continuous complementarity of the primer to the target generally refers to the nucleotide sequence in the primer where each base pairs in order with the corresponding ordered bases in the target sequence, and gaps, additional sequences, or unpaired bases in the sequence are not considered to be continuously complementary. In some embodiments, the primer does not contain any additional sequences that are not continuously complementary to the target sequence (e.g., at the 5' and / or 3' ends, or within the primer), such as additional sequences present in tailed primers or circularized primers, and / or additional sequences that provide a cleavage agent recognition site (e.g., nicking enzyme recognition site). In some embodiments, the amplification reaction components do not contain primers that contain additional sequences (i.e., sequences other than those that are continuously complementary to the target sequence), such as tailed primers, circularized primers, primers capable of forming a stem-loop structure, hairpin structures, and / or additional sequences that provide a cleavage agent recognition site (e.g., nicking enzyme recognition site), etc.

[0212] In some embodiments, primers 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 modifiers that alter primer binding characteristics. In some embodiments, primers can contain detectable molecules or entities (e.g., fluorophores, radioisotopes, colorimetric agents, particles, enzymes, etc.).

[0213] Polymerase

[0214] The amplification reaction components (e.g., one or more amplification reagents) can contain one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides against a nucleic acid target sequence (e.g., where a primer anneals to it) to extend the 3'-hydroxyl terminus of a primer molecule (e.g., an amplification primer as described herein). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that are active at elevated reaction temperatures (e.g., above 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C). Hyperthermophilic polymerases can be referred to as hyperthermophilic DNA polymerases. The polymerase may or may not have strand displacement ability. In some embodiments, the polymerase can incorporate from about 1 to about 50 nucleotides in a single synthesis, e.g., incorporate about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in a single synthesis, or an amount or range between any two of these values.

[0215] The amplification reaction components can contain one or more DNA polymerases selected from the following: 9°N DNA polymerase; 9°Nm TM DNA polymerase; Therminator TM DNA polymerase; Therminator TM IIDNA polymerase; Therminator TM IIIDNA polymerase; Therminator TM γDNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (Escherichia coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exonuclease); T4 DNA polymerase; T7 DNA polymerase; Deep VentR TM (exonuclease) DNA polymerase; DeepVentR TM DNA polymerase; DyNAzyme TM EXT DNA; DyNAzyme TMII Hot Start DNA Polymerase; Phusion TM High Fidelity DNA Polymerase; DNA Polymerase; (Exo)DNA Polymerase; RepliPHI TM Phi29 DNA Polymerase; rBst DNA Polymerase, large fragment (IsoTherm TM DNA Polymerase); MasterAmp TM AmpliTherm TM DNA Polymerase; Taq DNA Polymerase; Tth DNA Polymerase; Tfl DNA Polymerase; Tgo DNA Polymerase; SP6 DNA Polymerase; Tbr DNA Polymerase; DNA Polymerase β; and ThermoPhi DNA Polymerase.

[0216] 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 polymerase can have a half-life of about 5 hours to 10 hours at 95 °C, or a half-life of about 1 hour to 3 hours at 100 °C. For example, the amplification reaction components can include one or more hyperthermophilic DNA polymerases from archaea (e.g., hyperthermophilic DNA polymerases from Thermococcus or hyperthermophilic DNA polymerases from Thermococcaceaen archaean). The amplification reaction components can include one or more hyperthermophilic DNA polymerases from Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0217] In some embodiments, the amplification reaction components comprise a hyperthermophilic DNA polymerase or a functional fragment thereof. A functional fragment generally retains one or more functions of the full-length polymerase, such as the ability to polymerize DNA (e.g., in an amplification reaction). In some cases, the functional fragment performs its function (e.g., polymerizes DNA in an amplification reaction) at a level of at least about 50%, at least about 75%, at least about 90%, at least about 95% of the full-length polymerase functional level. The level of polymerase activity can be evaluated, for example, using a detectable nucleic acid amplification method, such as the methods described herein. In some embodiments, the amplification reaction components comprise a hyperthermophilic 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.

[0218] In some embodiments, the amplification reaction components (e.g., one or more amplification reagents) comprise a polymerase comprising an amino acid sequence that is at least about 90% identical to a hyperthermophilic polymerase or a functional fragment thereof. In some embodiments, the amplification reaction components comprise a polymerase comprising an amino acid sequence that is 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.

[0219] The polymerase can have reverse transcriptase activity. In such embodiments, the amplification reaction can amplify an RNA target, for example, in a single step, without using a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase activity include Bst (large fragment), 9°N DNA polymerase, 9°Nm TM DNA polymerase, Therminator TM 、Therminator TM II, etc. The amplification reaction components can comprise 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 comprise a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0220] 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, compared to an unmodified polymerase, a modified polymerase with low exonuclease activity can have 10% or less exonuclease activity, e.g., less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase has no or low 5' to 3' exonuclease activity, and / or no or low 3' to 5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand-dependent exonuclease activity, and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of the polymerase include one or more amino acid substitutions at positions 141 and / or 143 and / or 458 of SEQ ID NO:31 (e.g., D141A, E143A, E143D, and A485L) or at positions corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:31.

[0221] Detection and Quantification

[0222] The methods described herein can include detecting and / or quantifying nucleic acid amplification products. For example, amplification products can be detected and / or quantified 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., colorimetry, turbidimetry), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension methods (e.g., iPLEX TM) Molecular Inversion Probe (MIP) technology from Affymetrix, Restriction Fragment Length Polymorphism (RFLP analysis), Allele-Specific Oligonucleotide (ASO) analysis, Methylation-Specific PCR (MSPCR), Pyrosequencing analysis, acycloprime analysis, Reverse Dot Blot, GeneChip microarray, Dynamic Allele-Specific Hybridization (DASH), Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA) probes, AlphaScreen, SNPstream, Genetic Bit Analysis (GBA), Multiplex Microsequencing, SNaPshot, GOOD assay, Microarray Microsequencing, Array Primer Extension (APEX), Microarray Primer Extension, Tag Array, Encoded Microspheres, Template-Directed Incorporation (TDI), Colorimetric Oligonucleotide Ligation Assay (OLA), Sequence-Encoded OLA, Microarray Ligation, Ligase Chain Reaction, Padlock probes, Invader assay, Hybridization using at least one probe, Hybridization using at least one fluorescently labeled probe, Cloning and sequencing, Use and quantitative real-time polymerase chain reaction (QRT-PCR) of hybridization probes, Nanopore sequencing, Chips, and combinations thereof. In some embodiments, detecting nucleic acid amplification products includes using real-time detection methods (i.e., detecting and / or continuously monitoring the products during the amplification process). In some embodiments, detecting nucleic acid amplification products includes using endpoint detection methods (i.e., detecting the products after completion or cessation of the amplification process). Nucleic acid detection methods can also employ labeled nucleotides incorporated directly into the target sequence or into probes containing target-complementary sequences. Such labels can be radioactive and / or fluorescent in nature and can be resolved in any of the ways discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more of the following detection methods. In some embodiments, the detection methods can be used in conjunction with measurement of signal intensity and / or generation (or reference) of a standard curve and / or lookup table for quantifying nucleic acid amplification products.

[0223] Detecting nucleic acid amplification products can include using molecular beacon technology. The term molecular beacon generally refers to a detectable molecule, where the detectable property of the molecule is detectable under certain conditions such that the molecule can function as a specific and informative signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and time or velocity through a known size opening. A molecular beacon for detecting a nucleic acid molecule can be, for example, a hairpin-shaped oligonucleotide that contains a fluorophore at one end and a quencher dye at the other end. The loop of the hairpin can contain a probe sequence complementary to the target sequence, and the stem is formed by annealing of complementary arm sequences located on either side of the probe sequence. The fluorophore and quencher molecule can be covalently linked at 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 quencher molecule are in close proximity to each other, thus preventing FRET. When the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, and the loop structure is transformed into a stable and more rigid conformation, resulting in the separation of the fluorophore and quencher molecule, thereby generating fluorescence. Due to the specificity of the probe, the generation of fluorescence is usually entirely due to the synthesis of the expected amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is the same or complementary to the sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is not the same or complementary to the sequence in the target nucleic acid (e.g., hybridizes to a sequence added to the amplification product by tailing the amplification primer or ligation). Molecular beacons are highly specific and can discriminate single nucleotide polymorphisms. Molecular beacons can also be synthesized with different colored fluorophores and different target sequences, enabling the simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction). For quantitative amplification processes, the molecular beacon can specifically bind to the amplified target after each amplification cycle, and since the unhybridized molecular beacon is dark, it is not necessary to separate the probe-target hybrid to quantitatively determine the amount of the amplification product. The signal generated is proportional to the amount of the amplification product. Detection using molecular beacons can be done in real time or as an end-point detection method.

[0224] Detecting nucleic acid amplification products can include using lateral flow. The use of lateral flow generally includes the use of a lateral flow device, which includes but is not limited to strip assays and thin layer chromatography plates with various appropriate coatings. Fixed on the flow path are various binding reagents for the sample, binding partners, or conjugates involving binding partners for the sample and the signal generation system.

[0225] Detecting nucleic acid amplification products can include using FRET, which is an energy transfer mechanism between two chromophores (donor and acceptor molecules). Briefly, the donor fluorophore molecule is excited at a specific excitation wavelength. When the donor molecule returns to its ground state, the subsequent emission of the donor molecule can transfer the excitation energy to the acceptor molecule through long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored and varies with the distance between the donor and acceptor, the overlap of the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be used to quantify molecular dynamics, for example, in the DNA-DNA interactions described for molecular beacons. To monitor the production of a specific product, the probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe-target hybridization results in a change in the distance or orientation of the donor and acceptor, and a change in FRET is observed.

[0226] Detecting nucleic acid amplification products can include using fluorescence polarization (FP). The FP technique is based on the principle that when excited by linearly polarized light, a fluorescently labeled compound will emit fluorescence, the degree of polarization of which is inversely proportional to its rotation rate. Thus, when a molecule with a fluorescent label such as a tracer-nucleic acid conjugate is excited by linearly polarized light, the emitted light remains highly polarized because the fluorophore is restricted from rotating between the time the light is absorbed and emitted. When the free tracer compound (i.e., not bound to nucleic acid) is excited by linearly polarized light, it rotates much faster than the corresponding tracer-nucleic acid conjugate and the molecules are more randomly oriented, and thus, the emitted light is depolarized. Thus, fluorescence polarization provides a quantitative method for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0227] Detecting nucleic acid amplification products can include using surface capture, for example, achieved by immobilizing a specific oligonucleotide onto a surface, thereby producing a biosensor that is both highly sensitive and selective. Example surfaces that can be used to attach the probe include gold and carbon. Detecting nucleic acid amplification products can include using 5' to 3' exonuclease hydrolysis of the probe (e.g., TAQMAN). For example, a TAQMAN probe is a hydrolysis probe that can increase the specificity of a quantitative amplification method (e.g., quantitative PCR). The TAQMAN probe principle relies on 1) the 5' to 3' exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to a complementary target sequence and 2) fluorophore-based detection. The resulting fluorescent signal allows for the quantitative measurement of the accumulation of amplification products during the exponential phase of amplification, and the TAQMAN probe can significantly increase the specificity of detection.

[0228] Detecting nucleic acid amplification products can include using intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes exhibit enhanced fluorescence when bound to DNA or RNA). The dyes can include DNA or RNA intercalating fluorophores, including but not limited to 82, acridine orange, ethidium bromide, Hoechst dyes, propidium iodide, I (asymmetric cyanine dye), II, TOTO (thiazole orange dimer) and YOYO (oxazole yellow dimer). Detecting nucleic acid amplification products can include using absorbance methods (e.g., colorimetry, turbidimetry). In some embodiments, detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260 nm) to concentration. The direct measurement of nucleic acids can be converted to concentration using the Beer Lambert law, which relates absorbance to concentration using the measured path length and extinction coefficient. Detecting nucleic acid amplification products can include using electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or using mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and quantity of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample can be ionized and the resulting ions are separated in an electric field and / or magnetic field according to their mass-to-charge ratio, and the detector measures the mass-to-charge ratio of the ions. Mass spectrometry methods include, for example, MALDI, MALDI-TOF and electrospray. These methods can be used in combination with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can be high-throughput due to high-speed signal acquisition and automated analysis from solid surfaces.

[0229] Detecting nucleic acid amplification products can include using nucleic acid sequencing. The entire sequence or a partial sequence of the amplification product can be determined, and the determined nucleotide sequence can be referred to as a read. For example, linear amplification products can be directly analyzed without further amplification (e.g., by using single molecule sequencing methods). In some embodiments, the linear amplification products are further amplified and then analyzed (e.g., using ligation sequencing or pyrosequencing methods). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, ligation sequencing, pyrosequencing, synthesis sequencing, single molecule sequencing, multiplex sequencing, solid-phase single nucleotide sequencing and nanopore sequencing. Detecting nucleic acid amplification products can include using digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., Company).

[0230] Lysis Buffer

[0231] Lytic Agent

[0232] As disclosed herein, the lytic agent may include a detergent. The detergent may include one or more of a cationic surfactant, anionic surfactant, nonionic surfactant, and zwitterionic surfactant. The anionic surfactant may include NH4 + 、K + 、Na + or Li + as a counterion. The cationic surfactant may contain I - 、Br - or Cl - as a counterion.

[0233] The lytic agent provided herein is capable of acting as a denaturing agent. As used herein, the "denaturing agent" or "denaturant" shall be given its ordinary meaning and includes any compound or material that will cause the 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 denaturants 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 may include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sodium dodecyl sarcosinate, mild nonionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(dioleyloxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaine (Zwittergent), 3-(3-chloramidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-chloramidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO). Organic, water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkylene diols (especially C2-C4 alkylene diols such as ethylene glycol) can be used as denaturing agents. Phospholipids can be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants such as dihexanoyl phosphatidylcholine or diheptanoyl phosphatidylcholine.

[0234] Suitable surfactant levels can be from about 0.1% to about 25%, about 0.25% to about 10%, or 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).

[0235] Useful anionic surfactants herein include water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl group, and water-soluble salts of sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of anionic surfactants of this type.

[0236] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having one long alkyl chain containing about 8 to 18 carbon atoms, such as lauryl trimethyl ammonium chloride; cetylpyridinium chloride; benzalkonium chloride; cetyl trimethyl ammonium bromide; diisobutyl phenoxyethyl dimethyl benzyl ammonium chloride; coconut alkyl trimethyl ammonium nitrite; cetylpyridinium fluoride; etc. In the compositions disclosed herein, certain cationic surfactants can also act as fungicides.

[0237] Suitable nonionic surfactants useful in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds produced by the condensation of an alkylene oxide group (essentially hydrophilic) with an organic hydrophobic compound that can be aliphatic and / or aromatic in nature. Examples of suitable nonionic surfactants include poloxamers; sorbitan derivatives, such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of fatty alcohols or alkylphenols; products produced by the condensation of the reaction product of ethylene oxide with propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides, and mixtures of these materials. These materials can be used to stabilize foams without causing excessive viscosity in consumer product compositions.

[0238] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary, phosphonium, and sulfonium compounds, where the aliphatic group can be straight-chain or branched, and where one aliphatic substituent contains about 8 to 18 carbon atoms, and one contains an anionic water-soluble group, such as carboxyl, sulfonate, sulfate, phosphate, or phosphonate.

[0239] Exemplary anionic, single-chain surfactants include alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, and saturated or unsaturated fatty acids and their salts. The portion of the cationic surfactant that contains the polar head group can include, for example, quaternary ammonium, pyridinium, sulfonium, and / or phosphonium groups. For example, the polar head group can include trimethylammonium. Exemplary cationic, single-chain surfactants include alkyltrimethylammonium halides, alkyltrimethylammonium tosylates, and N-alkylpyridinium halides.

[0240] Reducing Agent

[0241] The lysis buffer and / or reagent composition (e.g., dry composition) can contain one or more reducing agents. A "reducing agent" can be a compound or a group of compounds. As used herein, a "reducing agent", also referred to as a "reductant", "reductive agent", or "reduction equivalent", can be an element or compound that donates electrons to another substance. In particular, a reducing agent is typically a compound that breaks disulfide bonds by reduction, thereby overcoming those tertiary protein folds and quaternary protein structures (oligomeric subunits) that are stabilized by disulfide bonds. Examples of suitable reducing agents include, but are not limited to, 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythritol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-mercaptoacetate, sulfite, mercaptoacetic acid, and HED. In some embodiments of the methods, compositions, and kits provided herein, the lysis buffer and / or reagent composition (e.g., dry composition) does not contain one or more reducing agents.

[0242] Reagent Composition

[0243] The reagent compositions described herein (e.g., dry compositions) can be provided in a "dry form" or a form not suspended in a liquid medium. The "dry form" of the composition can include dry powder, lyophilized composition, spray-dried or precipitated composition. The "dry form" composition can contain one or more lyoprotectants, such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethyl cellulose. As used herein, the terms "lyophilize", "lyophilized", and "freeze-dried" refer to the process of first freezing the material to be dried and then removing the ice or frozen solvent by sublimation in a vacuum environment. A "lyophilizate" refers to the lyophilized material.

[0244] The reagent composition (e.g., the dry composition) can be frozen or lyophilized or spray-dried. The reagent composition can be dried thermally. The reagent composition can contain one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). 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, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The lyophilized reagent can include poly rA, EGTA, EDTA, Tween 80, and / or Tween 20.

[0245] The frozen or lyophilized or spray-dried or thermally dried composition or the aqueous composition for preparing the frozen or lyophilized or spray-dried composition can contain one or more of the following: (i) non-aqueous solvents, such as ethylene glycol, glycerol, dimethyl sulfoxide, and dimethylformamide. (ii) Surfactants, such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic F127 (polyoxyethylene-polyoxypropylene copolymer) also known as poloxamer, poloxamine, and sodium dodecyl sulfate. (iii) Disaccharides, such as trehalose, sucrose, lactose, and maltose. (iv) Polymers (which can have different MWs), such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, and albumin. (v) Amino acids, such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and γ-aminobutyric acid.

[0246] A reagent composition (e.g., a dry composition) can comprise one or more protecting agents and one or more amplification reagents. The one or more protecting agents can include cyclodextrin compounds. Cyclodextrin (CD) can be used to complex with a lysing agent (e.g., SDS). Cyclodextrin (CD) can be a truncated cone-like cyclic oligosaccharide having a hydrophobic inner cavity and a hydrophilic outer surface. The most commonly used natural cyclodextrins include 6, 7, and 8 glucose units, called α, β, and γ-CD. Natural CDs can have solubility. Chemically modified CDs such as hydroxypropyl derivatives increase the solubility in an aqueous medium by up to 50%. is a trade name for WACKER cyclodextrin derivatives, including various α, β, and γ-CD derivatives. β-CD can form a strong inclusion complex with sodium dodecyl sulfate (SDS) (stronger than α-CD and β-CD), and the stoichiometric ratio is mainly 1:1. The binding constant of β-CD with SDS can range from 2100M -1 to 2500M -1 .

[0247] Kit

[0248] 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 comprise: a lysis buffer comprising one or more lysing agents capable of lysing a biological entity to release the sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of containing the target nucleic acid sequence, optionally one or more of the lysing agents include a detergent, and wherein the detergent includes one or more of a cationic surfactant, anionic surfactant, nonionic surfactant, and zwitterionic surfactant. The kit can comprise: a reagent composition comprising one or more amplification reagents, the one or more amplification reagents comprising one or more components for amplification for amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplification include: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of generating a nucleic acid amplification product. In some embodiments, the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer.

[0249] The kit can include: at least one component providing real-time detection activity for the nucleic acid amplification product. The real-time detection activity can be provided by a molecular beacon. The real-time detection activity can be provided by the signal generating oligonucleotide provided herein. The reagent composition (e.g., a dry composition) can comprise a reverse transcriptase and / or a reverse transcription primer.

[0250] The molar ratio of one or more protecting agents to one or more amplification reagents can be between about 10:1 and about 1:10 (e.g., about 2:1). In some embodiments, one or more additives include Tween 20, Triton X-100, Tween 80, a nonionic detergent (e.g., a nonionic surfactant), or any combination thereof. In some embodiments, one or more protecting agents comprise a cyclodextrin compound. In some embodiments, one or more lysis reagents constitute from about 0.001% (w / v) to about 1.0% (w / v) (e.g., about 0.2% (w / v)) of the treated sample. In some embodiments, one or more lysing agents include a detergent. The detergent can include one or more of a cationic surfactant, anionic surfactant, nonionic surfactant, and zwitterionic surfactant. In some embodiments, it can be advantageous for one or more protecting agents to be able to sequester one or more lysing agents, thereby preventing one or more amplification reagents from being denatured by one or more lysing agents.

[0251] The kit can include, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, as described herein. In the case of amplifying one target, a pair of primers (forward and reverse) can be included in the kit. In the case of amplifying multiple target sequences, more than one primer pair can be included in the kit. The kit can include control polynucleotides, and in the case of amplifying multiple target sequences, more than one control polynucleotide can be included in the kit.

[0252] An enzyme having hyperthermophilic DNA 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 having hyperthermophilic DNA polymerase activity can include the amino acid sequence of SEQ ID NO:31.

[0253] The nucleic acid amplification product can be about 20 to 40 bases in length. The nucleic acid amplification product can comprise: (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, wherein the spacer sequence is 1 to 10 bases in length.

[0254] Biological entities can include one or more of prokaryotic cells, eukaryotic cells, virus particles, exosomes, protoplasts, and microvesicles. Biological entities can include viruses, bacteria, fungi, protozoa, parts thereof, or any combination thereof. The target nucleic acid sequence can be a nucleic acid sequence of a virus, bacteria, fungi, or protozoa. The sample nucleic acid can be derived from a virus, bacteria, fungi, or protozoa.

[0255] The kit may also include one or more components in any number of separate vessels, chambers, containers, packets, test tubes, vials, microtiter plates, etc., or the components may be combined in various combinations in such containers. For example, the components of the kit may be present in one or more containers. In some embodiments, all components are provided in one container. In some embodiments, the enzyme (e.g., polymerase and / or reverse transcriptase) may be provided in a container separate from the primer. The components may be, for example, lyophilized, heat-dried, freeze-dried, or in a stabilizing buffer. In some embodiments, the polymerase and / or reverse transcriptase are in lyophilized or heat-dried form in a single container, and the primer is lyophilized, heat-dried, freeze-dried, or in buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase and the primer are in lyophilized or heat-dried form in a single container.

[0256] The kit may also include, for example, dNTPs used in the reaction, or modified nucleotides, vessels, cuvettes, or other containers for the reaction, or water or buffer for the vials for rehydrating the lyophilized or heat-dried components. For example, the buffer used may be suitable for both polymerase and primer annealing activities.

[0257] The kit may also include instructions for performing one or more of the methods described herein and / or a description of one or more of the components described herein. The instructions and / or description may be in printed form and may be included in a kit insert. The kit may also contain a written description of the Internet location providing such instructions or description. The kit may also include reagents for detection methods, such as reagents for FRET, lateral flow devices, test strips, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. Examples

[0258] Some aspects of the embodiments discussed above are further disclosed in detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.

[0259] Example 1

[0260] Molecular Beacon Characterization Experiment

[0261] In this example, first, the beacon performance was evaluated in the MB characterization experiment. 500 nM synthetic target was combined with 50 nM MB + / - 9°N. Figure 7 A non-limiting exemplary schematic of the signal-generating oligonucleotide provided herein is depicted. Figures 8A - 8BDepicts non-limiting exemplary data related to MB characterization. The vertical lines in the figure indicate the measured temperature. Differences were observed between the conditions with and without the extended signal, and a 9°N distortion of MB was found to result in an elevated baseline. The empirical Tm can be evaluated from the melting curve.

[0262] Example 2

[0263] Comparison of Neisseria gonorrhoeae Assay Designs

[0264] This example provides a comparison of an old Neisseria gonorrhoeae assay with a clean primer having 6 spacers to a new Neisseria gonorrhoeae assay with a clean primer having 4 spacers (Table 3). Figures 13A - 13B Depicts data related to 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 11mer to 13mer improved the assay performance, and the speed increased by more than 1 minute. Accordingly, primer Tm should be considered in the APA assay design.

[0265] Table 3: Neisseria gonorrhoeae Assay Components

[0266]

[0267] Example 3

[0268] Effect of Primer Length on APA Assay

[0269] This example examines the effect of primer length on APA assay performance. Table 4 provides various APA assay designs. The IDTOligoAnalyzer settings are as follows: [oligonucleotide], 0.5 uM; [Na + , 20 mM; [Mg ++ , 4 mM; [dNTP], 2 mM. Figure 14 Depicts data related to the effect of primer length on APA assay performance. It was found that longer primers tend to have poor amplification. It was found that if the product Tm >> assay Tm, the amplification of longer primers is poor (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 being bound by any particular theory, this may be due to poor strand displacement activity of 9°N.

[0270] Table 4: APA Assay Design

[0271]

[0272] Example 4

[0273] Case Study - Flu A PB2.2 Assay

[0274] Figure 17 Depicts non - limiting exemplary diagrams related to the Flu A APA assay design. In some embodiments, primers are positioned to avoid missing mismatched variant amplification. In some embodiments, the 3'-end of the primer is positioned to have a minimum of mismatched variants within the first 3 - 4 nt. In some embodiments, primers are positioned against spacers containing minimal mismatches. In some embodiments, no more than 1 mismatched base is allowed in the spacer region (for a total of fewer than three probes). Mismatched variants m3 and m4 can be detected using the P1 probe. In some embodiments, a conserved sequence (not shown) immediately upstream of the 5' of the RT primer is employed.

[0275] [[ID=1

[0276] ​

[0277] This example examined the performance of Chlamydia trachomatis DNA detection in an APA reaction containing molecular beacons and Syto 61 fluorescent dye. ​ Depicts non - limiting exemplary conventional molecular beacons for detecting Chlamydia trachomatis gDNA in an APA reaction. The molecular beacons are labeled with HEX at the 5'-end and with the IBFQ quencher at the 3'-end. ​ Depicts data related to the detection of Chlamydia trachomatis gDNA in an APA reaction using conventional molecular beacons in the HEX ( ​ ) and cy5 ( ​ ) channels. ​ Depicts data related to the real - time detection of four replicates of a no - target control (NTC, dashed curve) and 500 copies of Ct gDNA (solid curve) in a wet APA reaction. These results indicate that although the fluorescent dye detection shows strong target amplification (solid curve in the fluorescent cy5 channel), the conventional molecular beacons are unable to detect the amplification product in real - time.

[0278] ​

[0279] ​

[0280] This example examined the performance of synthetic DNA target detection in an APA reaction containing molecular beacons and Syto 61 fluorescent dye. ​ Depicts non - limiting exemplary asymmetric hairpin probes provided herein, and Table 5 provides the sequences of the assay components. ​ Depicts data related to the detection of a synthetic DNA target in an APA reaction using an asymmetric hairpin probe ( ​ ), followed by melting curve analysis (​ ) Evaluation of the Unwinding Derivative ​ ) ​ Shows the results of synthetic DNA target detection using an asymmetric hairpin probe. At 68 °C, the hairpin probe was unable to detect the synthetic oligonucleotide target at 150 nM (green and red curves). In the presence of 9° Nm, the extension of the synthetic target led to the formation of a real-time stable hybrid and an increase in the fluorescence signal. The melting curve indicated that the product did not form a stable hybrid at the assay temperature (Tm = 60 °C), while the extension of the target on the hairpin probe increased the Tm of the hybrid to 80 °C.

[0281] ​

[0282]

[0283]

[0284] ​

[0285] ​

[0286] This example examined the performance of the APA assay, in which the Solomon Islands strain of the Flu A virus was detected using a hairpin probe containing a 3-base pair stem and a 5'-terminal non-target overhang (lowercase letters represent artificial bases, while uppercase letters represent the target sequence) (Table 6). ​ Depicts a non-limiting exemplary asymmetric hairpin probe provided herein. The assay was designed to generate a 23-base DNA product that contains a 4-base spacer. Product P2 forms a 15-base hybrid with the hairpin probe. The Tm of the product was calculated to be 67.6 °C under the assay salt conditions (IDT Oligo analyzer), and the Tm of the probe / product hybrid was 60.7 °C. ​ Depicts data related to the limit of detection (LOD) study using the hairpin probe provided herein for the detection of the Flu A virus. Experiments were performed using the APA "hot start" method, in which both the sample and the lyophilized mixture were preheated to 63 °C and then combined. The reaction was carried out on a BioRad CFx thermal cycler at 67 °C for 10 minutes. These results show that the hairpin probes provided herein (e.g., signal-generating oligonucleotides containing a 5'-terminal domain) can be used for the real-time, sensitive detection of short amplicons.

[0287] ​

[0288]

[0289] ​

[0290] ​

[0291] This example examined the performance of an APA assay that included real-time detection of SARS-CoV-2 virus in a reaction using both a hairpin probe and the fluorescent DNA dye Syto 61. The assay was designed to produce a 28-base DNA product that contained a 4-base spacer. ​ Depicted are non-limiting exemplary signal-generating oligonucleotides provided herein, and Table 7 provides the sequences of assay components. This study was conducted using the APA "hot start" method, in which both the sample and the lyophilized mixture were preheated to 63 °C and then combined and incubated at 67 °C for 10 minutes on a CFx thermal cycler. ​ Depicted are data related to the real-time detection ​ 、 ​ ) of SARS-CoV-2 virus in a reaction using a hairpin probe ( ​ 、 ​ ) and the fluorescent DNA dye Syto 61 ( ​ ) and the melting curve evaluation ( ​ ). The results indicated that the 28-base assay product had a melting temperature of 67 °C, which did not form a stable hybrid with the hairpin probe. Extension of the product on the hairpin probe produced a strong fluorescent signal with a melting temperature of 76 °C.

[0292] ​

[0293]

[0294] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0295] Regarding the use of substantially any plural and / or singular terms herein, those skilled in the art may convert from plural to singular and / or from singular to plural, as appropriate for the context and / or application. For clarity, various singular / plural permutations may be set forth explicitly herein. As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Any reference to "or" herein is intended to cover "and / or" unless otherwise stated.

[0296] Those skilled in the art will understand that, generally speaking, the terms used herein, and especially the terms in the appended claims (e.g., the subject matter 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 "includes but is not limited to", etc.). Those skilled in the art will also understand that if a specific number of introduced claim recitations is intended, such intention will be expressly stated in the claim, and in the absence of such a statement, there is no such intention. For example, as an aid to understanding, the following appended claims may contain the use of the preambles "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to an embodiment containing only one such recitation, even when the same claim includes the preambles "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"); this applies equally to the use of the definite article to introduce claim recitations. In addition, even if a specific number of the introduced claim recitations is expressly stated, those skilled in the art will recognize that such a statement should be interpreted to mean at least the stated number (e.g., merely stating "two recitations" without further modifiers means at least two recitations, or two or more recitations). Further, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally such syntactic construction is intended in a sense that those skilled in the art will understand (e.g., "a system having at least one of A, B, and C" will include, but not be limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).In those cases where a convention similar to “at least one of A, B, or C, etc.” is used, typically such syntactic constructs are intended in a sense that would be understood by one of ordinary skill in the art (e.g., a “system having at least one of A, B, or C” would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One of ordinary skill in the art will also understand that, in fact, any transitional word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood as contemplating the possibilities of including one of the terms, either term, or both terms.

[0297] In addition, when features or aspects of the present disclosure are described in terms of a Markush group, one of ordinary skill in the art will recognize that the present disclosure is also thereby described in terms of any individual member of the Markush group or a subgroup of the members.

[0298] As one of ordinary skill in the art will understand, for any and all purposes, such as in providing a written description, all ranges disclosed herein also include any and all possible subranges and combinations of subranges thereof. Any recited range can be readily identified as fully describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As one of ordinary skill in the art will also understand, all language such as “up to,” “at least,” “greater than,” “less than,” etc. includes the recited number and refers to ranges that can then be broken down into subranges as discussed above. Finally, as one of ordinary skill in the art will understand, a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0299] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to one of ordinary skill in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to limit the true scope and spirit indicated by the appended claims.

Claims

1. A method for detecting a target nucleic acid sequence in a sample, comprising: amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby producing a nucleic acid amplification product; and The nucleic acid amplification product is detected using a signal generating oligonucleotide, wherein the signal generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product, and wherein: 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' subdomain and the 3' subdomain can 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 the nucleic acid amplification product, The signal generating oligonucleotide comprises a 5' terminal domain having a length of about 1 nt to about 6 nt and located 5' of the 5' subdomain, and The 5' terminal domain is not capable of hybridizing with the 3' end of the nucleic acid amplification product, Optionally, wherein the signal generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, 5' subdomain and / or 3' subdomain.

2. A method for detecting a target nucleic acid sequence in a sample, comprising: amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby producing a nucleic acid amplification product; and The nucleic acid amplification product is detected using a signal generating oligonucleotide, wherein the signal generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product, and wherein: 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' subdomain and the 3' subdomain can 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 the 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, wherein: The signal generating oligonucleotide comprises a 5' terminal domain having a length of about 1 nt to about 15 nt and located 5' of the 5' subdomain, and The 5' terminal domain cannot hybridize to the 3' end of the nucleic acid amplification product.

3. The method according to any one of claims 1 to 2, wherein: (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 one, two, three, four, five, six, seven or eight LNA nucleotides; (c) The loop domain contains one or more LNA nucleotides. Optionally, the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for the nucleic acid amplification product. Optionally, enhancing the specificity of the signal-generating oligonucleotide for the nucleic acid amplification product includes increasing the mismatch discrimination between the nucleic acid amplification product and the mismatch product. Optionally, the mismatch product includes a non-template control product and / or a non-target genotype; (d) The terminal 3'-nucleotide of the signal-generating oligonucleotide is an LNA nucleotide. Optionally, the LNA nucleotide reduces or prevents digestion of the signal-generating oligonucleotide and / or removal of the quencher associated with the 3'-end of the signal-generating oligonucleotide. Optionally, it also reduces or prevents digestion by the exonuclease activity of the polymerase; (e) The 5'-subdomain and / or the 3'-subdomain contains one or more LNA nucleotides. Optionally, the one or more LNA nucleotides enhance the stability of the paired stem domain. Optionally, the paired stem domain contains base pairing of at least one pair of opposing LNA nucleotides; (f) The nucleotides located in the 5'-terminal domain are not capable of intramolecular nucleobase pairing; and / or less than about 5 nt, 4 nt, 3 nt, 2 nt or 1 nt of the 5'-terminal domain is complementary to the 3'-end of the nucleic acid amplification product; and / or (g) The signal-generating oligonucleotide does not contain nucleotides located 3' of the 3'-subdomain.

4. The method according to any one of claims 1-3, wherein the signal-generating oligonucleotide contains a label. Optionally, the label includes a quencheable label. Optionally, the quencheable label is a fluorophore.

5. The method according to any one of claims 1-4, wherein the signal-generating oligonucleotide contains a quencher. Optionally: 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.

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

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

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

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

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

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

12. The method according to any one of claims 1 - 11, wherein extending the nucleic acid amplification product hybridized with the signal - generating oligonucleotide with an enzyme having polymerase activity is capable of disrupting the intramolecular nucleotide base pairing between the 5'-subdomain and the 3'-subdomain, thereby unwinding the paired stem domain.

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

14. The method according to any one of claims 1 - 13, wherein when: (i) the signal - generating oligonucleotide hybridizes with the nucleic acid amplification product; and / or (ii) the nucleic acid amplification product is extended to generate an extended nucleic acid amplification product hybridized with the signal - generating oligonucleotide, the label generates a detectable signal. Optionally, the signal is fluorescence.

15. The method according to any one of claims 1 - 14, wherein 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 include a constant temperature of about 30°C to about 72°C, further optionally about 55°C to about 75°C, optionally about 56°C to about 68°C, and further optionally about 66°C to about 68°C; wherein the amplification is carried out at the optimal temperature of an enzyme having hyperthermophilic polymerase activity. Optionally, the optimal temperature is about 66°C to about 68°C, and further optionally the amplification is carried out at a constant temperature; wherein the nucleic acid amplification product has a melting temperature within at least about 5 °C of the constant temperature; and / or 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 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.

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

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

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

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

20. The method according to any one of claims 1-19, 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 method according to any one of claims 1-20, wherein the signal generating oligonucleotide is configured such that: the paired stem domain is stable at the constant temperature in the absence of the nucleic acid amplification product, and when the nucleic acid amplification product hybridizes to the loop domain, the paired stem domain can be unwound, optionally by altering 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, 5' subdomain and / or 3' subdomain.

22. The method according to any one of claims 1-21, 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 the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement, optionally the spacer sequence has a length of about 4 nt to about 7 nt and / or has a GC content of less than about 50%.

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

24. The method according to any one of claims 1-23, wherein: (a)(i) The signal-generating oligonucleotide has a length of about 10 nt to about 100 nt; (ii) the second region, the spacer region and / or the first region have a length of about 1 nt to about 25 nt; and / or (iii) the 5'-subdomain, the 3'-subdomain, the loop domain and / or the 5'-terminal domain have a length of about 1 nt to about 25 nt; (b) The 5'-terminal domain has a length of about 1 nt to about 6 nt, the loop domain has a length of about 4 nt to about 15 nt, and the paired stem domain has a length of about 3 bp to about 8 bp; (c) The nucleic acid amplification product has a length of about 25 nt to about 35 nt; (d) The target nucleic acid sequence has a length of no more than about 20 nt to no more than about 90 nt, optionally the target nucleic acid sequence has a length of about 30 nt; and / or (e) The spacer sequence comprises a portion of the target nucleic acid sequence, optionally the spacer sequence is 1 to 10 bases in length, optionally the spacer sequence has a length of about 4 nt to about 7 nt and / or has a GC content of less than about 50%.

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

26. The method according to any one of claims 1-25, wherein amplifying the target nucleic acid sequence comprises: amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplification comprises contacting a nucleic acid comprising the target nucleic acid sequence with the following: (i) Forward primer and reverse primer, wherein the forward primer is capable of hybridizing with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with the sequence of the second strand of the target nucleic acid sequence; and (ii) An enzyme having hyperthermophilic DNA polymerase activity to produce the nucleic acid amplification product.

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

28. The method according to any one of claims 1-27, wherein amplifying the target nucleic acid sequence comprises: (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 hyperthermophilic DNA polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises the target nucleic acid sequence, and wherein 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 amplification comprises contacting the dsDNA with: (i) Forward primer and reverse primer, wherein the forward primer is capable of hybridizing with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with the sequence of the second strand of the target nucleic acid sequence; and (ii) An enzyme having hyperthermophilic DNA polymerase activity to produce the nucleic acid amplification product.

29. The method according to any one of claims 1-28, wherein the forward primer and / or the reverse primer: is configured to have a Tm of less than about 45°C; has a length of about 5 nt to about 25 nt, optionally a length of about 10 nt to about 14 nt; is configured to produce a nucleic acid amplification product having a length of about 25 nt to about 35 nt and a melting temperature within at least about 5°C of the constant temperature; comprises one or more phosphorothioate bonds; and / or has a GC content of about 30% to about 55%.

30. The method according to any one of claims 1-29, wherein: the 3' region of the forward primer and / or the reverse primer does not contain thymine bases, optionally the 3' region contains the first, second, third, and / or fourth nucleotides starting from the 3' end; the 5' region of the forward primer and / or the reverse primer does not contain more than 3 nt complementary to the spacer sequence, the region adjacent thereto, its complement, or any combination thereof, optionally the 5' region contains the first, second, third, and / or fourth nucleotides starting from the 5' end; The forward primer and / or reverse primer comprises a phosphorothioate bond between the first and second nucleotides starting from the 3'-end of the forward primer and / or reverse primer, optionally the phosphorothioate bond being capable of reducing or preventing polymerase-mediated degradation; The forward primer and / or reverse primer comprises a phosphorothioate bond between the second and third nucleotides starting from the 3'-end of the forward primer and / or reverse primer; The 3'-region of the forward primer and / or reverse primer does not contain more than 2 phosphorothioate bonds, optionally the 3'-region contains the first, second, third, and / or fourth nucleotides starting from the 3'-end; The forward primer and / or reverse primer comprises one or more phosphorothioate bonds in a region containing a GC dinucleotide repeat, optionally the one or more phosphorothioate bonds being capable of destabilizing base pairing; The presence of one or more LNA nucleotides in the loop domain, 5'-subdomain, and / or 3'-subdomain increases the sensitivity and / or specificity of the detection of the 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 LNA nucleotides; and / or The presence of the 5'-terminal domain in the signal-generating oligonucleotide increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide contains a blunt-ended hairpin structure.

31. The method according to any one of claims 1-30, wherein: The method comprises determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample; Determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample comprises determining the presence, absence, and / or amount of dsDNA and / or nucleic acid containing 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 containing the target nucleic acid sequence in the sample.

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

33. The method according to any one of claims 1-32, wherein: (a) The one or more polymerase terminators are located in the loop domain, the first region, the second region, and / or the spacer region, optionally the 5'-subdomain, the paired stem domain, and / or the 3'-subdomain do not contain one or more polymerase terminators; (b) The one or more polymerase terminators comprise one or more 2'-O-methyl (2'OM) RNA nucleotides; (c) The one or more polymerase terminators comprise one or more of an abasic site, a stabilized abasic site, a chemically trapped abasic site, or any combination thereof; (d)(i) The chemically trapped abasic site includes an abasic site reactive with an alkoxyamine or sodium borohydride; (ii) The abasic site includes an apurinic site, an apyrimidinic 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 terminators comprise: one or more RNA bases, one or more 2'-methoxyethyl riboses (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-deoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more isodeoxycytidine (iso-dC), one or more isodeoxyguanosine (iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH 26 OPO3] groups, or any combination thereof; (f) The one or more polymerase terminators comprise one or more steric blocking groups, optionally the one or more steric blocking groups increase the Tm of the nucleic acid amplification product / signal generating oligonucleotide duplex; and / or (g) The polymerase terminator comprises a modification incorporated between two bases of the signal generating oligonucleotide.

34. The method according to any one of claims 1-33, wherein the modification: (i) is a naphthylene azo compound, optionally Zen or iFQ; (ii) has the following structure: Among them, The linking groups L1 and L2 positioning the modification at an internal position of the signal generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy group; R1-R5 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 an attachment point for a ligand; and X is a nitrogen or carbon atom, wherein, if X is a carbon atom, the fourth substituent attached to this carbon atom can be hydrogen or a C1-C8 alkyl group; (iii) has the following structure: Among them, the linking groups L1 and L2 that position the modification at an internal position of the signal-generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl or alkoxy group; R1, R2, R4, R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group or electron-donating group; R6, R7, R9-R 12 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group or electron-donating group; R8 is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy or electron-withdrawing group; and X is a nitrogen or carbon atom, wherein if X is a carbon atom, the fourth substituent attached to this carbon atom can be hydrogen or a C1-C8 alkyl group; and / or (iv) has the following structure: Optionally, wherein R8 is NO2.

35. The method according to any one of claims 1-34, Among them, when the forward primer binds to the signal generating oligonucleotide to form a first undesired duplex, the one or more polymerase terminators are capable of terminating the polymerase extension of the forward primer of the first undesired duplex towards the 5' end of the signal generating oligonucleotide, optionally, the one or more polymerase terminators are capable of terminating the polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide.

36. The method according to any one of claims 1-35, Among them, when the reverse primer binds to the signal generating oligonucleotide to form a second undesired duplex, the one or more polymerase terminators are capable of terminating the polymerase extension of the reverse primer of the second undesired duplex towards the 5' end of the signal generating oligonucleotide, optionally, the one or more polymerase terminators are capable of terminating the polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide.

37. The method according to any one of claims 1-36, Among them, When the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired double-stranded body, the one or more polymerase terminators are capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired double-stranded body towards the 5'-end of the signal-generating oligonucleotide. Optionally, the one or more polymerase terminators are capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired double-stranded body beyond the one or more polymerase terminators of the signal-generating oligonucleotide. Optionally, the exogenous nucleic acid is selected from the group consisting of: sample nucleic acid, a primer configured to hybridize with a second target nucleic acid sequence, a primer configured to hybridize with an internal control, and any combination thereof.

38. The method according to any one of claims 1-37. Among them, If the forward primer binds to the signal-generating oligonucleotide to form a first undesired double-stranded body, the extension of the forward primer of the first undesired double-stranded body towards the 5'-end of the signal-generating oligonucleotide by an enzyme having hyperthermophilic DNA polymerase activity produces a first undesired extension product. Wherein the first undesired extension product is capable of being amplified by an enzyme having hyperthermophilic DNA polymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product. And Wherein the one or more polymerase terminators are capable of terminating the polymerase extension of the forward primer of the first undesired double-stranded body to produce a first stalled extension product. Wherein the first stalled extension product cannot be amplified by an enzyme having hyperthermophilic DNA polymerase activity in the presence of the forward primer and the reverse primer to produce the first undesired amplification product.

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

40. The method according to any one of claims 1-39. Among them, If the reverse primer binds to the signal-generating oligonucleotide to form a second undesired double-stranded body, the extension of the reverse primer of the second undesired double-stranded body towards the 5'-end of the signal-generating oligonucleotide by an enzyme having hyperthermophilic DNA polymerase activity produces a second undesired extension product. Wherein the second undesired extension product is capable of being amplified by an enzyme having hyperthermophilic DNA polymerase activity in the presence of the reverse primer to form a second undesired amplification product. And Wherein the one or more polymerase terminators are capable of terminating the polymerase extension of the reverse primer of the second undesired double-stranded body to produce a second stalled extension product. Wherein the second stalled extension product cannot be amplified by an enzyme having hyperthermophilic DNA polymerase activity in the presence of the reverse primer to produce the second undesired amplification product.

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

42. The method according to any one of claims 1-41, Among them, if the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the exogenous nucleic acid of the third undesired duplex is extended by an enzyme having hyperthermophilic DNA polymerase activity towards the 5'-end of the signal-generating oligonucleotide to generate a third undesired extension product, wherein the third undesired extension product is capable of being amplified by an enzyme having hyperthermophilic DNA polymerase activity in the presence of the reverse primer to form a third undesired amplification product; and wherein the one or more polymerase terminators are capable of terminating the polymerase extension of the exogenous nucleic acid of the third undesired duplex to produce a third stalled extension product, wherein the third stalled extension product is not capable of being amplified by an enzyme having hyperthermophilic DNA polymerase activity in the presence of the reverse primer to produce the third undesired amplification product.

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

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

45. The method according to any one of claims 1-44, wherein: 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 the detection of the false positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

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

47. The method according to any one of claims 1-46, wherein: the generation of the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate false positive signals; and / or the signal generating oligonucleotide that hybridizes to the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate false positive signals.

48. The method according to any one of claims 1-47, wherein: 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 unwanted amplification product, the second unwanted amplification product, and / or the third unwanted amplification product reach a detectable level; and / or 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 the false positive signal reaches a detectable level.

49. The method according to any one of claims 1-48, wherein: compared to a comparable method in which the signal generating oligonucleotide does not contain the one or more polymerase terminators, the appearance of the detectable level of the false positive signal, the first unwanted amplification product, the second unwanted amplification product, and / or the third unwanted 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; the false positive signal, the first unwanted amplification product, the second unwanted amplification product, and / or the third unwanted amplification product do not reach a detectable level within at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the start of the amplification step; and / or compared to a comparable method in which the signal generating oligonucleotide does not contain one or more polymerase terminators, the generation of the false positive signal, the first unwanted amplification product, the second unwanted amplification product, and / or the third unwanted amplification product is reduced by at least about 1.1-fold.

50. The method according to any one of claims 1-49, 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. The method according to any one of claims 1-50, the method comprising: contacting a sample containing a biological entity with a lysis buffer to produce a treated sample, wherein the lysis buffer contains one or more lysing agents capable of lysing the biological entity to release the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of containing the target nucleic acid sequence; and contacting a reagent composition with the treated sample to produce the amplification reaction mixture, wherein the reagent composition contains one or more amplification reagents.

52. The method according to any one of claims 1-51, wherein Amplifying the target nucleic acid sequence includes generating the nucleic acid amplification product at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes; and / or The detection is performed within less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes starting from the time when the reagent composition contacts the treated sample.

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

2.

54. The method according to any one of claims 1 - 53, wherein the sample nucleic acid includes sample ribonucleic acid and / or sample deoxyribonucleic acid, optionally the sample nucleic acid includes cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof.

55. The method according to any one of claims 1 - 54, wherein the one or more amplification reagents include: Reverse transcriptase; An enzyme having hyperthermophilic DNA polymerase activity, optionally, the enzyme having hyperthermophilic DNA polymerase activity has reverse transcriptase activity; Forward primer; Reverse primer; Reverse transcription primer; and / or dNTP.

56. The method according to any one of claims 1 - 55, wherein the reagent composition is lyophilized, heat - dried, and / or contains one or more additives, and the one or more additives include: Tween 20, Triton X - 100, and / or Tween 80; Amino acids; Sugars or sugar alcohols, optionally the sugars or sugar alcohols include sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or Polymers, optionally the polymers include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides, or any combination thereof, Optionally, contacting the reagent composition with the treated sample includes dissolving the reagent composition in the treated sample.

57. The method according to any one of claims 1 - 56, wherein the one or more lysis reagents include: About 0.001% (w / v) to about 1.0% (w / v) of the treated sample, optionally about 0.2% (w / v) of the treated sample; and / or Detergents, optionally the detergents include one or more of cationic surfactants, anionic surfactants, non - ionic surfactants, and amphoteric surfactants.

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

59. The method according to any one of claims 1-58, wherein the amplification: is carried out for a period of about 5 minutes to about 60 minutes, optionally the amplification is carried out for a period of about 15 minutes; and / or is carried out under isothermal amplification conditions without a helicase, without a single-strand binding protein, without a cleavage agent, and without a recombinase.

60. The method according to any one of claims 1-59, (a) wherein the amplification 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), optionally the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); (b) wherein the enzyme having hyperthermophilic DNA polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:31 or a functional fragment thereof, optionally the enzyme having hyperthermophilic DNA polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:31, further optionally the enzyme having hyperthermophilic DNA polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:31, optionally the enzyme having hyperthermophilic DNA polymerase activity has low exonuclease activity or no exonuclease activity; and / or (c) wherein the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase and the enzyme having hyperthermophilic DNA polymerase activity, optionally the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophilic DNA polymerase activity, and the forward primer and the reverse primer, further optionally the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophilic DNA polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

61. The method according to any one of claims 1-60, wherein: the biological entity includes one or more of prokaryotic cells, eukaryotic cells, virus particles, exosomes, protoplasts, and microvesicles; the biological entity includes viruses, bacteria, fungi, protozoa, parts thereof, or any combination thereof; and / or the target nucleic acid sequence is a nucleic acid sequence of a virus, bacterium, fungus, or protozoan, optionally the sample nucleic acid is derived from a virus, bacterium, fungus, or protozoan.

62. The method according to any one of claims 1-61, wherein: The virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus; The bacteria include one or more of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes; The fungi include one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or The protozoa include one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.

63. The method according to any one of claims 1-62, wherein the sample is a biological sample or an environmental sample, wherein the environmental sample is or is obtained from: a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a salt water sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof; and / or wherein the biological sample is or is obtained from: a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof.

64. The method according to any one of claims 1-63, (a) wherein the amplification step comprises multiplex amplification of two or more target nucleic acid sequences, and wherein the detection step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally the two or more target nucleic acid sequences are specific for two or more different organisms, and further optionally the two or more different organisms include one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C; (b) wherein the amplification does not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA), optionally the amplification does not include LAMP; (c) wherein the amplification comprises one or more of the following: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA, optionally the amplification does not include LAMP; and / or (d) wherein the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) heat denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) addition of a ribonuclease inhibitor to the treated sample; (vii) addition of a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more lysis agents from the treated sample or the amplification reaction mixture; (xii) heat denaturation and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or amplification reaction mixture.

65. A signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to a nucleic acid amplification product, and wherein: 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' subdomain and the 3' subdomain is capable of forming 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 the nucleic acid amplification product, the signal-generating oligonucleotide comprises a 5' terminal domain having a length of about 1 nt to about 6 nt and located 5' of the 5' subdomain, and the 5' terminal domain is not capable of hybridizing to the 3' end of the nucleic acid amplification product.

66. The signal-generating oligonucleotide according to claim 65, wherein the signal-generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, 5' subdomain, and / or 3' subdomain.

67. A signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to a nucleic acid amplification product, and wherein: 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' subdomain and the 3' subdomain is capable of forming 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 the nucleic acid amplification product, and the signal-generating oligonucleotide comprises one or more locked nucleic acid (LNA) nucleotides in the loop domain, 5' subdomain, and / or 3' subdomain.

68. The signal-generating oligonucleotide according to claim 67, wherein: The signal-generating oligonucleotide comprises a 5'-terminal domain that is about 1 nt to about 15 nt in length and is located 5' of the 5'-subdomain; and / or the 5'-terminal domain is not capable of hybridizing to the 3'-end of the nucleic acid amplification product.

69. The signal-generating oligonucleotide according to any one of claims 65-68, wherein: (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 one, two, three, four, five, six, seven, or eight LNA nucleotides; (iii) the loop domain comprises one or more LNA nucleotides, optionally the one or more LNA nucleotides enhance the specificity and / or affinity of the signal-generating oligonucleotide for the nucleic acid amplification product, and further optionally enhancing the specificity of the signal-generating oligonucleotide for the nucleic acid amplification product includes increasing the mismatch discrimination between the nucleic acid amplification product and a mismatch product, optionally the mismatch product includes a non-template control product and / or a non-target genotype; (iv) the terminal 3'-nucleotide of the signal-generating oligonucleotide is an LNA nucleotide, 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, and further optionally reduces or prevents digestion by the exonuclease activity of a polymerase; (v) the 5'-subdomain and / or the 3'-subdomain comprises one or more LNA nucleotides, optionally the one or more LNA nucleotides enhance the stability of the paired stem domain, and further optionally the paired stem domain comprises base pairing of at least one pair of opposing LNA nucleotides; (vi) the nucleotides located in the 5'-terminal domain are not capable of intramolecular nucleobase pairing; and / or less than about 5 nt, 4 nt, 3 nt, 2 nt, or 1 nt of the 5'-terminal domain is complementary to the 3'-end of the nucleic acid amplification product; and / or (vii) the signal-generating oligonucleotide does not comprise nucleotides located 3' of the 3'-subdomain.

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

71. The signal-generating oligonucleotide according to any one of claims 65-70, wherein the signal-generating oligonucleotide comprises a quencher, optionally: 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.

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

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

74. The signal-generating oligonucleotide according to any one of claims 65-73, wherein: When intermolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain forms a paired stem domain, the quencher and the label are in close proximity; When the signal-generating oligonucleotide does not contain a paired stem domain, the quencher and the label are not in close proximity; The nucleic acid amplification product is produced by amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other, optionally amplifying the target nucleic acid sequence under isothermal amplification conditions, optionally the isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, further optionally about 55°C to about 75°C, optionally about 56°C to about 68°C, and further optionally 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 producing an extended nucleic acid amplification product hybridized to the signal-generating oligonucleotide, optionally the extended nucleic acid amplification product comprises a complement of the 5' terminal domain; and / or Extending the nucleic acid amplification product hybridized to the signal-generating oligonucleotide with an enzyme having polymerase activity can disrupt the intermolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain, thereby unwinding the paired stem domain.

75. The signal-generating oligonucleotide according to any one of claims 65-74, wherein the label is capable of generating a detectable signal under the following conditions: (i) 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, optionally the signal is fluorescence.

76. The signal-generating oligonucleotide according to any one of claims 65-75, wherein under the following conditions: (i) 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, the label generates a detectable signal, optionally the signal is fluorescence.

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

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

80. A signal generating oligonucleotide according to any one of claims 65 - 79, wherein in the absence of extension of the nucleic acid amplification product, the nucleic acid amplification product is not capable of forming a stable duplex with the signal generating oligonucleotide.

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

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

83. A signal generating oligonucleotide according to any one of claims 65 - 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. A signal generating oligonucleotide according to any one of claims 65 - 83, wherein the signal generating oligonucleotide is configured such that: the paired stem domain is stable at the constant temperature in the absence of the nucleic acid amplification product, and when the nucleic acid amplification product hybridizes to the loop domain, the paired stem domain can be unwound, optionally by altering 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, 5'-subdomain and / or 3'-subdomain.

85. A signal generating oligonucleotide according to any one of claims 65 - 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) An intervening 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, optionally the intervening sequence has a length of about 4 nt to about 7 nt and / or has a GC content of less than about 50%.

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

87. The signal-generating oligonucleotide according to any one of claims 65-86, wherein: (a) (i) The signal-generating oligonucleotide has a length of about 10 nt to about 100 nt; (ii) the second region, the spacer region and / or the first region has a length of about 1 nt to about 25 nt; and / or (iii) the 5'-subdomain, the 3'-subdomain, the loop domain and / or the 5'-terminal domain has a length of about 1 nt to about 25 nt; (b) The 5'-terminal domain has a length of about 1 nt to about 6 nt, the loop domain has a length of about 4 nt to about 15 nt, and the paired stem domain has a length of about 3 bp to about 8 bp; (c) The nucleic acid amplification product has a length of about 25 nt to about 35 nt; (d) The target nucleic acid sequence has a length of no more than about 20 nt to no more than about 90 nt, optionally the target nucleic acid sequence has a length of about 30 nt; and / or (e) The intervening sequence comprises a portion of the target nucleic acid sequence, optionally the intervening sequence is 1 to 10 bases in length, optionally the intervening sequence has a length of about 4 nt to about 7 nt and / or has a GC content of less than about 50%.

88. The signal-generating oligonucleotide according to any one of claims 65-87, wherein: (a) The forward primer is capable of hybridizing to the sequence of the first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to the sequence of the second strand of the target nucleic acid sequence, optionally the nucleic acid amplification product is produced by amplifying the target nucleic acid sequence with the forward primer and the reverse primer; (b) The signal-generating oligonucleotide includes 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 contains one or more polymerase terminators and / or one or more phosphorothioate linkages. Optionally, the first region, the second region, and / or the spacer region contains one or more polymerase terminators.

89. The signal-generating oligonucleotide according to any one of claims 65 - 88, wherein: (i) The one or more polymerase terminators are located in the loop domain, the first region, the second region, and / or the spacer region. Optionally, the 5'-subdomain, the paired stem domain, and / or the 3'-subdomain do not contain one or more polymerase terminators; (ii) The one or more polymerase terminators contain one or more 2'-O-methyl (2'OM) RNA nucleotides; (iii) The one or more polymerase terminators contain one or more abasic sites, stable abasic sites, chemically trapped abasic sites, or any combination thereof; (iv)(a) The chemically trapped abasic site includes an abasic site that reacts with an alkoxyamine or sodium borohydride; (b) The abasic site includes an apurinic site, an apyrimidinic 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 terminators comprise: one or more RNA bases, one or more 2'-methoxyethyl riboses (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-deoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more isodeoxycytidine (iso-dC), one or more isodeoxyguanosine (iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH 26 OPO3] groups, or any combination thereof; (vi) The one or more polymerase terminators contain one or more steric blocking groups. Optionally, 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 terminator contains a modification incorporated between two bases of the signal-generating oligonucleotide.

90. The signal-generating oligonucleotide according to any one of claims 65 - 89, wherein the modification: (i) is a naphthylazo compound, optionally Zen or iFQ; (ii) has the following structure: Among them, The linking groups L1 and L2 that position the modification at an internal position of the signal-generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, or alkoxy group; R1 - R5 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 an attachment point for a ligand; and X is a nitrogen or carbon atom, wherein, if X is a carbon atom, the fourth substituent attached to this carbon atom can be hydrogen or a C1 - C8 alkyl group; (iii) has the following structure: Among them, the linking groups L1 and L2 that position the modification at an internal position of the signal-generating oligonucleotide are independently an alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl or alkoxy group; R1, R2, R4, R5 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group or electron-donating group; R6, R7, R9-R 12 are independently hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy, electron-withdrawing group or electron-donating group; R8 is hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl, alkylaryl, alkoxy or electron-withdrawing group; and X is a nitrogen or carbon atom, wherein if X is a carbon atom, the fourth substituent attached to this carbon atom can be hydrogen or a C1-C8 alkyl group; and / or (iv) has the following structure: Optionally, wherein R8 is NO2.

91. The signal-generating oligonucleotide according to any one of claims 65 - 90, Among them, When the forward primer binds to the signal - generating oligonucleotide to form a first undesired double - strand, the one or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesired double - strand towards the 5' end of the signal - generating oligonucleotide. Optionally, the one or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesired double - strand beyond the one or more polymerase terminators of the signal - generating oligonucleotide.

92. The signal - generating oligonucleotide according to any one of claims 65 - 91, Among them, When the reverse primer binds to the signal - generating oligonucleotide to form a second undesired double - strand, the one or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesired double - strand towards the 5' end of the signal - generating oligonucleotide. Optionally, the one or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesired double - strand beyond the one or more polymerase terminators of the signal - generating oligonucleotide.

93. The signal - generating oligonucleotide according to any one of claims 65 - 92, Among them, When an exogenous nucleic acid binds to the signal - generating oligonucleotide to form a third undesired double - strand, the one or more polymerase terminators can terminate the polymerase extension of the exogenous nucleic acid of the third undesired double - strand towards the 5' end of the signal - generating oligonucleotide. Optionally, the one or more polymerase terminators can terminate the polymerase extension of the exogenous nucleic acid of the third undesired double - strand beyond the one or more polymerase terminators of the signal - generating oligonucleotide.

94. A kit for detecting a target nucleic acid sequence in a sample, the kit comprising: (a) The signal - generating oligonucleotide according to any one of claims 1 - 93; (b) A lysis buffer containing one or more lysis agents capable of lysing a biological entity to release the sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of containing the target nucleic acid sequence. Optionally, the one or more lysis agents include a detergent, and wherein the detergent includes one or more of a cationic surfactant, anionic surfactant, non - ionic surfactant, and zwitterionic surfactant; and / or (c) A reagent composition containing one or more amplification reagents, the amplification reagents containing one or more components for amplification for amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplification include: (i) A forward primer and a reverse primer, wherein the forward primer can hybridize with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer can hybridize with the sequence of the second strand of the target nucleic acid sequence; and / or (ii) An enzyme with hyperthermophilic DNA polymerase activity capable of generating nucleic acid amplification products. Optionally, the enzyme with hyperthermophilic DNA polymerase activity has an amino acid sequence that is 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 DNA polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

31. Optionally further, the enzyme with hyperthermophilic DNA polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:

31.

95. The kit according to claim 94, wherein the forward primer and / or the reverse primer: is configured to have a Tm of less than about 45°C; has a length of about 5 nt to about 25 nt, optionally a length of about 10 nt to about 14 nt; is configured to generate a nucleic acid amplification product having a length of about 25 nt to about 35 nt and a melting temperature within at least about 5°C of the constant temperature; comprises one or more phosphorothioate bonds; and / or has a GC content of about 30% to about 55%.

96. The kit according to any one of claims 94 - 95, wherein the 3' region of the forward primer and / or the reverse primer does not contain thymine bases. Optionally, the 3' region contains the first, second, third, and / or fourth nucleotides starting from the 3' end; wherein the 5' region of the forward primer and / or the reverse primer does not contain more than 3 nt complementary to the spacer sequence, the region adjacent thereto, its complement, or any combination thereof. Optionally, the 5' region contains the first, second, third, and / or fourth nucleotides starting from the 5' end; wherein the forward primer and / or the reverse primer comprises a phosphorothioate bond between the first and second nucleotides starting from the 3' end of the forward primer and / or the reverse primer. Optionally, the phosphorothioate bond can reduce or prevent polymerase-mediated degradation; wherein the forward primer and / or the reverse primer comprises a phosphorothioate bond between the second and third nucleotides starting from the 3' end of the forward primer and / or the reverse primer; wherein the 3' region of the forward primer and / or the reverse primer does not contain more than 2 phosphorothioate bonds. Optionally, the 3' region contains the first, second, third, and / or fourth nucleotides starting from the 3' end; wherein the forward primer and / or the reverse primer comprises one or more phosphorothioate bonds in a region containing a GC dinucleotide repeat. Optionally, the one or more phosphorothioate bonds can destabilize base pairing; wherein the presence of one or more LNA nucleotides in the loop domain, 5' subdomain, and / or 3' subdomain increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product to at least about 1.1-fold compared to a comparable method where the signal-generating oligonucleotide does not contain LNA nucleotides; The presence of the 5'-terminal domain in the signal-generating oligonucleotide increases the sensitivity and / or specificity of the detection of the nucleic acid amplification product to at least about 1.1-fold compared to a comparable method where 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.

97. The kit according to any one of claims 94-96, wherein the nucleic acid amplification product is about 20 to 40 bases in length, and 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 the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length.

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

99. The kit according to any one of claims 94 - 98, wherein the reagent composition is freeze-dried and / or heat-dried and comprises one or more additives, and wherein the one or more additives comprise: Amino acids; Sugars or sugar alcohols, optionally the sugars or sugar alcohols comprise sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof; and / or Polymers, optionally the polymers comprise polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides or any combination thereof.

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