Modified molecular beacons for improved detection specificity

Abstract

The present disclosure includes methods, compositions, and kits for use in detecting a target nucleic acid sequence in a sample. The method can include the use of a signal-generating oligonucleotide that can hybridize to a nucleic acid amplification product and includes one or more polymerase stoppers. In some embodiments, assays using the protected signal-generating oligonucleotides provided herein exhibit reduced non-specific product formation and fewer false positives.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 374,772, filed September 7, 2022; and U.S. Provisional Application No. 63 / 374,774, filed September 7, 2022, the entire contents of which are expressly incorporated herein by reference in their entirety. Sequence Listing Reference This application is filed with an electronic Sequence Listing. The Sequence Listing is provided in file number 68EB-317332-WO, created September 6, 2023, and is 115,267 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. The present disclosure relates generally to methods and compositions for amplifying (eg, isothermal amplification) nucleic acids. [Background technology]

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

[0003] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture under isothermal amplification conditions, thereby generating a nucleic acid amplification product; and detecting the nucleic acid amplification product using a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product and comprises one or more polymerase stoppers. The method may include the steps of: contacting a sample containing a biological entity with a lysis buffer to generate a processed sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entity and releasing sample nucleic acid contained therein, the sample nucleic acid being suspected of containing the target nucleic acid sequence. The method may include the steps of: contacting a reagent composition with the processed sample to generate an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0004] Disclosed herein are 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, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the loop domain comprises one or more polymerase stoppers. In some embodiments, the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. In some embodiments, the nucleic acid amplification products are generated by amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other. In some embodiments, the nucleic acid amplification products are generated by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0005] The signal-generating oligonucleotide may comprise a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, one or more polymerase stoppers are located within the loop domain. In some embodiments, the 5' subdomain, paired stem domain, and / or 3' subdomain do not comprise one or more polymerase stoppers. In some embodiments, the nucleic acid amplification product comprises (1) the sequence of a forward primer and its reverse complement, (2) the sequence of a reverse primer and its reverse complement, and (3) a spacer sequence flanked by (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 is 1 to 10 bases in length. In some embodiments, the signal-generating oligonucleotide comprises a first region comprising at least a portion of the sequence of the reverse primer. In some embodiments, the signal-generating oligonucleotide comprises a second region comprising a sequence complementary to at least a portion of the forward primer. In some embodiments, the signal-generating oligonucleotide comprises a spacer region comprising at least a portion of the spacer sequence. One or more of the first region, second region, and / or spacer region may comprise one or more polymerase stoppers. The first region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. The second region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. In some embodiments, the spacer region comprises a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer.

[0006] The signal-generating oligonucleotide can be from about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the forward primer and / or the reverse primer is from about 5 nucleotides to about 25 nucleotides in length. In some embodiments, the second region, the spacer region, and / or the first region is from about 1 nucleotide to about 25 nucleotides in length. In some embodiments, the 5' subdomain, the 3' subdomain, the loop domain, the 5' terminal domain, and / or the 3' terminal domain is from about 1 nucleotide to about 25 nucleotides in length. In some embodiments, the signal-generating oligonucleotide comprises a 5' terminal domain that is 5' of the 5' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a 3' terminal domain that is 3' of the 5' subdomain. In some embodiments, the 5' terminal domain and / or the 3' terminal domain does not comprise one or more polymerase stoppers.

[0007] In some embodiments, the first region comprises at least a portion of the 5' subdomain and / or the loop domain. In some embodiments, the spacer region comprises at least a portion of the loop domain. In some embodiments, the second region comprises at least a portion of the loop domain and / or the 3' subdomain. In some embodiments, the 5' subdomain comprises at least a portion of the first region and / or the spacer region. In some embodiments, the loop domain comprises at least a portion of the spacer region, the first region, and / or the second region. In some embodiments, the 3' subdomain comprises at least a portion of the second region and / or the spacer region.

[0008] The one or more polymerase stoppers may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. In some embodiments, the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemically captured abasic site, or any combination thereof. In some embodiments, the stable abasic site comprises 1',2'-dideoxy. In some embodiments, the chemically captured abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride. In some embodiments, the abasic site comprises an apurinic base site, an apyrimidinic base site, or both. In some embodiments, the abasic site is generated by an alkylating agent or an oxidizing agent. In some embodiments, the one or more polymerase stoppers are one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more a plurality of 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3(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.

[0009] In some embodiments, the signal-generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids. In some embodiments, the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. In some embodiments, the signal-generating oligonucleotide comprises a label, e.g., a quenchable label (e.g., a fluorophore). In some embodiments, the signal-generating oligonucleotide comprises a quencher. In some embodiments, the label is in the 3'-terminal domain and the quencher is in the 5'-terminal domain, and / or the label is in the 5'-terminal domain and the quencher is in the 3'-terminal domain.

[0010] In some embodiments, after the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, one or more polymerase stoppers can stop the polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, one or more polymerase stoppers can stop the polymerase extension of the forward primer of the first undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, after the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, one or more polymerase stoppers can stop the polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, one or more polymerase stoppers can stop the polymerase extension of the reverse primer of the second undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, after the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers can stop the polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide. In some embodiments, the one or more polymerase stoppers can stop the polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, the exogenous nucleic acid is selected from the group including a sample nucleic acid, a primer configured to hybridize to a second target nucleic acid sequence, a primer configured to hybridize to an internal standard, or any combination thereof.

[0011] In some embodiments, the sample nucleic acid comprises a nucleic acid comprising a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence comprises the following steps: amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, wherein the nucleic acid comprising the target nucleic acid sequence is contacted with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the nucleic acid is double-stranded DNA. In some embodiments, the nucleic acid is a product of a reverse transcription reaction. In some embodiments, the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid. In some embodiments, the amplifying step comprises generating a nucleic acid by a reverse transcription reaction. In some embodiments, the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. In some embodiments, the step of amplifying a target nucleic acid sequence comprises the following steps: (c1) contacting a sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying step comprises contacting the dsDNA with (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity, thereby producing a nucleic acid amplification product.

[0012] In some embodiments, when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a first undesired extension product. In some embodiments, the first undesired extension product can be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product. In some embodiments, one or more polymerase stoppers can stop polymerase extension of the forward primer of the first undesired duplex to generate a first extension stall product. In some embodiments, the first extension stall product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to generate a first undesired amplification product. In some embodiments, one or more polymerase stoppers can terminate polymerase extension of the forward primer of a first undesired duplex past one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a second undesired extension product. In some embodiments, the second undesired extension product can be amplified by an enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to form a second undesired amplification product. In some embodiments, one or more polymerase stoppers can terminate polymerase extension of the reverse primer of the second undesired duplex to generate a second extension stall product. In some embodiments, the second extension stalled product cannot be amplified by an enzyme with hyperthermophile polymerase activity in the presence of a reverse primer to produce a second undesired amplification product.In some embodiments, the one or more polymerase stoppers can terminate polymerase extension of the reverse primer of the second undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when an exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a third undesired extension product. In some embodiments, the third undesired extension product can be amplified by an enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to form a third undesired amplification product. In some embodiments, the one or more polymerase stoppers can terminate polymerase extension of the exogenous nucleic acid of the third undesired duplex to generate a third extension stall product. In some embodiments, the third extension stalled product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to produce a third undesired amplification product, hi some embodiments, the one or more polymerase stoppers can stop polymerase extension of the third undesired duplex exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

[0013] In some embodiments, the detecting step comprises contacting the nucleic acid amplification product with a 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 label is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product. In some embodiments, the label generates a signal (e.g., fluorescence) after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product. In some embodiments, the detecting step comprises detecting the signal of the label before the amplification reaction, after the amplification reaction, or both. In some embodiments, detecting the nucleic acid amplification product comprises detecting a signal generated by the label of the signal-generating oligonucleotide. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, detecting the signal comprises detecting fluorescence emitted by the label. In some embodiments, the method comprises 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 comprises determining the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. The presence, absence, and / or amount of the signal may indicate 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 signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample.

[0014] In some embodiments, the label is capable of generating a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. In some embodiments, the label generates a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. In some embodiments, the signal and the false-positive signal are indistinguishable. In some embodiments, the generation of the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, detection of a false-positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

[0015] The presence of one or more polymerase stoppers in the signal-generating oligonucleotide can increase the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in a sample by at least about 1.1 times compared to a signal-generating oligonucleotide that does not contain one or more polymerase stoppers. In some embodiments, the generation of the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, the signal-generating oligonucleotide hybridized to the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, the nucleic acid amplification product reaches a detectable level 1, 2, 5, 10, 15, or 20 minutes before, or at least about 1, 2, 5, 10, 15, or 20 minutes before, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reaches a detectable level. In some embodiments, the signal reaches a detectable level 1, 2, 5, 10, 15, or 20 minutes before, or at least about 1, 2, 5, 10, 15, or 20 minutes before, the false positive signal reaches a detectable level. In some embodiments, the appearance of detectable levels of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product is delayed by 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes, or by at least about 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes, compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers. In some embodiments, the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach detectable levels for 5 minutes, 10 minutes, 15 minutes, or 20 minutes, or for at least about 5 minutes, 10 minutes, 15 minutes, or 20 minutes, after the amplifying step begins.In some embodiments, the occurrence of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product is reduced by at least about 1.1-fold compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers.

[0016] In some embodiments, amplifying the target nucleic acid sequence comprises producing a detectable level of nucleic acid amplification product within 20 minutes, 15 minutes, or 10 minutes, or within about 20 minutes, 15 minutes, or 10 minutes, hi some embodiments, the detecting step is performed less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the treated sample.

[0017] In some embodiments, the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. In some embodiments, the pH of the lysis buffer is about 1.0 to about 10.0 (e.g., about 2.2). In some embodiments, the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid. In some embodiments, the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, the one or more amplification reagents comprise a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity; and / or dNTPs. In some embodiments, the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity, a forward primer, a reverse primer, and a reverse transcription primer.

[0018] The reagent composition may be lyophilized, heat-dried, and / or may include one or more additives. In some embodiments, the one or more additives include Tween 20, Triton X-100, and / or Tween 80; amino acids; sugars or sugar alcohols; and / or polymers. The sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the polymers include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. In some embodiments, contacting the reagent composition with the processed sample includes dissolving the reagent composition in the processed sample. In some embodiments, the one or more lysis reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) of the processed sample (e.g., about 0.2% (wt / vol) of the processed sample); and / or a detergent (e.g., one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant). In some embodiments, the method is performed in a single reaction vessel; does not include a step of using any enzymes other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include a step of using any enzymes other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein.

[0019] The target nucleic acid sequence may comprise a length of about 20 nucleotides or less to about 90 nucleotides or less (e.g., about 30 nucleotides). In some embodiments, the forward primer, reverse primer, and / or reverse transcription primer are about 8 to 16 bases in length. In some embodiments, the nucleic acid amplification product is about 20 to 40 bases in length. In some embodiments, the spacer sequence comprises a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1 to 10 bases in length. In some embodiments, the isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, e.g., about 55°C to about 75°C or about 56°C to about 67°C. In some embodiments, the amplifying step is carried out for a time period of about 5 minutes to about 60 minutes (e.g., a time period of about 15 minutes). In some embodiments, the amplifying step is carried out under isothermal amplification conditions that are helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free. In some embodiments, the amplifying step is carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immuno-amplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). In some embodiments, the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0020] In some embodiments, the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof. In some embodiments, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase and the enzyme with hyperthermophilic polymerase activity. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, and the forward and reverse primers. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

[0021] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the biological entity includes a virus, a bacterium, a fungus, a protozoan, a portion thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoan. The virus can be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus.In some embodiments, the bacteria is Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella pneumophila, Legionella pneumophila, or any of a number of other bacteria. dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes. In some embodiments, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum.In some embodiments, the protozoa include one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp. The sample can be a biological sample or an environmental sample. In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

[0022] The amplifying step comprises multiplex amplification of two or more target nucleic acid sequences, and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally the two or more target nucleic acid sequences being specific to two or more different organisms, and further optionally the two or more different organisms comprising one or more of SARS-CoV-2, influenza A, influenza B and / or influenza C. In some embodiments, the amplifying step may include and / or exclude one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplifying step does not include LAMP. In some embodiments, the methods do not include one or more of the following: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture.

[0023] The present disclosure includes a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit includes a signal-generating oligonucleotide disclosed herein. The kit can include a lysis buffer containing one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence, and optionally, the one or more lysis agents include a detergent, and the detergent can include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit may include a reagent composition comprising one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions, the one or more components for amplification comprising: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, wherein the enzyme optionally has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof, optionally has an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO: 7, and further optionally is a polymerase comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer.

[0024] Disclosed herein is a method for detecting Neisseria gonorrhea in a sample. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17. The method can include, if the sample contains Neisseria gonorrhea, generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can include determining the presence or amount of the amplicon as an indication of the presence of Neisseria gonorrhea in the sample.

[0025] In some embodiments, at least one primer pair comprises a first primer comprising the sequence of SEQ ID NO: 2 or 17 and a second primer comprising the sequence of SEQ ID NO: 3 or 16. In some embodiments, at least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae is SEQ ID NO: 2 and 3, SEQ ID NO: 2 and 16, SEQ ID NO: 17 and 3, or SEQ ID NO: 17 and 16. In some embodiments, determining the presence or amount of an amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more signal-generating oligonucleotides, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14. In some embodiments, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14. In some embodiments, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

[0026] Disclosed herein is a method for detecting Chlamydia trachomatis (C. trachomatis) in a sample. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of C. trachomatis, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOS: 20-23, 25-26, 28-29, and 31, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOS: 20-23, 25-26, 28-29, and 31. The method can include, if the sample contains C. trachomatis, generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can include determining the presence or amount of the amplicon as an indication of the presence of C. trachomatis in the sample.

[0027] In some embodiments, at least one primer pair comprises a first primer comprising the sequence of SEQ ID NO: 20, 22, 25, or 28, and a second primer comprising the sequence of SEQ ID NO: 21, 23, 26, 29, or 31. In some embodiments, at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis is SEQ ID NO: 20 and 21, SEQ ID NO: 20 and 23, SEQ ID NO: 20 and 26, SEQ ID NO: 20 and 29, SEQ ID NO: 20 and 31, SEQ ID NO: 22 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 22 and 26, SEQ ID NO: 22 and 29, SEQ ID NO: 22 and 31, SEQ ID NO: 25 and 21, SEQ ID NO: 25 and 23, SEQ ID NO: 25 and 26, SEQ ID NO: 25 and 29, SEQ ID NO: 25 and 31, SEQ ID NO: 28 and 21, SEQ ID NO: 28 and 23, SEQ ID NO: 28 and 26, SEQ ID NO: 28 and 29, or SEQ ID NO: 28 and 31. In some embodiments, determining the presence or amount of an amplicon of a target nucleic acid sequence comprises contacting the amplicon with one or more signal-generating oligonucleotides, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32. In some embodiments, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32. In some embodiments, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

[0028] Disclosed herein includes methods for detecting influenza B virus in a sample. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of influenza B virus, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 36-37 and 41, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOs: 36-37 and 41. The method can include, if the sample contains influenza B virus, generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can include determining the presence or amount of the amplicon as an indication of the presence of influenza B virus in the sample.

[0029] In some embodiments, at least one primer pair comprises a first primer comprising the sequence of SEQ ID NO: 36 and a second primer comprising the sequence of SEQ ID NO: 37 or 41. In some embodiments, at least one primer pair capable of hybridizing to an influenza B virus target nucleic acid sequence is SEQ ID NO: 36 and 37, or SEQ ID NO: 36 and 41. In some embodiments, determining the presence or amount of an amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more signal-generating oligonucleotides, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. In some embodiments, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. In some embodiments, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. The method can include contacting sample ribonucleic acid of the sample with a reverse transcriptase and a primer that exhibits at least about 85% identity to SEQ ID NO:38.

[0030] In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a wastewater sample, a saltwater sample, a sample exposed to air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is or is derived from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof. In some embodiments, the sample is contacted with a reagent composition comprising at least one primer pair to produce an amplification reaction mixture. The method can include contacting a sample containing a biological entity with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entity to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence; and contacting the processed sample with a reagent composition comprising at least one primer pair to produce an amplification reaction mixture, the reagent composition comprising one or more amplification reagents. In some embodiments, the one or more amplification reagents comprise a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity, optionally having reverse transcriptase activity; a reverse transcription primer; and / or dNTPs.

[0031] In some embodiments, generating an amplicon of the target nucleic acid sequence comprises amplifying the target nucleic acid sequence in an amplification reaction mixture under amplification conditions, thereby generating an amplicon of the target nucleic acid sequence. In some embodiments, the amplifying is carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA), and optionally, the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR). In some embodiments, the amplifying step 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 (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA), and optionally, the amplifying step does not include LAMP. In some embodiments, the amplifying step comprises one or more of APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA, and optionally, the amplifying step does not comprise LAMP.

[0032] In some embodiments, the methods do not include one or more of: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture.

[0033] The determining step can include contacting the amplicon of the target nucleic acid sequence with a signal-generating oligonucleotide for hybridization, and optionally includes the use of a real-time detection method. In some embodiments, the label of the signal-generating oligonucleotide can generate a signal after the signal-generating oligonucleotide hybridizes to the amplicon of the target nucleic acid sequence. In some embodiments, the label generates a signal after the signal-generating oligonucleotide hybridizes to the amplicon of the target nucleic acid sequence, and optionally the signal is fluorescent.

[0034] Disclosed herein are compositions for detecting Neisseria gonorrhea in a sample. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea, wherein each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17. In some embodiments, the at least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea comprises a primer comprising the sequence of SEQ ID NOS: 2 or 17 and a primer comprising the sequence of SEQ ID NOS: 3 or 16. The compositions can include one or more signal-generating oligonucleotides, each of which comprises a sequence selected from the group consisting of SEQ ID NOS: 1 and 12-14, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOS: 1 and 12-14. In some embodiments, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14. In some embodiments, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

[0035] Disclosed herein are compositions for detecting Chlamydia trachomatis in a sample. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 20-23, 25-26, 28-29, and 31, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOs: 20-23, 25-26, 28-29, and 31. In some embodiments, the at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis comprises a primer comprising the sequence set forth in SEQ ID NO: 20, 22, 25, or 28, and a primer comprising the sequence set forth in SEQ ID NO: 21, 23, 26, 29, or 31. The composition may comprise one or more signal-generating oligonucleotides, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32. In some embodiments, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32. In some embodiments, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

[0036] Disclosed herein are compositions for detecting influenza B virus in a sample. In some embodiments, the composition comprises at least one primer pair capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 36-37 and 41, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOs: 36-37 and 41. In some embodiments, the at least one primer pair capable of hybridizing to a target nucleic acid sequence of influenza B virus comprises a primer comprising the sequence of SEQ ID NO: 36 and a primer comprising the sequence of SEQ ID NO: 37 or 41. The composition may comprise a primer exhibiting at least about 85% identity to SEQ ID NO: 38. The composition may comprise one or more signal-generating oligonucleotides, each of which comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. In some embodiments, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. In some embodiments, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

[0037] In some embodiments, the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the loop domain comprises one or more polymerase stoppers. In some embodiments, the 5' subdomain, paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. In some embodiments, the signal-generating oligonucleotide comprises a 5' terminal domain located 5' of the 5' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a 3' terminal domain located 3' of the 5' subdomain. In some embodiments, the 5' terminal domain and / or the 3' terminal domain do not comprise one or more polymerase stoppers.

[0038] The one or more polymerase stoppers may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. In some embodiments, the one or more polymerase stoppers comprise an abasic site, a stable abasic site, a chemically captured abasic site, or any combination thereof. In some embodiments, the stable abasic site comprises 1',2'-dideoxy. In some embodiments, the chemically captured abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride. In some embodiments, the abasic site comprises an apurinic base site, an apyrimidinic base site, or both. In some embodiments, the abasic site is generated by an alkylating agent or an oxidizing agent. In some embodiments, the one or more polymerase stoppers comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 5-aminomethyl- ... or a plurality of 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3(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.

[0039] In some embodiments, the signal-generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids. In some embodiments, the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. In some embodiments, the signal-generating oligonucleotide comprises a label, e.g., a quenchable label (e.g., a fluorophore). In some embodiments, the signal-generating oligonucleotide comprises a quencher. In some embodiments, the label is in the 3'-terminal domain and the quencher is in the 5'-terminal domain, and / or the label is in the 5'-terminal domain and the quencher is in the 3'-terminal domain.

[0040] In some embodiments, provided are signal-generating oligonucleotides or primers up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, the signal-generating oligonucleotides or primers comprising a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17.

[0041] In some embodiments, provided are signal-generating oligonucleotides or primers up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, the signal-generating oligonucleotides or primers comprising a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32.

[0042] In some embodiments, provided are signal-generating oligonucleotides or primers up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of influenza B virus, the signal-generating oligonucleotides or primers comprising a sequence selected from the group consisting of SEQ ID NOs: 36-45, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 36-45. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 36-45. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 36-45. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 36-45.

[0043] In some embodiments, provided are signal-generating oligonucleotides or primers up to about 100 nucleotides in length that are capable of hybridizing to a target nucleic acid sequence of influenza A virus, the signal-generating oligonucleotides or primers comprising a sequence selected from the group consisting of SEQ ID NOs: 33-35, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 33-35. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 33-35. In some embodiments, the signal-generating oligonucleotides or primers comprise a sequence selected from the group consisting of SEQ ID NOs: 33-35. In some embodiments, the signal-generating oligonucleotides or primers consist of a sequence selected from the group consisting of SEQ ID NOs: 33-35.

[0044] In some embodiments, compositions are provided that include two or more of the signal-generating oligonucleotides and / or primers provided herein. The compositions may include: a lysis buffer containing one or more lysis agents capable of lysing a biological entity to release sample nucleic acid contained therein, the sample nucleic acid suspected of containing a target nucleic acid sequence, optionally the one or more lysis agents including a detergent, optionally the detergent including one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; and / or a reagent composition containing one or more amplification reagents including one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the one or more components for amplifying include an enzyme having hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, optionally having 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 having an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:7, and further optionally, the enzyme is a polymerase that includes the amino acid sequence of SEQ ID NO:7. [Brief explanation of the drawings]

[0045] [Figure 1A-1B] 1A-1B show non-limiting, exemplary schematics of isothermal amplification reactions provided herein. [Figures 2A-2C] 2A-2C are diagrams showing non-limiting exemplary primers and probes (molecular beacons) for an archaeal polymerase amplification (APA)-based Neisseria Gonorrhea assay. FIG. 2A depicts the position of the primers and molecular beacons relative to the template. FIG. 2B depicts the molecular beacon of FIG. 2A in unbound hairpin formation. FIG. 2C depicts a non-limiting exemplary protective molecular beacon (where "M" indicates a polymerase stopper, e.g., a 2'OM modified base) in unbound hairpin formation. [Figure 3]FIG. 2B shows non-limiting exemplary non-specific product formation that can occur in the APA-based Neisseria gonorrhoeae assay depicted in FIGS. 2A-2B. [Figure 4] 2A-2B show non-limiting exemplary inhibition of non-specific product formation in the APA-based Neisseria gonorrhoeae assay depicted in FIGS. 2A-2B using a protection probe (e.g., the protection probe shown in FIG. 2C). [Figures 5A-5E] Figure 5 shows data demonstrating nonspecific product formation (no 2'OM protection) in the FluB PB2 assay. The FluB PB2 reverse primer was set at 600 nM (Figure 5A), 500 nM (Figure 5B), 400 nM (Figure 5C), 300 nM (Figure 5D), and 200 nM (Figure 5E). [Figures 6A-6D]

[0033] Figure 6 shows data related to the prevention of nonspecific product formation in the FluB PB2 assay by 2'OM-modified beacons. Depicted are results generated using an unmodified control molecular beacon (Figure 6A; LNA3.13), 2'OM-modified version 1 (Figure 6B; LNA3.13m1), 2'OM-modified version 2 (Figure 6C; LNA3.13m2), and 2'OM-modified version 3 (Figure 6D; LNA3.13m3). [Figures 7A-7D] Figure 7 shows data related to false positive evaluation for Neisseria gonorrhoeae and Chlamydia trachomatis performed on NTC 10% urine samples (Figures 7A-7B) and NTC vaginal swab samples (Figures 7C-7D) using 8U 9dN polymerase (Figures 7A, 7C) and 12U 9dN polymerase (Figures 7B, 7D). [Figures 8A-8D] Figure 8A shows data related to false-positive evaluation in the Chlamydia trachomatis assay (Figures 8A-8B) and Neisseria gonorrhoeae assay (Figures 8C-8D) performed on NTC 15% urine samples using the nominal probe (Figure 8A, Figure 8C) and protective probe (Figure 8B, Figure 8D). Figure 8B contains one false amplification associated with a suspected Chlamydia trachomatis contamination event. [Figures 9A-9D]Figures 9A-9B show data related to false-positive assessment in the Chlamydia trachomatis assay (Figures 9A-9B) and Neisseria gonorrhoeae assay (Figures 9C-9D) performed on vaginal swab samples using the nominal probe (Figures 9A, 9C) and the protective probe (Figures 9B, 9D). [Figures 10A-10D] Figure 10 shows data related to false-positive evaluations in the Chlamydia trachomatis assay (Figures 10A-10B) and Neisseria gonorrhoeae assay (Figures 10C-10D) performed on NTC urine samples using protection probes. Figure 10D contains one false amplification associated with a suspected Neisseria gonorrhoeae contamination event. [Figures 11A-11B] 11A and 11B show data related to false positive assessment in the Chlamydia trachomatis (FIG. 11A) and Neisseria gonorrhoeae (FIG. 11B) assays performed on NTC vaginal swab samples using protection probes. [Figure 12] FIG. 1 shows non-limiting exemplary protective molecular beacons (where "M" indicates a polymerase stopper, e.g., a 2'OM modified base) in unbound hairpin formation used in the Chlamydia trachomatis assay. [Figures 13A-13C] Figure 13 shows data related to RNA base incorporation for protection of molecular beacons in FluA assays. FluA assays were performed with NTC only-Primer Drops (Figures 13A-13B) or probes were screened with targets (Figure 13C). DETAILED DESCRIPTION OF THE INVENTION

[0046] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments set forth in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form part of this disclosure. All patents, published patent applications, other publications, and GenBank sequences and other databases referenced herein are incorporated by reference in their entirety with respect to the relevant art.

[0047] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0048] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture under isothermal amplification conditions, thereby generating nucleic acid amplification products; and detecting the nucleic acid amplification products using a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification products and comprises one or more polymerase stoppers. The method can include contacting a sample containing biological entities with a lysis buffer to generate a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence. The method can include the steps of: contacting a reagent composition with the processed sample to generate an amplification reaction mixture, the reagent composition comprising one or more amplification reagents.

[0049] Disclosed herein are signal-generating oligonucleotides, e.g., signal-generating oligonucleotides capable of hybridizing to nucleic acid amplification products. In some embodiments, the signal-generating oligonucleotides comprise a 5' subdomain and a 3' subdomain, and optionally a loop domain between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the loop domain comprises one or more polymerase stoppers. In some embodiments, the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. In some embodiments, the nucleic acid amplification product is generated by amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other. In some embodiments, the nucleic acid amplification product is generated by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0050] Disclosed herein is a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises a signal-generating oligonucleotide disclosed herein. The kit may include: a lysis buffer comprising one or more lysis agents capable of lysing a biological entity to release a sample nucleic acid comprised herein, the sample nucleic acid suspected of containing a target nucleic acid sequence, and optionally, the one or more lysis agents comprising a detergent. The kit may include a reagent composition comprising one or more amplification reagents comprising one or more components for amplifying the target nucleic acid sequence under isothermal amplification conditions.

[0051] Disclosed herein are methods for detecting Neisseria gonorrhea in a sample. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17. The method can include, if the sample contains Neisseria gonorrhea, generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can include determining the presence or amount of the amplicon as an indication of the presence of Neisseria gonorrhea in the sample.

[0052] Disclosed herein is a method for detecting Chlamydia trachomatis in a sample. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOS: 20-23, 25-26, 28-29, and 31, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOS: 20-23, 25-26, 28-29, and 31. The method can include, if the sample contains Chlamydia trachomatis, generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can also include determining the presence or amount of the amplicon as an indication of the presence of Chlamydia trachomatis in the sample. Disclosed herein includes methods for detecting influenza B virus in a sample. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of influenza B virus, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 36-37 and 41, or a sequence exhibiting at least about 85% identity to any one of the sequences set forth in SEQ ID NOs: 36-37 and 41. The method can include, if the sample contains influenza B virus, generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can include determining the presence or amount of the amplicon as an indication of the presence of influenza B virus in the sample.

[0053] Disclosed herein are compositions for detecting Neisseria gonorrhea in a sample. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea, wherein each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17, or a sequence that exhibits at least about 85% identity to any one of the sequences set forth in SEQ ID NOS: 2-3 and 16-17. Disclosed herein are compositions for detecting Chlamydia trachomatis in a sample. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 20-23, 25-26, 28-29, and 31, or a sequence that exhibits at least about 85% identity to any one of the sequences set forth in SEQ ID NOs: 20-23, 25-26, 28-29, and 31.

[0054] Disclosed herein includes compositions for detecting influenza B virus in a sample. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 36-37 and 41, or a sequence that exhibits at least about 85% identity to any one of the sequences set forth in SEQ ID NOs: 36-37 and 41. In some embodiments, a signal-generating oligonucleotide or primer up to about 100 nucleotides in length is provided that is capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, and that comprises a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17.

[0055] In some embodiments, a signal-generating oligonucleotide or primer is provided that is up to about 100 nucleotides in length and capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, the signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32. In some embodiments, a signal-generating oligonucleotide or primer is provided that is up to about 100 nucleotides in length and capable of hybridizing to a target nucleic acid sequence of influenza B virus, the signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 36-45, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 36-45.

[0056] In some embodiments, provided are signal-generating oligonucleotides or primers up to about 100 nucleotides in length that are capable of hybridizing to a target nucleic acid sequence of an influenza A virus, the signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 33-35, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 33-35. In some embodiments, provided are compositions comprising two or more of the signal-generating oligonucleotides provided herein.

[0057] Methods and compositions for pathogen detection As described herein, nucleic acid amplification and detection reactions can be performed to determine the presence, absence, type, and / or level of a pathogen, such as Neisseria gonorrhoeae, Chlamydia trachomatis, and / or influenza virus (e.g., influenza B virus), in a sample. In some embodiments, the presence, absence, and / or level of one or more of Neisseria gonorrhoeae, Chlamydia trachomatis, and / or influenza virus is determined by detecting one or more target sequences of the target organism using methods known in the art, such as DNA amplification. In some embodiments, multiplex reactions can be performed to detect the presence, absence, or level of two or more of Neisseria gonorrhoeae, Chlamydia trachomatis, and influenza virus.

[0058] In some embodiments, a method for detecting Neisseria gonorrhea in a sample is provided. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea, and each primer in the at least one primer pair comprises a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to any one of the sequences of SEQ ID NOs: 2-3 and 16-17. If the sample contains Neisseria gonorrhea, the method can include generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. The method can include determining the presence or amount of the amplicon as an indication of the presence of Neisseria gonorrhea in the sample. At least one primer pair can include a first primer comprising the sequence of SEQ ID NO: 2 or 17 and a second primer comprising the sequence of SEQ ID NO: 3 or 16. In some embodiments, the at least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea is SEQ ID NO: 2 and 3, SEQ ID NO: 2 and 16, SEQ ID NO: 17 and 3, or SEQ ID NO: 17 and 16. Determining the presence or amount of an amplicon of a target nucleic acid sequence can include contacting the amplicon with one or more signal-generating oligonucleotides, each of which comprises a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14, or a sequence exhibiting at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.Each of the one or more signal-generating oligonucleotides may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

[0059] In some embodiments, a method for detecting Chlamydia trachomatis in a sample is provided, comprising contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, and each primer in the at least one primer pair comprises a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to any one of the sequences of SEQ ID NOs: 20-23, 25-26, 28-29, and 31. The method can include generating an amplicon (e.g., a nucleic acid amplification product) of a target nucleic acid sequence if the sample contains Chlamydia trachomatis. The method can also include determining the presence or amount of the amplicon as an indication of the presence of Chlamydia trachomatis in the sample. At least one primer pair can include a first primer comprising the sequence of SEQ ID NO: 20, 22, 25, or 28, and a second primer comprising the sequence of SEQ ID NO: 21, 23, 26, 29, or 31. In some embodiments, at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis is SEQ ID NOs:20 and 21, SEQ ID NOs:20 and 23, SEQ ID NOs:20 and 26, SEQ ID NOs:20 and 29, SEQ ID NOs:20 and 31, SEQ ID NOs:22 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:22 and 26, SEQ ID NOs:22 and 29, SEQ ID NOs:22 and 31, SEQ ID NOs:25 and 21, SEQ ID NOs:25 and 23, SEQ ID NOs:25 and 26, SEQ ID NOs:25 and 29, SEQ ID NOs:25 and 31, SEQ ID NOs:28 and 21, SEQ ID NOs:28 and 23, SEQ ID NOs:28 and 26, SEQ ID NOs:28 and 29, or SEQ ID NOs:28 and 31.Determining the presence or amount of an amplicon of a target nucleic acid sequence can include contacting the amplicon with one or more signal-generating oligonucleotides, each of which comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, or a sequence exhibiting at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32. Each of the one or more signal-generating oligonucleotides can comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

[0060] In some embodiments, a method for detecting influenza B virus in a sample is provided. In some embodiments, the method includes contacting the sample with at least one primer pair, wherein the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of influenza B virus, and each primer in the at least one primer pair comprises any one of the sequences set forth in SEQ ID NOs: 36-37 and 41, or a sequence exhibiting at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to any one of the sequences set forth in SEQ ID NOs: 36-37 and 41. The method may include generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence if the sample contains influenza B virus. The method may include determining the presence or amount of the amplicon as an indication of the presence of influenza B virus in the sample. At least one primer pair may comprise a first primer comprising the sequence of SEQ ID NO: 36 and a second primer comprising the sequence of SEQ ID NO: 37 or 41. In some embodiments, at least one primer pair capable of hybridizing to a target nucleic acid sequence of influenza B virus is SEQ ID NO: 36 and 37, or SEQ ID NO: 36 and 41. Determining the presence or amount of an amplicon of a target nucleic acid sequence can include contacting the amplicon with one or more signal-generating oligonucleotides, each of which comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45, or a sequence exhibiting at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.Each of the one or more signal-generating oligonucleotides can comprise a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. The method can include contacting a sample ribonucleic acid of the sample with a reverse transcriptase and a primer exhibiting at least about 85% identity to SEQ ID NO: 38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values).

[0061] In some embodiments, a sample is contacted with a reagent composition comprising at least one primer pair to produce an amplification reaction mixture. The method may include the following steps: contacting a sample containing a biological entity with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of dissolving the biological entity and releasing sample nucleic acid contained therein, the sample nucleic acid suspected of containing a target nucleic acid sequence; and contacting the processed sample with a reagent composition comprising at least one primer pair to produce an amplification reaction mixture, the reagent composition comprising one or more amplification reagents. Producing an amplicon of the target nucleic acid sequence may include amplifying the target nucleic acid sequence in the amplification reaction mixture under amplification conditions, thereby producing an amplicon of the target nucleic acid sequence. The determining step may include contacting the amplicon of the target nucleic acid sequence with a signal-generating oligonucleotide for hybridization, optionally including the use of a real-time detection method. The label on the signal-generating oligonucleotide is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the amplicon of the target nucleic acid sequence. In some embodiments, the label generates a signal after the signal-generating oligonucleotide hybridizes to an amplicon of the target nucleic acid sequence, and optionally, the signal is fluorescent.

[0062] In some embodiments, compositions for detecting Neisseria gonorrhea in a sample are provided. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhea, wherein each primer in the at least one primer pair comprises a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to any one of the sequences set forth in SEQ ID NOs: 2-3 and 16-17. In some embodiments, the at least one primer pair capable of hybridizing to the target nucleic acid sequence of Neisseria gonorrhea comprises a primer comprising the sequence of SEQ ID NO: 2 or 17 and a primer comprising the sequence of SEQ ID NO: 3 or 16. The composition may comprise one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14, or a sequence exhibiting at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14. Each of the one or more signal-generating oligonucleotides may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

[0063] In some embodiments, a composition for detecting Chlamydia trachomatis in a sample is provided, comprising at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one primer pair comprises a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to any one of the sequences set forth in SEQ ID NOs: 20-23, 25-26, 28-29, and 31. In some embodiments, at least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis includes a primer comprising the sequence of SEQ ID NO: 20, 22, 25, or 28, and a primer comprising the sequence of SEQ ID NO: 21, 23, 26, 29, or 31. The composition may comprise one or more signal-generating oligonucleotides, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, or a sequence exhibiting at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32. Each of the one or more signal-generating oligonucleotides may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

[0064] In some embodiments, compositions for detecting influenza B virus in a sample are provided. In some embodiments, the compositions include at least one primer pair capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one primer pair comprises a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to any one of the sequences of SEQ ID NOs: 36-37 and 41. In some embodiments, the at least one primer pair capable of hybridizing to the target nucleic acid sequence of influenza B virus comprises a primer comprising the sequence of SEQ ID NO: 36 and a primer comprising the sequence of SEQ ID NO: 37 or 41. The composition may include a primer that exhibits at least about 85% identity to SEQ ID NO: 38 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values). The composition may comprise one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45, or a sequence exhibiting at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. Each of the one or more signal-generating oligonucleotides may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

[0065] In some embodiments, signal-generating oligonucleotides or primers up to about 100 nucleotides in length are provided that are capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, and that include a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17. In some embodiments, the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17. The signal-generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17.

[0066] In some embodiments, signal-generating oligonucleotides or primers up to about 100 nucleotides in length are provided that are capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, and that include a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32. In some embodiments, the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence that exhibits at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32. The signal-generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32.

[0067] In some embodiments, signal-generating oligonucleotides or primers up to about 100 nucleotides in length are provided that are capable of hybridizing to a target nucleic acid sequence of influenza B virus, and that include a sequence selected from the group consisting of SEQ ID NOs: 36-45, or a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 36-45. In some embodiments, the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 36-45, or a sequence that exhibits at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 36-45. The signal-generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 36-45.

[0068] In some embodiments, signal-generating oligonucleotides or primers up to about 100 nucleotides in length are provided that are capable of hybridizing to a target nucleic acid sequence of an influenza A virus, and that include a sequence selected from the group consisting of SEQ ID NOs: 33-35, or a sequence that exhibits at least about 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to a sequence selected from the group consisting of SEQ ID NOs: 33-35. In some embodiments, the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 33-35, or a sequence that exhibits at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) identity to a sequence selected from the group consisting of SEQ ID NOs: 33-35. The signal-generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 33-35.

[0069] In some embodiments, compositions are provided that include two or more of the signal-generating oligonucleotides provided herein. Oligonucleotides (e.g., amplification primers or signal-generating oligonucleotides) that contain one, two, three, four, or more mismatch or universal nucleotides to SEQ ID NOs: 1-45 or their complements, such as oligonucleotides that are at least 80% identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) to SEQ ID NOs: 1-45 or their complements, are also provided.

[0070] Protection Probe Disclosed herein are methods and compositions comprising modified molecular beacons (e.g., "protection probes"), which, in some embodiments, can improve assay specificity. The protection probes disclosed herein can be used in assays that isothermally amplify regions of interest within a target DNA (or cDNA) template using archaeal polymerase amplification ("APA") for real-time analyte detection. In some embodiments, the protection probes provided herein include a polymerase stopper (e.g., one or more 2'-O-methyl RNA bases ("2'OM")) within the molecular beacon probe to reduce nonspecific product formation (and subsequent false-positive signals). While not intending to be bound by any particular theory, in some embodiments, modifying specific bases within a molecular beacon construct can prevent unwanted "read-through" of the probe molecule. Currently available methods do not utilize 2'OM bases for this purpose. Spacer modifications (e.g., C3 spacers) can be used to this same effect, although it has been shown that C3 spacers are incompatible with the molecular beacons used in the APA assays described herein. While not intending to be bound by any particular theory, in some embodiments, the compositions and methods provided herein take advantage of the inability of 9dN (DNA-dependent DNA polymerase) to read RNA templates. Specifically, it is theorized that when a 2'-O-methyl base (i.e., a methylated RNA base) is encountered within a given template, the enzyme's processivity is halted (e.g., the enzyme is unable to successfully "read" this position within the DNA template). 2'-O-methyl RNA can be found in small RNAs (e.g., tRNA) and is a naturally occurring post-transcriptional modification of RNA. Oligonucleotides containing 2'-O-methyl RNA can be directly synthesized. This modification can increase the melting temperature of RNA:RNA duplexes while also causing a slight change in RNA:DNA stability. Additionally, this modification can exhibit stability against single-stranded ribonuclease attack, generally being 5-10 times less sensitive to deoxyribonucleases than DNA.2'OM modifications can be used in antisense oligonucleotides to improve stability and binding affinity to targets. In addition to the 2'OM modifications described herein, the following alternative base modifications within the context of molecular beacons used in APA have also been tested: C3 spacer modifications; abasic site modifications; and unmethylated RNA bases. Based on the resulting data, 2'OM modifications provide the greatest protection (without rendering the molecular beacon incompatible with APA).

[0071] Figures 2A-2C depict non-limiting exemplary primers and probes (molecular beacons) for rapid nucleic acid amplification and detection assays provided herein, including Neisseria gonorrhoeae assays based on archaeal polymerase amplification (APA). Figure 2A depicts the position of the primers and molecular beacons relative to the template. Figure 2B depicts the molecular beacon of Figure 2A in unbound hairpin formation. The spacer region, indicated by the arrow, corresponds to the region of the template between the forward primer binding site and the reverse primer binding site. In some embodiments, one or more bases of the probes provided herein are LNA bases (indicated by a circle in Figure 2B). Figure 2C depicts a protected molecular beacon (where "M" represents a polymerase stopper, e.g., a 2'OM-modified base) in unbound hairpin formation. Figure 3 depicts non-limiting exemplary nonspecific product formation that may occur in the APA-based Neisseria gonorrhoeae assay depicted in Figures 2A-2B (and without the use of the 2'OM protected probe described herein). A theoretically possible unintended nonspecific interaction between the reverse primer and the molecular beacon is depicted. This is an inherent problem for some embodiments of the APA assay design provided herein due to the intentional overlap of the primer / probe footprints. In this example, the assay uses a molecular beacon that completely overlaps with the reverse primer. Polymerase extension of the reverse primer bound to the molecular beacon can generate a product that is capable of exponential amplification by the reverse primer. Figure 4 depicts a non-limiting exemplary prevention of nonspecific product formation in the APA-based Neisseria gonorrhoeae assay depicted in Figures 2A-2B using a protection probe containing a polymerase stopper as described herein (e.g., the protection probe shown in Figure 2C). A possible unintended nonspecific interaction between the reverse primer and the molecular beacon is depicted therein. By preventing read-through of the embedded primer footprint within the molecular beacon, the methods and compositions provided herein using protection probes can prevent the formation of unintended extension products.The 2'OM can serve as a control point to enable this readthrough inhibition. Without intending to be bound by any particular theory, the location of this modification determines the level of inhibition of nonspecific product formation—it is placed at the very beginning of the primer overlap region to stop readthrough immediately (i.e., so that the overlapping bases are not replicated). In some embodiments, one or more polymerase stoppers are located at or near the 3' end of the first region (directly adjacent to the spacer region) and within the loop domain.

[0072] Unintended interactions between signal-generating oligonucleotides (e.g., molecular beacons) and forward and / or reverse primers, resulting in nonspecific product formation, can be an inherent and significant problem for some embodiments of the amplification / detection assays provided herein due to the intentional overlap of primer / probe footprints—the signal-generating oligonucleotide (e.g., molecular beacon) may include a first region comprising at least a portion of the sequence of the reverse primer and / or a second region comprising a sequence complementary to at least a portion of the forward primer. Additionally, due to the hairpin nature of the signal-generating oligonucleotides provided herein, the repetition of the two 3'-terminal nucleotides of the reverse primer in the stem-loop may result in unintended reverse primer / probe interactions. Thus, these inherent elements of some of the amplification / detection assays based on the signal-generating oligonucleotides provided herein may result in nonspecific product formation (and thereby false-positive signals). However, the methods and compositions provided herein solve these problems in the art and result in assays with reduced nonspecific product formation, reduced false-positive signals, and / or increased likelihood of accurately determining the presence, absence, and / or amount of target nucleic acid sequences in a sample. The primer and probe sequences for the Neisseria gonorrhoeae assay described above are shown in Table 1 below. Additionally, Figure 12 and Table 2 provide primer and protection probe sequences for the Chlamydia trachomatis assay. Table 3 provides non-limiting exemplary primers and probes for FluA and FluB assays. In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 5' modification (e.g., 5TEX615, FAM). In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 3' modification (e.g., 3IAbRQSp, IBFQ).

[0073] [Table 1]

[0074] [Table 2]

[0075] [Table 3]

[0076] In some embodiments, a method for detecting a target nucleic acid sequence in a sample is provided. In some embodiments, the method includes the steps of: amplifying a target nucleic acid sequence in an amplification reaction mixture under isothermal amplification conditions, thereby generating nucleic acid amplification products; and detecting the nucleic acid amplification products using a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing to the nucleic acid amplification products and comprises one or more polymerase stoppers. The method may include the steps of: contacting a sample containing a biological entity with a lysis buffer to generate a processed sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entity to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence. The method may include the steps of: contacting a reagent composition with the processed sample to generate an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0077] In some embodiments, a signal-generating oligonucleotide is provided. In some embodiments, the signal-generating oligonucleotide is capable of hybridizing to a nucleic acid amplification product. In some embodiments, the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal-generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain. In some embodiments, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the loop domain comprises one or more polymerase stoppers. In some embodiments, the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. In some embodiments, the nucleic acid amplification product is generated by amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other. In some embodiments, the nucleic acid amplification product is generated by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0078] 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 possible to form a paired stem domain. One or more polymerase stoppers may be within the loop domain. In some embodiments, the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise one or more polymerase stoppers. The nucleic acid amplification product can include: (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.

[0079] The signal-generating oligonucleotide may include a first region comprising at least a portion of the sequence of the reverse primer. The signal-generating oligonucleotide may include a second region comprising a sequence complementary to at least a portion of the forward primer. The signal-generating oligonucleotide may include a spacer region comprising at least a portion of the sequence of the spacer sequence. The first region, second region, and / or spacer region may include one or more polymerase stoppers. The first region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. The second region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. The spacer region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. The first region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. The second region may comprise a sequence complementary to at least the two 3'-terminal nucleotides of the forward primer and / or reverse primer. The spacer region may comprise a sequence complementary to at least the two 3' terminal nucleotides of the forward primer and / or the reverse primer.

[0080] The signal-generating oligonucleotide may be about 10 nucleotides to about 100 nucleotides in length. The forward primer and / or reverse primer may be about 5 nucleotides to about 25 nucleotides in length. The second region, spacer region, and / or first region may be about 1 nucleotide to about 25 nucleotides in length. The 5' subdomain, 3' subdomain, loop domain, 5' terminal domain, and / or 3' terminal domain may be about 1 nucleotide to about 25 nucleotides in length. The signal-generating oligonucleotide may comprise a 5' terminal domain located 5' of the 5' subdomain. The signal-generating oligonucleotide may comprise a 3' terminal domain located 3' of the 5' subdomain. In some embodiments, the 5' terminal domain and / or the 3' terminal domain do not comprise one or more polymerase stoppers. The first region may comprise at least a portion of the 5' subdomain and / or the loop domain. The spacer region may comprise at least a portion of the loop domain. The second region may comprise at least a portion of the loop domain and / or the 3' subdomain. The 5' subdomain may comprise at least a portion of the first region and / or the spacer region. The loop domain may comprise at least a portion of the spacer region, the first region, and / or the second region. The 3' subdomain may comprise at least a portion of the second region and / or the spacer region. In some embodiments, one or more polymerase stoppers are located near or at the 3' end of the first region (directly adjacent to the spacer region) and within the loop domain.

[0081] As used herein, a "polymerase stopper" is a molecule (e.g., a modified nucleotide) that can terminate or inhibit polymerization. In some embodiments, at least one of the one or more polymerase stoppers is a 2'-O-methylated nucleotide. Non-limiting examples of 2'-O-methylated nucleotides include 2'-O-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methylguanosine. The one or more polymerase stoppers may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. The one or more polymerase stoppers may comprise one or more of an abasic site, a stable abasic site, a chemically captured abasic site, or any combination thereof. A stable abasic site may comprise a 1',2'-dideoxy. A chemically captured abasic site may comprise an abasic site reacted with an alkoxyamine or sodium borohydride. Abasic sites can include apurinic, apyrimidinic, or both. Abasic sites can be generated by alkylating or oxidizing agents.In some embodiments, the one or more polymerase stoppers comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 5-aminomethyl- ... or multiple 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3(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] group, or any combination thereof. The protection probes provided herein can include one or more 2'-O-methylribonucleosides, such as 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylpseudouridine, 2'-O-methylguanosine, 2'-O-methyl-5-methyluridine, and / or 2'-O-methyluridine.

[0082] The signal-generating oligonucleotide may contain one or more phosphorothioate linkages and / or one or more locked nucleic acids. The signal-generating oligonucleotide may be a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. The signal-generating oligonucleotide may contain a label. The label may include a quenchable label (e.g., a fluorophore). The signal-generating oligonucleotide may contain a quencher. The label may be in the 3'-terminal domain and the quencher may be in the 5'-terminal domain. The label may be in the 5'-terminal domain and the quencher may be in the 3'-terminal domain.

[0083] After the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, the one or more polymerase stoppers may be capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the forward primer of the first undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. After the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, the one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. After the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers may be capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

[0084] The sample nucleic acid may include a nucleic acid comprising a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence includes the following steps: amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, the step including contacting the nucleic acid comprising the target nucleic acid sequence with i) a forward primer and a reverse primer, the forward primer being capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the reverse primer being capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. The nucleic acid may be double-stranded DNA. The nucleic acid may be a product of a reverse transcription reaction. The nucleic acid may be a product of a reverse transcription reaction generated from a sample ribonucleic acid. The amplifying step may include generating a nucleic acid by a reverse transcription reaction. The sample nucleic acid may include a sample ribonucleic acid, and the method may include contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. In some embodiments, the step of amplifying a target nucleic acid sequence comprises the following steps: (c1) contacting a sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying step comprises contacting the dsDNA with (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophile polymerase activity, thereby producing a nucleic acid amplification product.

[0085] In some embodiments, when the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a first undesired extension product. The first undesired extension product may be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product. One or more polymerase stoppers may terminate polymerase extension of the forward primer of the first undesired duplex to generate a first extension stall product. In some embodiments, the first extension stall product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to generate a first undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the forward primer of the first undesired duplex past the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme with hyperthermophile polymerase activity generates a second undesired extension product. The second undesired extension product may be amplified by an enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to form a second undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex to generate a second extension stall product. In some embodiments, the second extension stalled product cannot be amplified by an enzyme with hyperthermophile polymerase activity in the presence of a reverse primer to produce a second undesired amplification product.The one or more polymerase stoppers may be capable of terminating polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide. In some embodiments, when an exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity generates a third undesired extension product. The third undesired extension product may be amplified by an enzyme having hyperthermophile polymerase activity in the presence of the reverse primer to form a third undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex to generate a third extension stall product. In some embodiments, the third extension stalled product cannot be amplified by an enzyme having hyperthermophile polymerase activity in the presence of a reverse primer to produce a third undesired amplification product. The one or more polymerase stoppers may be capable of terminating polymerase extension of the third undesired duplex exogenous nucleic acid beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

[0086] The detecting step may include contacting the nucleic acid amplification product with a signal-generating oligonucleotide for hybridization. Detecting the nucleic acid amplification product may include using a real-time detection method. The label may be capable of generating a signal after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product. In some embodiments, the label generates a signal (e.g., fluorescence) after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product. The detecting step may include detecting the signal of the label before the amplification reaction, after the amplification reaction, or both. Detecting the nucleic acid amplification product may include detecting a signal generated by the label of the signal-generating oligonucleotide. The label may be a fluorophore, and the signal may be fluorescence. Detecting the signal may include detecting fluorescence emitted by the label. The method may include determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample. Determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample may include determining the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the presence, absence, and / or amount of the 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 signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample.

[0087] The label may be capable of generating a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. In some embodiments, the label generates a false-positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product. The signal and the false-positive signal may be indistinguishable. In some embodiments, the generation of the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, detection of a false-positive signal reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample.

[0088] In some embodiments, the presence of one or more polymerase stoppers in the signal-generating oligonucleotide increases the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in a sample by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any two of these values) compared to a signal-generating oligonucleotide that does not contain one or more polymerase stoppers. In some embodiments, production of the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, a signal-generating oligonucleotide hybridized to the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal. In some embodiments, the nucleic acid amplification product reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes, or a number or range between any two of these values, before the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reach a detectable level. In some embodiments, the signal reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes, or a number or range between any two of these values, before the false positive signal reaches a detectable level. The appearance of detectable levels of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product may be delayed by at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes, or a number or range between any two of these values, compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers. In some embodiments, the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach detectable levels for at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the amplifying step begins.The occurrence of a false positive signal, a first undesired amplification product, a second undesired amplification product, and / or a third undesired amplification product may be reduced by at least about 1.1-fold (e.g., 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or a number or range between any two of these values) compared to a comparable method in which the signal-generating oligonucleotide does not include one or more polymerase stoppers.

[0089] Amplifying the target nucleic acid sequence can include producing a detectable level of nucleic acid amplification product within about 20 minutes, about 15 minutes, or about 10 minutes. The detecting step can be performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition is contacted with the treated sample.

[0090] The lysis buffer may contain one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. The pH of the lysis buffer may be about 1.0 to about 10.0 (e.g., about 2.2). The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample nucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, the one or more amplification reagents include a reverse transcriptase; an enzyme having hyperthermophilic polymerase activity; and / or dNTPs. In some embodiments, the one or more amplification reagents include an enzyme having reverse transcriptase activity, an enzyme having hyperthermophilic polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer. The reagent composition may be lyophilized, heat-dried, and / or include one or more additives. In some embodiments, the one or more additives include Tween 20, Triton X-100, and / or Tween 80; an amino acid; a sugar or sugar alcohol; and / or a polymer. The sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof. Contacting the reagent composition with the processed sample can include dissolving the reagent composition in the processed sample. In some embodiments, the one or more solubility reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) of the processed sample (e.g., about 0.2% (wt / vol) of the processed sample); and / or include a detergent (e.g., one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant).

[0091] In some embodiments, the method is performed in a single reaction vessel; does not include the use of any enzymes other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include the use of any enzymes other than an enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein.

[0092] The target nucleic acid sequence can have a length of about 20 nucleotides or less to about 90 nucleotides or less (e.g., about 30 nucleotides). The forward primer, reverse primer, and / or reverse transcription primer can be about 8 to 16 bases long. The nucleic acid amplification product can be about 20 to 40 bases long. The spacer sequence can comprise a portion of the target nucleic acid sequence. The spacer sequence can be 1 to 10 bases long. Isothermal amplification conditions can include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, and optionally about 56°C to about 67°C. The amplifying step can be carried out for a period of about 5 minutes to about 60 minutes (e.g., a period of about 15 minutes). The amplifying step can be carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions. The amplifying step may be carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). The PCR may be real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0093] The enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that may be at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof. The enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that may be at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. The enzyme with hyperthermophilic polymerase activity may be a polymerase comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase and the enzyme with hyperthermophilic polymerase activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, and the forward and reverse primers. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme with hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

[0094] In some embodiments, the amplifying step includes and / or does not include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplifying step does not include LAMP. In some embodiments, the methods do not include one or more of the following: (i) diluting the treated sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the treated sample; (iv) sonicating the treated sample; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the treated sample; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the treated sample or amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the treated sample or amplification reaction mixture.

[0095] The term "isothermal amplification reaction" shall be given its ordinary meaning and shall include reactions in which the temperature does not change significantly during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10°C, e.g., not more than 5°C or not more than 2°C, during the main enzymatic reaction step in which amplification occurs. Depending on the method of isothermal amplification of nucleic acids, different enzymes can be used for amplification. Isothermal amplification compositions and methods are described in PCT application published as WO2017176404, the entire contents of which are incorporated herein by reference. In some embodiments, the methods and compositions described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer is resistant to the formation of precipitates over a period of time under storage conditions (e.g., a storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer resists precipitation are described in International Application No. PCT / US23 / 61980, filed February 3, 2023, entitled "NON-OPAQUE LYTIC BUFFER COMPOSITION FORMULATIONS," the entire contents of which are incorporated herein by reference.

[0096] Some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than acid and / or low pH conditions. Compositions, kits, and methods for nucleic acid detection in which nucleic acid strands are dissociated under low pH conditions (e.g., by contact with an acidic lysis buffer) to facilitate subsequent rapid amplification and detection are described in International Application No. PCT / US23 / 61978, filed February 3, 2023, entitled "METHOD FOR SEPARATING GEOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS," the entire contents of which are incorporated herein by reference.

[0097] In some embodiments, the methods and compositions described herein can include a lysis buffer and / or reagent composition. A lysis buffer comprising a lysis agent and a reducing agent, and a reagent composition comprising one or more protectants (e.g., cyclodextrin compounds) capable of sequestering the amplification agent and the lysis agent are described in International Application No. PCT / US22 / 21015, filed March 18, 2022, entitled "ISOTHERMAL AMPLIFICATION OF PATHOGENS," the entire contents of which are incorporated herein by reference.

[0098] Some embodiments of the methods and compositions described herein can, in some embodiments, be used in conjunction with systems, methods, compositions, and kits for monitoring amplification reactions, as described in U.S. Provisional Patent Application No. 63 / 374,835, filed September 7, 2022, entitled "HAIRPIN INTERNAL CONTROL FOR ISOTHERMAL NUCLEIC ACID AMPLIFICATION," the entire contents of which are incorporated herein by reference.

[0099] Some embodiments of the methods and compositions described herein may include probes that melt at a temperature different from the optimal APA reaction temperature to allow for multiplexed targets and / or internal standards. Compositions, kits, and methods for multiplexed nucleic acid detection are described in U.S. Provisional Patent Application No. 63 / 374,831, entitled "ARCHEAL POLYMERASE AMPLIFICATION," filed September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0100] Nucleic acids, subjects, samples and nucleic acid processing Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein. This term refers to nucleic acids of any composition, including DNA (e.g., complementary DNA (cDNA) and genomic DNA (gDNA)), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single- or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be found in plasmids, phages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or in vivo. It may be or be derived from other nucleic acids that can be replicated or replicated in vitro or in a host cell, cell, cell nucleus, mitochondria, or cell cytoplasm. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.The term can also include nucleotide analogs, single-stranded polynucleotides ("sense" or "antisense," "plus" or "minus" strand, "forward" or "reverse" reading frame, "forward" or "reverse" strand), and RNA or DNA equivalents, derivatives, variants, and analogs synthesized from double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain and generally includes regions preceding and following the coding region (leader and trailer) involved in transcription / translation of the gene product and regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). In RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed as the number of bases.

[0101] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid may be referred to as a target sequence, a target polynucleotide, and / or a target polynucleotide sequence, and may include double-stranded and single-stranded nucleic acid molecules. The target nucleic acid may be, for example, DNA or RNA. If the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may include a single-stranded target sequence. If the target nucleic acid is double-stranded, the target nucleic acid generally comprises a first strand and a second strand. The first strand and the second strand may be referred to as the forward strand and the reverse strand, and are generally complementary to each other. If the target nucleic acid is single-stranded, the complementary strand may be generated, for example, by polymerization and / or reverse transcription, to make the target nucleic acid double-stranded and have a first / forward strand and a second / reverse strand.

[0102] A target sequence can refer to either the sense or antisense strand of a nucleic acid sequence, a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence can also be a subsequence within a larger polynucleotide. For example, a target sequence can be a short sequence (e.g., 20-50 bases) within a nucleic acid fragment, chromosome, or plasmid targeted for amplification. In some embodiments, a target sequence can refer to a sequence within a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence can refer to the entire sequence targeted for amplification, or to a subsequence within the target nucleic acid to which the oligonucleotide binds. An amplification product can be a larger molecule containing the target sequence as well as at least one other sequence or other nucleotide. An amplification product can be approximately the same length as the target sequence, e.g., exactly the same length as the target sequence. An amplification product can comprise or consist of the target sequence. The target sequence length and / or guanosine cytosine (GC) concentration (percentage) may depend, in part, on the temperature at which the amplification reaction is performed, which may in turn depend, in part, on the stability of the polymerase used in the reaction. Trial assays may be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, if the polymerase is stable up to 60°C-65°C, the target sequence may be, for example, 19-50 nucleotides in length, or, for example, about 40-50, 20-45, 20-40, or 20-30 nucleotides in length. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0103] Target nucleic acids may include, for example, genomic nucleic acids, plasmid nucleic acids, mitochondrial nucleic acids, cellular nucleic acids, extracellular nucleic acids, bacterial nucleic acids, and viral nucleic acids. In some embodiments, target nucleic acids may include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, genes, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA, or synthetic oligonucleotides. Genomic nucleic acids may include any nucleic acid derived from any genome, for example, animal genomes, plant genomes, insect genomes, viral genomes, and bacterial genomes (e.g., genomes present in spores). In some embodiments, genomic target nucleic acids are present within a specific genomic locus or multiple genomic loci. A genomic locus may include any or a combination of open reading frame DNA, non-transcribed DNA, intronic sequences, exonic sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequences considered to be associated with a given genomic locus.

[0104] The target sequence may comprise one or more types of repetitive elements (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as a repetitive element (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellite repeats, minisatellite repeats, etc.). For example, the target sequence may occur multiple times as a repetitive element, and one, some, or all occurrences of the target sequence within the repetitive element can be amplified (e.g., using a single pair of primers) using the methods described herein. In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as duplicates and / or paralogs. The target nucleic acid may include a microRNA. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21-23 nucleotides in length), single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNAs and are partially complementary to them. The target nucleic acid may also include microRNA precursors, such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop RNAs that are further processed into miRNAs. The target nucleic acid may also include small interfering RNAs (siRNAs), which are short (e.g., about 20-25 nucleotides in length), at least partially double-stranded RNA molecules involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0105] Nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, isolated from a sample obtained from a subject. The subject can be any living or non-living organism, including, but not limited to, humans, non-human animals, plants, bacteria, fungi, viruses, or protists. Any human or non-human animal can be selected, including, but not limited to, mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), suidae (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), ursidae (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female, and the subject can be of any age (eg, embryo, fetus, infant, child, adult).

[0106] A sample or test sample may be any specimen isolated or obtained from a subject or a portion thereof. Non-limiting examples of specimens include fluids or tissues derived from a subject, including, but not limited to, blood or blood products (such as serum or plasma), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), serum, plasma, urine, aspirate, biopsy sample, intestinal puncture sample, cells (e.g., blood cells) or portions thereof (e.g., mitochondria, nuclei, or extracts), female reproductive tract washings, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, mammary fluid, hard tissue (e.g., liver, spleen, kidney, lung, or ovary), or the like, or combinations thereof. The term blood, as conventionally defined, includes whole blood, blood products, or any fraction of blood, such as serum, plasma, or buffy coat. Plasma refers to the fraction of whole blood obtained by centrifugation of blood that has been treated with an anticoagulant. Serum refers to the aqueous portion of the fluid that remains after a blood sample has clotted. Fluid or tissue samples are often collected according to standard protocols commonly followed by hospitals or clinics. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 milliliters) is often collected and can be stored according to standard procedures before or after preparation.

[0107] The sample can include spores, viruses, cells, nucleic acids, and / or samples containing any free nucleic acid from prokaryotes or eukaryotes. For example, the methods described herein can be used to detect nucleic acids outside of spores (e.g., no lysis is required). The sample can be isolated from any material suspected of containing the target sequence, such as from a subject as described above. In some embodiments, the target sequence is present in air, plants, soil, or other material suspected of containing biological organisms.

[0108] Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from biological samples can be used, including art-known DNA preparation methods and various commercially available reagents or kits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit, or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wisconsin), and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, New Jersey), or combinations thereof. U.S. Patent No. 7,888,006 provides a DNA purification method, but does not disclose the compositions (e.g., lysis buffer, protectant) and methods provided herein.

[0109] In some embodiments, a cell lysis procedure is performed. Cell lysis may be performed before initiating the amplification reaction described herein (e.g., to release DNA and / or RNA from cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical methods (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or a combination thereof), physical methods (e.g., French press and sonication, etc.), or electrolytic lysis. For example, chemical methods generally involve disrupting cells using a lysing agent, extracting nucleic acids from the cells, followed by treatment with a chaotropic salt. In some embodiments, cell lysis involves the use of a detergent (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis involves the use of an ionic detergent (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods, such as freeze / thaw trituration and the use of a cell press, may also be useful. High-salt lysis procedures can also be used. For example, alkaline lysis procedures can be used. The latter procedure traditionally incorporates the use of a phenol-chloroform solution, although an alternative phenol-chloroform-free procedure involving three solutions can also be used. In the latter procedure, for example, one solution may contain 15 mM Tris (pH 8.0), 10 mM EDTA, and 100 μg / ml RNase A, a second solution may contain 0.2 N NaOH and 1% SDS, and a third solution may contain 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and components described herein.

[0110] Nucleic acids for performing the methods described herein can be provided without processing a sample containing the nucleic acid. For example, nucleic acids for performing the amplification methods described herein can be provided without prior nucleic acid purification. In some embodiments, target sequences are amplified directly from a sample (e.g., without any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids for performing the methods described herein are provided after processing a sample containing the nucleic acid. For example, nucleic acids can be extracted, isolated, purified, or partially purified from a sample. The term "isolated" generally refers to nucleic acids that have been removed from their original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed) and thus have been altered from their original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid" can refer to nucleic acids removed from a subject (e.g., a human subject). Isolated nucleic acids can be provided that have fewer non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. A composition containing isolated nucleic acids may contain no more than about 50%-99% of non-nucleic acid components. A composition comprising an isolated nucleic acid may be free of more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of non-nucleic acid components. The term "purified" generally refers to a nucleic acid that contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. A composition comprising a purified nucleic acid may be free of more than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of other non-nucleic acid components.

[0111] Nucleic acids can be provided for performing the methods described herein without modifying the nucleic acid, for example, by denaturing, digesting, nicking, unwinding, incorporating and / or ligating heterologous sequences, adding epigenetic modifications, adding labels (e.g., 32P, 33 P, 125 I, or 35 Examples of such labels include radioactive labels such as S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, and fluorescent dyes. Thus, in some embodiments, unmodified nucleic acids are amplified.

[0112] The methods disclosed herein for detecting target nucleic acid sequences (single-stranded or dsDNA and / or RNA) in a sample can detect target nucleic acid sequences (e.g., DNA or RNA) with high sensitivity. In some embodiments, the methods can be used to detect target DNA / RNA present in a sample containing multiple RNAs / DNAs (including a target RNA / DNA and multiple non-target RNAs / DNAs), where the target RNA / DNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 It is present in one or more copies per non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meaning and shall refer to DNA, or RNA, or a combination of DNA and RNA.

[0113] The detection threshold of a method for detecting target RNA / DNA in a sample can be, for example, 10 nM or lower. The term "detection threshold" shall be given its ordinary meaning and shall describe the minimum amount of target RNA / DNA that must be present in a sample for detection to occur. As an illustrative example, if the detection threshold is 10 nM, a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the methods of the present disclosure provide a method for detecting target RNA / DNA at a concentration of 5 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower, 0.05 nM or lower, 0.01 nM or lower, 0.005 nM or lower, 0.001 nM or lower, 0.0005 nM or lower, 0.0001 nM or lower, 0.00005 nM or lower, 0.00001 nM or lower, or 0.00001 nM. or lower, 10 pM or lower, 1 pM or lower, 500 fM or lower, 250 fM or lower, 100 fM or lower, 50 fM or lower, 500 aM (attomolar) or lower, 250 aM or lower, 100 aM or lower, 50 aM or lower, 10 aM or lower, or 1 aM or lower. In some embodiments, the disclosed compositions or methods exhibit attamolar (aM), femtomolar (fM), picomolar (pM), and / or nanomolar (nM) detection sensitivity.

[0114] A sample may contain sample nucleic acids (e.g., multiple sample nucleic acids). The term "multiple" is used herein to mean two or more. Thus, in some embodiments, a sample contains two or more (e.g., three or more, five or more, ten or more, twenty or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, or five thousand or more) sample nucleic acids (e.g., DNA / RNA). The disclosed methods can be used as highly sensitive methods for detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, a sample contains 5, 10, 20, 25, 50, 100, 500, 10, 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 seeds or 10 7 The sample may contain DNA / RNA from 50 or more species, each differing in sequence from the others. In some embodiments, the sample contains DNA / RNA from cells (e.g., eukaryotic, mammalian, or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0115] As used herein, the term "sample" shall be given its ordinary meaning and shall include any sample containing RNA and / or DNA (e.g., for determining whether target DNA and / or target RNA is present in a population of RNA and / or DNA). A sample may be a biological sample or an environmental sample. A sample may be derived from any source; for example, a sample may be a synthetic combination of purified DNA and / or RNA. A sample may be a cell lysate, a DNA / RNA-enriched cell lysate, or DNA / RNA isolated and / or purified from a cell lysate. A sample may be derived from a patient (e.g., for diagnostic purposes). A sample may be derived from permeabilized cells, crosslinked cells, tissue sections, or combinations thereof. A sample may be derived from tissue prepared by crosslinking followed by delipidation and adjustment to a uniform refractive index. A sample may contain a target nucleic acid (e.g., target DNA / RNA) and multiple species of non-target DNA / RNA. In some embodiments, the target DNA / RNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 Present in the sample at 1 copy per non-target DNA / RNA.

[0116] Patient-related samples include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, and samples that have been manipulated in some way after procurement (e.g., treatment with reagents); washed; or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA). Samples may include biological samples, including, but not limited to, clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, and bone marrow. Biological samples may also include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), such as samples containing RNA obtained from such cells (e.g., RNA-containing cell lysates or other cell extracts). In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof.

[0117] The source of the sample may be a diseased (or suspected) cell, fluid, tissue, or organ, or may be a normal (non-diseased) cell, fluid, tissue, or organ. In some embodiments, the source of the sample is a cell, tissue, or organ infected (or suspected) with a pathogen. For example, the source of the sample may be an individual, which may be infected or uninfected - and the sample may be any biological sample collected from an individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, stool sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., buccal swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal secretion, tears, buffy coat, mucosal sample, epithelial cell sample (e.g., epithelial cell scraping), etc.), and cultures thereof. The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens may be viruses, fungi, helminths, protozoans, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites, etc. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny headworms, and tapeworms (Cestodes). Protozoan infections include infections by Giardia spp., Trichomonas spp., African trypanosomiasis, amebic dysentery, babesiosis, balantidiosis, Chagas disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens, such as parasitic / protozoan pathogens, include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza virus, dengue fever, West Nile virus, herpes virus, yellow fever virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, and papillomavirus. Pathogenic viruses include papovaviruses (e.g., HPV, polyomavirus); hepadnaviruses; herpes viruses (e.g., HSV (e.g., HSV I, HSV II), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, and pityriasis rosea). Rosea, Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; variola virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; and Phycodnaviridae. Non-limiting examples of pathogens include Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus pneumoniae, Cryptococcus neoformans, Histoplasma capsulatum, and Haemophilus influenzae.influenzae B, Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, human serum parvo-like virus, respiratory syncytial virus, measles virus, adenovirus, human T-cell leukemia virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma brucei rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria species (e.g., chicken cecal coccidia (tenella)), Onchocercavolvulus), Leishmania species (e.g., Leishmania tropica), Streptococcus pneumoniae, Pneumocystis carinii, Trichophyton rubrum, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora species, SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), herpes simplex, herpesvirus 6, herpesvirus 7, JC virus, and type A Influenza, influenza B, influenza C, rotavirus, human adenovirus, human enterovirus, hantavirus, Legionella dumophila, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsii, Ehrlichia species (e.g., Ehrlichia chaffeensis), Borrelia burgdorferi, Yersinia pestis, Chlamydia pneumoniae, Trichinella spiralis spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma species (e.g., Mycoplasma arthritidis), M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae.

[0118] amplification Methods for amplifying nucleic acids are provided herein. In some embodiments, nucleic acids are amplified using a suitable amplification process. Nucleic acid amplification typically involves enzymatic synthesis of nucleic acid amplicons (copies) containing sequences complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is performed in a single vessel, a single chamber, and / or a single volume (i.e., adjacent volumes). In some embodiments, the amplification method and the detection method (e.g., the detection methods described herein) are performed in a single vessel, a single chamber, and / or a single volume (i.e., adjacent volumes).

[0119] The terms "amplify," "amplification," "amplification reaction," or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification can also refer to an "exponential" increase in the target nucleic acid. "Amplifying" can also refer to a linear increase in the number of target nucleic acids, but is distinct from a single, single primer extension step. In some embodiments, a limited amplification reaction, also known as preamplification, can be performed. Preamplification is a method in which a limited amount of amplification occurs because a small number of cycles, e.g., 10 cycles, are performed. Preamplification allows for some amplification but stops amplification before the exponential phase, typically producing approximately 500 copies of the desired nucleotide sequence. Preamplification can be used to limit inaccuracies associated with reactant depletion in a particular amplification reaction and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a single primer extension step can be performed prior to linear or exponential amplification.

[0120] A general description of the amplification process is provided herein. For example, when a primer (e.g., an oligonucleotide described herein) and a target nucleic acid are contacted, complementary sequences anneal or hybridize to each other. The primer can anneal to the target nucleic acid at or near (e.g., adjacent, abutting, etc.) the sequence of interest. A primer annealed to a target may be referred to as a primer-target hybrid, a hybridized primer-target, or a primer-target duplex. The terms "near" or "adjacent" when referring to a nucleotide sequence of interest refer to the distance (e.g., number of bases) or region between the end of the primer and one or more nucleotides (e.g., a nucleotide sequence) of the target. Generally, adjacent refers to a range of about 1 nucleotide to about 50 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides) from the nucleotide or nucleotide sequence of interest. In some embodiments, a set of primers (e.g., a pair of primers, a forward primer and a reverse primer, a first oligonucleotide and a second oligonucleotide) anneals within about 1-20 nucleotides of a nucleotide or nucleotide sequence of interest to generate an amplification product. In some embodiments, the primers anneal within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended along the target (i.e., template strand) by a polymerase to generate a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplification product is generated. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step.

[0121] Components of an amplification reaction (e.g., one or more amplification reagents) can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, and multiple primer sets for multiplex amplification), a nucleic acid target (e.g., a target nucleic acid derived from a sample), one or more polymerases, nucleotides (e.g., dNTPs), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, a monovalent ion, and a divalent ion). The amplification reaction may further include one or more of a reverse transcriptase, a reverse transcription primer, and one or more detection agents. Nucleic acid amplification can be performed in the presence of natural nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derivatized nucleotides. Natural nucleotides generally refer to adenylate, guanylate, cytidylate, thymidylate, or uridylate. Derivatized nucleotides generally are nucleotides other than natural nucleotides. Ribonucleoside triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, or U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be referred to herein as A, G, C, T, or U, without specific reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs can be used, such as analogs containing a detectable label (e.g., a fluorescent label or a colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, such as 32 P, 33 P, 125 I, or 35 The amplification can be performed in the presence of a radioactive label such as S; an enzyme label such as alkaline phosphatase; a fluorescent label such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, an antigen, a hapten, or a fluorescent dye. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., TriLink's CleanAmp™ dNTPs).

[0122] The one or more amplification reagents may include non-enzymatic and enzymatic components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions. In some embodiments, the non-enzymatic components do not include proteins (e.g., nucleic acid-binding proteins), enzymes, or proteins with enzymatic activity, such as polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. In some embodiments, the enzymatic components consist of a polymerase or a polymerase and a reverse transcriptase. Thus, such enzymatic components would exclude other proteins (e.g., nucleic acid-binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases.

[0123] In some embodiments, amplification conditions include enzymatic activity (e.g., enzymatic activity provided by a polymerase, or enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include enzymatic activity provided by enzymes other than the polymerase and / or reverse transcriptase, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. The polymerase activity and reverse transcriptase activity may be provided by separate enzymes or separate enzyme types (e.g., a polymerase and a reverse transcriptase), or may be provided by a single enzyme or enzyme type (e.g., a polymerase). Nucleic acid amplification may include non-thermal cycling PCR. In some embodiments, nucleic acid amplification includes an isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process performed at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature is maintained essentially constant during the amplification reaction. Isothermal amplification conditions generally do not include a thermal cycling (i.e., cycling between upper and lower temperature limits) component to the amplification process. When amplifying under isothermal conditions, the reaction can be maintained at an essentially constant temperature, meaning that the temperature does not need to be maintained at exactly one temperature. For example, isothermal amplification processes may experience small temperature fluctuations (e.g., ±1-5°C) due to environmental or equipment-based variables. Often, the entire reaction volume is maintained at an essentially constant temperature, and isothermal reactions, as used herein, generally do not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0124] The isothermal amplification reaction herein can be carried out at an essentially constant temperature. In some embodiments, the isothermal amplification reaction herein is carried out at a temperature of about 55°C to about 75°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C, or a temperature approximately these values, or a value or range between any two of these values. In some embodiments, a temperature element (e.g., a heat source) is maintained at an essentially constant temperature, for example, about 75°C or less, about 70°C or less, about 65°C or less, or about 60°C or less.

[0125] The amplification process herein can be carried out for a certain length of time, for example, until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product can be detected by any suitable detection process and / or detection process described herein. The amplification process can be carried out within about 20 minutes or less, or within about 10 minutes or less. For example, the amplification process can be carried out within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or within any value or range between any two of these values. In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation during the amplification step (and / or other steps). In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that promote unwinding during the amplification step (and / or other steps). Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote unwinding can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, and proteins (e.g., enzymatic agents).

[0126] In some embodiments, the methods disclosed herein do not involve heat denaturation (e.g., heating a solution containing nucleic acids to an elevated temperature, such as 75°C, 80°C, 90°C, or 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of nucleic acids. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions provided herein do not include a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and / or a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions and methods provided herein do not include intercalating agents, alkylating agents, and / or chemicals such as formamide, glycerol, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods of the present disclosure do not include contacting nucleic acids with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding). In some embodiments, the amplification step (e.g., step (c)) does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase) and / or low pH conditions (e.g., contact with acid).

[0127] Nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-strand binding proteins), or any combination thereof. For example, nucleic acid targets can be amplified without exposure to helicases, including, but not limited to, DNA helicases and RNA helicases. Amplification conditions that do not include the use of helicases are helicase-free amplification conditions.

[0128] Nucleic acid targets can be amplified without exposure to recombinases, including, but not limited to, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX. In some embodiments, nucleic acid targets are amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY). Nucleic acid targets can be amplified without exposure to nucleic acid binding proteins (e.g., single-strand binding protein or single-stranded DNA binding protein (SSB)), such as T4 gp32. In some embodiments, nucleic acid targets are amplified without exposure to topoisomerases. Nucleic acid targets can be amplified with or without exposure to agents or conditions that destabilize nucleic acids. As used herein, the term "destabilization" shall be given its ordinary meaning and shall refer to the disruption of the overall organization and geometric orientation (e.g., double helix structure) of nucleic acid molecules by one or more of tilt, rotation, twist, slip, and flip effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, page 17). Destabilization generally does not refer to the melting or separation (e.g., denaturation) of nucleic acid strands. Nucleic acid destabilization can be achieved by exposure to agents, such as intercalating or alkylating agents and / or chemicals, such as formamide, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, the nucleic acid target is amplified without exposure to a ligase and / or an RNA replicase.

[0129] In some embodiments, nucleic acid targets can be amplified without cleavage or digestion. For example, nucleic acid targets can be amplified without prior exposure to one or more cleavage agents, resulting in an intact nucleic acid being amplified. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents during amplification. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents after amplification. Amplification conditions that do not include the use of a cleavage agent may be referred to herein as cleavage agent-free amplification conditions. The term "cleavage agent" generally refers to an agent, possibly a chemical or enzyme, that can cleave nucleic acids at one or more specific or non-specific sites. Specific cleavage agents often specifically cleave specific sites according to a specific nucleotide sequence. Cleavage agents can include endonucleases (e.g., restriction enzymes and nicking enzymes); exonucleases (DNAses, RNAses (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases); and chemical cleavage agents.

[0130] Nucleic acid targets can be amplified without the use of restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without prior exposure to restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes during amplification. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNAse, RNAse (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases. In some embodiments, nucleic acids are amplified without exonuclease treatment before, during, and / or after amplification. Amplification conditions that do not include the use of exonucleases are exonuclease-free amplification conditions. In some embodiments, the nucleic acid is amplified without DNAse and / or RNAse treatment. In some embodiments, the nucleic acid is amplified without RNAse H treatment.

[0131] Amplified nucleic acids may be referred to herein as nucleic acid amplification products or amplicons. In some embodiments, amplification products include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and the like, as well as combinations of the foregoing. Amplification products typically have a nucleotide sequence that is identical or substantially identical to the sequence of a sample nucleic acid (e.g., a target sequence) or its complement. A "substantially identical" nucleotide sequence in an amplification product will generally have a high degree of sequence identity (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity) to the nucleotide sequence being amplified or its complement, with variations being the result of poor polymerase fidelity or other variables.

[0132] The nucleic acid amplification product may include a polynucleotide that is contiguously complementary or substantially identical to a target sequence in the sample nucleic acid. Contiguously complementary generally refers to, for example, a nucleotide sequence in a first strand in which each base is paired in order (e.g., reading from 5' to 3') with a correspondingly ordered base in a second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered contiguously complementary. In other words, contiguously complementary generally refers to every contiguous base in the nucleotide sequence of the first strand being complementary to the corresponding contiguous base in the nucleotide sequence of the second strand. For example, a first strand having the sequence 5'-ATGCATGCATGC-3' (SEQ ID NO: 9) would be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 10) if every contiguous base in the first strand is complementary to every corresponding contiguous base in the second strand. However, a first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO: 11) would not be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 10) because the sequence of six adenines (six As) in the middle of the first strand would not pair with bases in the second strand. Contiguous complementary sequences are optionally about 5 to about 25 contiguous bases in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is contiguous complementary to or substantially identical to a target sequence in the sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequence (e.g., at the 5' and / or 3' end or within the product) that is not contiguous with or substantially identical to the target sequence, such as additional sequences incorporated into the amplification product by tail primers or ligation, and / or additional sequences that provide cleavage agent recognition sites (e.g., nicking enzyme recognition sites). Generally, unless the target sequence includes tandem repeats, the amplification product will not include products in the form of tandem repeats.

[0133] The nucleic acid amplification product can comprise sequences complementary to or substantially identical to one or more primers used in the amplification reaction, hi some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is contiguous to or identical to a first primer sequence and a second nucleotide sequence that is contiguous to or identical to a second primer sequence.

[0134] Nucleic acid amplification products can include spacer sequences. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is contiguous with or substantially identical to a portion of a target sequence in a sample nucleic acid and is flanked by sequences in the amplification product that are complementary to or substantially identical to one or more primers used in the amplification reaction. The spacer sequence flanked by sequences in the amplification product is generally located between a first sequence (complementary to or substantially identical to the first primer) and a second sequence (complementary to or substantially identical to the second primer). Thus, an amplification product typically includes a first sequence, followed by a spacer sequence, followed by a second sequence. The spacer sequence is generally neither complementary nor substantially identical to the sequence of the primers. The spacer sequence may be or include approximately 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists of or consists essentially of a first nucleotide sequence that is contiguously complementary to or identical to the first primer sequence, a second nucleotide sequence that is contiguously complementary to or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence that is not contiguously complementary to or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product), and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other mechanism incorporated into the amplification product. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguously complementary to or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product), and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other mechanism incorporated into the amplification product.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or another extra base (e.g., at the 5' and / or 3' end or within the product) introduced into the product due to, for example, errors or promiscuity in the amplification process.

[0135] Nucleic acid amplification products may be up to 50 bases in length, including 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number or range of bases between any two of these values. In some embodiments, nucleic acid amplification products of a given target sequence have the same or substantially the same length (e.g., within 1-10 bases). Thus, nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel) and generally do not produce multiple signals representing multiple lengths (e.g., a ladder or smear on an electrophoresis gel). In multiplex reactions, nucleic acid amplification products of different target sequences may have different lengths.

[0136] The methods and components described herein can be used for multiplex amplification, which generally refers to the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence). For example, multiplex amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample. For example, the amplifying step can include multiplex amplification of two or more target nucleic acid sequences, and the detecting step can include multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. The two or more target nucleic acid sequences can be specific to two or more different organisms (e.g., one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C). Multiplex amplification can also refer to the simultaneous or sequential amplification of one or more sequences present in multiple samples. For example, multiplex amplification can be used to amplify at least two amplifiable target sequences (e.g., the amplification reaction includes appropriate primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is configured to detect at least two target sequences, but only one of the target sequences is present in the sample being tested, so that both sequences are amplifiable, but only one sequence is amplified. In some embodiments, when two target sequences are present, the amplification reaction results in the amplification of both target sequences. A multiplex amplification reaction including appropriate primers and enzymes can result in the amplification of one, some, or all of the target sequences. In some embodiments, the amplification reaction is configured to detect two sequences using a pair of primers, one sequence being the target sequence and one sequence being a control sequence (e.g., a synthetic sequence that is amplifiable with the same primers as the target sequence but has spacer bases or a sequence different from the target). In some embodiments, the amplification reaction is configured to detect multiple sets of sequences using corresponding primer pairs, each set including a target sequence and a control sequence.

[0137] Primer Nucleic acid amplification is generally performed in the presence of one or more primers. A primer is generally characterized as an oligonucleotide comprising a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid at or near (e.g., adjacent to) a specific region of interest (i.e., target sequence). A primer can, for example, enable specific determination of the nucleotide sequence of a target nucleic acid or detection of the target nucleic acid or a characteristic thereof (e.g., the presence or absence of a sequence). A primer can be naturally occurring or synthetic. The term specific or specific generally refers to the binding or hybridization of one molecule, such as a primer for a target polynucleotide, with another molecule. That is, the term specific or specific refers to the recognition, contact, and formation of a stable complex between two molecules, compared to substantially less recognition, contact, or complex formation between either of those two molecules and other molecules. The term annealing or hybridization generally refers to the formation of a stable complex between two molecules. The terms primer, oligo, or oligonucleotide can be used interchangeably herein when referring to a primer.

[0138] Primers can be designed and synthesized using any suitable process and may be of any length suitable for hybridizing to a target sequence and carrying out the amplification processes described herein. Primers are often designed according to the sequence of the target nucleic acid. In some embodiments, primers may be about 5 to about 30 bases in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases in length. Primers may be composed of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides), or mixtures thereof. Modifications and modified bases include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite™, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker™, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6dT, Uni-Link™ amino modifier), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG), thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); fluorophores (e.g., Freedom™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO™ dyes, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy750 (NHS ester)); Iowa Black® dark quencher modifications (e.g., Iowa Black® FQ, Iowa Black® RQ); dark quencher modifications (e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl);spacers (C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate (PS) bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases);and click chemistry modifications. In some embodiments, modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS linkages, 3' phosphate groups, and spacer bases (such as C3 spacers or other spacer bases). For example, a primer may contain one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is a post-transcriptional modification of RNA commonly found in tRNA and other small RNA molecules. Primers containing 2'-O-methyl RNA bases can be directly synthesized. This modification can, for example, increase the Tm of an RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in a primer to, for example, increase stability and binding affinity with the target sequence. In some embodiments, a primer can contain one or more phosphorothioate (PS) linkages (e.g., PS linkage modifications). PS linkages can substitute a sulfur atom for a non-bridging oxygen in the phosphate backbone of the primer. This modification typically renders the internucleotide linkage resistant to nuclease degradation. PS bond can be introduced between approximately the last 3-5 nucleotides at the 5' or 3' end of the primer to inhibit exonuclease degradation, for example. In some embodiments, PS bond inclusion throughout the primer can help reduce endonuclease attack. Primers may contain, for example, a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases and, in certain cases, can be used to block extension by DNA polymerase. In some embodiments, primers contain one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated internally or at the 5' end of the primer. Multiple C3 spacers can be added to either end of the primer to introduce long hydrophilic spacer arms for attachment of, for example, fluorophores or other pendant groups.

[0139] A primer may comprise DNA bases, RNA bases, or both, and one or more of the DNA and RNA bases may be modified or unmodified. For example, a primer may be a mixture of DNA bases and RNA bases. A primer may comprise DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, a primer comprises unmodified DNA bases. In some embodiments, a primer comprises modified DNA bases. A primer may comprise RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, a primer comprises unmodified RNA bases. In some embodiments, a primer comprises modified RNA bases. In some embodiments, a primer does not comprise RNA bases. In some embodiments, a primer does not comprise DNA bases. In some embodiments, a primer does not comprise a cleavage agent recognition site (e.g., does not comprise a nicking enzyme recognition site). In some embodiments, a primer does not comprise a tail (e.g., does not comprise a tail comprising a nicking enzyme recognition site).

[0140] In some embodiments, all or a portion of the primer sequence may be complementary or substantially complementary to the target nucleic acid. Substantially complementary, in the context of sequences, generally refers to nucleotide sequences that will hybridize to each other. The stringency of hybridization conditions can be varied to allow for varying amounts of sequence mismatch. The target sequence and primer sequence may be at least 75% complementary to each other, including, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to each other. A primer that is substantially complementary to a target nucleic acid sequence is typically also substantially identical to the complement of the target nucleic acid sequence (i.e., the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid may be at least 75% identical in sequence, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each other.

[0141] In some embodiments, the primers comprise a pair of primers. A pair of primers may comprise a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid). In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, amplification of a target sequence is performed using a pair of primers, and no additional primers or oligonucleotides are included in the amplification of the target sequence (e.g., the amplification reaction components do not include additional primer pairs for a given target sequence, nested primers, bumper primers, oligonucleotides other than primers, probes, etc.). In some embodiments, the primers consist of a pair of primers. In some embodiments, the amplification reaction may include additional primer pairs for amplifying different target sequences, such as in multiplex amplification. In some embodiments, the primers consist of a pair of primers, but in some embodiments, the amplification reaction may include additional primers, oligonucleotides, or probes for a detection process that is not considered part of the amplification. In some embodiments, primers are used in sets. An amplification primer set may comprise a pair of forward and reverse primers for a given target sequence. In the case of multiplex amplification, the primers that amplify a first target sequence are considered to be a primer set, and the primers that amplify a second target sequence are considered to be a different primer set.

[0142] Nucleic acids (e.g., amplification products, sample nucleic acids, target nucleic acid sequences) described herein can comprise a first strand and a second strand that are complementary to each other. Amplification reaction components may comprise or consist of a first primer (first oligonucleotide) that is complementary to a target sequence in the first strand (e.g., sense strand, forward strand) of the sample nucleic acid, and a second primer (second oligonucleotide) that is complementary to a target sequence in the second strand (e.g., antisense strand, reverse strand) of the sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) comprises a first polynucleotide that is contiguously complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide that is contiguously complementary to the target sequence in the second strand of the sample nucleic acid. Contiguously complementary, with respect to primer-target, generally refers to a nucleotide sequence of a primer in which each base pairs sequentially with a corresponding, ordered base in the target sequence, with no gaps, additional sequence, or unpaired bases present within the sequence considered contiguously complementary. In some embodiments, the primer does not include any additional sequence (e.g., at the 5' and / or 3' end or within the primer) that is not contiguous with the target sequence, such as additional sequence present in a tail primer or loop primer, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site). In some embodiments, the amplification reaction components do not include primers that include additional sequence (i.e., sequence other than sequence that is contiguous with the target sequence), such as a tail primer, a loop primer, a step-loop structure, a primer that can form a hairpin structure, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site).

[0143] In some embodiments, a primer can contain modifications such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifier that alters the binding properties of the primer. In some embodiments, a primer can include a detectable molecule or entity (e.g., a fluorophore, a radioisotope, a colorimetric reagent, a particle, an enzyme, etc.).

[0144] polymerase Amplification reaction components (e.g., one or more amplification reagents) can include one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides extending the 3' hydroxyl end of a primer molecule, such as an amplification primer described herein, into a nucleic acid target sequence (e.g., to which the primer anneals). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that can exhibit activity at high reaction temperatures (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C). Hyperthermophilic polymerases are sometimes referred to as hyperthermophilic polymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, the polymerase can incorporate from about 1 to about 50 nucleotides in a single synthesis, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range of nucleotides between any two of these values ​​in a single synthesis.

[0145] Amplification reaction components include 9°N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA polymerase; Therminator™ II DNA polymerase; Therminator™ III DNA polymerase; Therminator™ γ DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo); T4 DNA polymerase; T7 DNA polymerase; DeepVentR™ (exo) DNA polymerase; DeepVentR™ DNA polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start DNA polymerase; Phusion™ High-Fidelity The polymerase may include one or more DNA polymerases selected from DNA polymerase; VentR® DNA polymerase; VentR® (exo) DNA polymerase; RepliPHI™ Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm™ DNA polymerase); MasterAmp™ AmpliTherm™ DNA polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase beta; and ThermoPhi DNA polymerase.

[0146] In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermostable at high temperatures). The half-life of the hyperthermophilic DNA polymerase may be about 5-10 hours at 95°C and about 1-3 hours at 100°C. For example, the amplification reaction components may include one or more hyperthermophilic DNA polymerases from Archaea (e.g., a hyperthermophilic DNA polymerase from Thermococcus or a hyperthermophilic DNA polymerase from Thermococcaceaen archaean). In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from the genus Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0147] In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase or a functional fragment thereof. A functional fragment generally retains one or more functions of a full-length polymerase, such as the ability to polymerize DNA (e.g., in an amplification reaction). In some cases, the functional fragment performs a function (e.g., polymerizing DNA in an amplification reaction) at a level that is at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the functional level of the full-length polymerase. The level of polymerase activity can be assessed using a detectable nucleic acid amplification method, such as those described herein. In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or a functional fragment of SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, an amplification reaction component (e.g., one or more amplification reagents) comprises a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase or a functional fragment thereof, hi some embodiments, an amplification reaction component comprises a polymerase comprising an amino acid sequence at least about 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or a functional fragment thereof.

[0148] The polymerase may have reverse transcriptase activity. In such embodiments, the amplification reaction can amplify an RNA target in a single step, for example, without the use of a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase activity include Bst (large fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, and Therminator™ II. The amplification reaction components can include one or more separate reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction can include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0149] In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases with no or low exonuclease activity are used during amplification. In some embodiments, the polymerase with no or low exonuclease activity comprises one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, a modified polymerase with low exonuclease activity may have 10% or less exonuclease activity compared to an unmodified polymerase, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase has no or low 5'-3' exonuclease activity and / or no or low 3'-5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand-dependent exonuclease activity and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of a polymerase include one or more amino acid substitutions at or corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:7 (e.g., D141A, E143A, E143D, and A485L).

[0150] Detection and Quantification The methods described herein can include detecting and / or quantifying the nucleic acid amplification product(s). The amplification product(s) can be detected and / or quantified, for example, by any suitable detection and / or quantification method described herein.Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, end-point), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating / binding dyes, absorbance methods (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension methods (e.g., iPLEX™), Affymetrix Molecular Inversion Probes (MIR), and the like. Probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, genetic bit The methods include, but are not limited to, GBA, multiplex minisequencing, SNaPshot, GOOD assay, microarray miniseq, arrayed primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed incorporation (TDI), colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probe, Invader assay, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips, and combinations thereof. In some embodiments, detecting the nucleic acid amplification product comprises the use of a real-time detection method (i.e., the product is detected and / or continuously monitored during the amplification process).In some embodiments, detecting nucleic acid amplification products involves the use of end-point detection methods (i.e., products are detected after the amplification process is completed or stopped). Nucleic acid detection methods can also employ the use of labeled nucleotides, either directly incorporated into the target sequence or incorporated into a probe containing a complementary sequence to the target. Such labels may be radioactive and / or fluorescent in nature and can be resolved in any of the manners discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, detection methods can be used in conjunction with measuring signal intensity and / or generating (or referencing) standard curves and / or look-up tables for quantification of nucleic acid amplification products.

[0151] Detection of nucleic acid amplification products may involve the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule whose detectable property is detectable under certain conditions, thereby enabling the molecule to function as a specific, useful signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or speed at which a molecule passes through an aperture of a known size. A molecular beacon for detecting nucleic acid molecules may be, for example, a hairpin-shaped oligonucleotide containing a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin may contain a probe sequence complementary to the target sequence, and the stem is formed by annealing complementary arm sequences located on either side of the probe sequence. The fluorophore and quenching molecule may be covalently linked to the opposite ends of each arm. Under conditions that prevent the oligonucleotide from hybridizing to its complementary target, or when the molecular beacon is free in solution, the fluorescent molecule and quenching molecule are in close proximity to each other, preventing FRET. When a molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, converting the loop structure into a stable, more rigid conformation, causing the fluorophore and quencher molecules to separate, resulting in fluorescence. Because the probe is specific, fluorescence generally occurs only with the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is identical to or complementary to a sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is neither identical to nor complementary to a sequence in the target nucleic acid (e.g., hybridizes to a tail amplification primer or a sequence added to the amplification product by ligation). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with different colored fluorophores and different target sequences, allowing for simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction).In the case of quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each cycle of amplification, and unhybridized molecular beacons are dark, so there is no need to isolate the probe-target hybrid to quantitatively determine the amount of amplification product. The signal obtained is proportional to the amount of amplification product. Detection using molecular beacons can be performed in real time or as an end-point detection method.

[0152] The detection of nucleic acid amplification products may involve the use of lateral flow. The use of lateral flow typically involves the use of lateral flow devices, including, but not limited to, dipstick assays and thin-layer chromatography plates with various appropriate coatings. Various binding reagents for the sample, binding partners for the sample or conjugates containing binding partners, and signal generating systems are immobilized in the flow channels. Detection of nucleic acid amplification products may involve the use of FRET, an energy transfer mechanism between two chromophores: a donor molecule and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emission of the donor molecule as it returns to its ground state can transfer the excitation energy to the acceptor molecule via long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored and is a function of the distance between the donor and acceptor, the overlap between the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be useful, for example, for quantifying the molecular dynamics of DNA-DNA interactions, as described for molecular beacons. To monitor the production of a specific product, a probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe-target hybridization changes the distance or orientation between the donor and acceptor, and a FRET change is observed.

[0153] Detection of nucleic acid amplification products may involve the use of fluorescence polarization (FP). FP techniques are based on the principle that when a fluorescently labeled compound is excited with linearly polarized light, it will emit fluorescence with a degree of polarization inversely proportional to its rotation rate. Thus, when a molecule, such as a fluorescently labeled tracer-nucleic acid conjugate, is excited with linearly polarized light, the fluorophore's rotation is constrained between light absorption and emission, resulting in a highly polarized emission. When a free tracer compound (i.e., not bound to a nucleic acid) is excited with linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, resulting in a more random molecular orientation and depolarized emitted light. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0154] Detection of nucleic acid amplification products may involve the use of surface capture, achieved, for example, by immobilizing specific oligonucleotides on a surface to create a biosensor with both high sensitivity and selectivity. Examples of surfaces that can be used to attach probes include gold and carbon. Detection of nucleic acid amplification products may involve the use of 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN). For example, TAQMAN probes are hydrolysis probes that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The principle of TAQMAN probes relies on 1) the 5'-3' exonuclease activity of Taq polymerase, which cleaves a dual-labeled probe upon hybridization to a complementary target sequence, and 2) fluorophore-based detection. The resulting fluorescent signal allows for quantitative measurement of the accumulation of amplification product during the exponential phase of amplification, and TAQMAN probes can significantly increase the specificity of detection.

[0155] Detecting nucleic acid amplification products can include the use of intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes exhibit enhanced fluorescence after binding to DNA or RNA). Dyes can include DNA or RNA intercalating fluorophores, including, but not limited to, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Detection of nucleic acid amplification products can also include the use of absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurements at 260 nm) to concentration. Direct measurements of nucleic acids can be converted to concentration using the Beer-Lambert law, which relates absorbance to concentration using the path length and extinction coefficient of the measurement. Detection of nucleic acid amplification products may involve the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and quantity of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample is ionized, and the resulting ions can be separated according to their mass-to-charge ratio in an electric and / or magnetic field, and the mass-to-charge ratio of the ions is measured by a detector. Mass spectrometry methods include, for example, MALDI, MALDI-TOF, and electrospray. These methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry methods (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can achieve high throughput due to fast signal acquisition from solid surfaces and automated analysis.

[0156] Detecting nucleic acid amplification products may involve the use of nucleic acid sequencing. The entire or partial sequence of the amplification product may be determined, and the determined nucleotide sequence may be referred to as a read. For example, linear amplification products may be directly analyzed without further amplification (e.g., by using single-molecule sequencing). In some embodiments, linear amplification products are subjected to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single-molecule sequencing, multiplex sequencing, solid-phase single-nucleotide sequencing, and nanopore sequencing. Detecting nucleic acid amplification products may involve the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm®).

[0157] Lysis buffer Soluble drugs As disclosed herein, the dissolving agent can include a detergent. The detergent can include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. Anionic surfactants have NH4 as a counterion. + , K. + , Na + , or Li + The cationic surfactant may contain I as a counter ion. - , Br - , or Cl - may also include:

[0158] The lysis agents provided herein may be capable of acting as denaturing agents. As used herein, "denaturing agent" or "denaturant" shall be given its ordinary meaning and include any compound or substance that will cause reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined by both the properties and concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturing agents include chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids, or combinations of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents can include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(dioleyoxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergents), 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). Organic, water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols, such as ethanol or isopropanol), or lower alkanediols (especially C2-C4 alkanediols, such as ethylene glycol) can be used as denaturing agents.The phospholipid may be a naturally occurring phospholipid such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or a synthetic phospholipid derivative or variant such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0159] Suitable surfactant levels may be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, it may be advantageous to use anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof). Anionic surfactants useful herein include the water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl radical, and the water-soluble salts of sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant.

[0160] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having a single long alkyl chain containing about 8 to 18 carbon atoms, such as lauryltrimethylammonium chloride, cetylpyridinium chloride, benzalkonium chloride, cetyltrimethylammonium bromide, di-isobutylphenoxyethyl-dimethylbenzylammonium chloride, alkyltrimethylammonium nitrite of coconut, cetylpyridinium fluoride, etc. Certain cationic surfactants can also act as antiseptics in the compositions disclosed herein.

[0161] Suitable nonionic surfactants that can be used in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds produced by the condensation of an organic hydrophobic compound, which can be aliphatic and / or aromatic, with an alkylene oxide group (hydrophilic nature).Examples of suitable nonionic surfactants include: poloxamers; sorbitan derivatives such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of aliphatic alcohols or alkylphenols; condensation products of ethylene oxide with the reaction product of propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides; and mixtures of such substances.These substances are useful for stabilizing foam without contributing to excessive viscosity increase of consumer product compositions.

[0162] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, where the aliphatic radical may be straight or branched chain, and one of the aliphatic substituents contains about 8 to 18 carbon atoms and one contains an anionic water-solubilizing group, such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of anionic short-chain surfactants include alkyl sulfates, alkyl sulfonates, alkylbenzene sulfonates, saturated or unsaturated fatty acids, and their salts. The polar head group-containing moiety in cationic surfactants can include, for example, quaternary ammonium, pyridinium, sulfonium, and / or phosphonium groups. For example, the polar head group can include trimethylammonium. Exemplary cationic short-chain surfactants include alkyltrimethylammonium halides, alkyltrimethylammonium tosylates, and N-alkylpyridinium halides.

[0163] reducing agent The lysis buffer and / or reagent composition (e.g., dried composition) can include one or more reducing agents. A "reducing agent" can be a compound or group of compounds. As used herein, a "reducing agent," also known as a "reductant," "reducing agent," or "reducing equivalent," can refer to an element or compound that donates electrons to another chemical species. In particular, reducing agents are generally compounds that cleave disulfide bonds by reduction, thereby overcoming tertiary protein folding and quaternary protein structure (multimeric subunits) stabilized by disulfide bonds. Examples of suitable reducing agents include, but are not limited to, 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and HED. In some embodiments of the methods, compositions, and kits provided herein, the lysis buffer and / or reagent composition (e.g., the dried composition) does not include one or more reducing agents.

[0164] Reagent Composition The reagent compositions (e.g., dry compositions) described herein can be provided in a "dry form," or a form not suspended in a liquid medium. A "dry form" of a composition can include a dry powder, a lyophilized composition, a spray-dried composition, or a precipitated composition. A "dry form" composition can include one or more lyoprotectants, such as sugars and their corresponding sugar alcohols, e.g., sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, e.g., arginine or histidine; lyotropic salts, e.g., magnesium sulfate; polyols, e.g., propylene glycol, glycerol, poly(ethylene glycol), or polypropylene glycol; and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethylcellulose. As used herein, the terms "lyophilization," "lyophilized," and "freeze-dried" refer to a process in which the material to be dried is first frozen, and then the ice or frozen solvent is removed by sublimation under reduced pressure. "Lyophile" refers to a lyphophilized material.

[0165] The reagent composition (e.g., a dried composition) may be frozen, lyophilized, or spray-dried. The reagent composition may be heat-dried. The reagent composition may include one or more additives (e.g., amino acids, polymers, sugars, or sugar alcohols). The sugars or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymers may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof. The lyophilized reagent may include poly rA, EGTA, EDTA, Tween 80, and / or Tween 20.

[0166] Frozen or lyophilized or spray-dried or heat-dried compositions, or aqueous compositions for preparing frozen or lyophilized or spray-dried compositions, may be prepared using the following: (i) non-aqueous solvents, such as ethylene glycol, glycerol, dimethyl sulfoxide, and dimethylformamide; (ii) surfactants, such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic acid, also known as poloxamer; The composition may include one or more of the following: F127 (polyoxyethylene-polyoxypropylene copolymer), poloxamine, and sodium dodecyl sulfate; (iii) dissacharides, such as trehalose, sucrose, lactose, and maltose; (iv) polymers (which may have different MW), such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, ficoll, and albumin; and (v) amino acids, such as one or more of glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and gamma-aminobutyric acid.

[0167] The reagent composition (e.g., a dried composition) can include one or more protecting agents and one or more amplification reagents. The one or more protecting agents can include a cyclodextrin compound. Cyclodextrins (CDs) can be used for complexation with soluble agents (e.g., SDS). Cyclodextrins (CDs) can be cyclic oligosaccharides resembling truncated cones with a hydrophobic interior cavity and a hydrophilic exterior surface. The most commonly used natural cyclodextrins contain 6, 7, and 8 glucose units, designated α-, β-, and γ-CD. Natural CDs can have solubility. Chemically modified CDs, such as hydroxypropyl derivatives, improve solubility in aqueous media by up to 50%. CAVASOL® is a trade name for cyclodextrin derivatives from WACKER, encompassing a variety of α-, β-, and γ-CD derivatives. β-CD can form a strong inclusion complex (stronger than α-CD and β-CD) with sodium dodecyl sulfate (SDS) in a predominantly 1:1 stoichiometry. The binding constant of β-CD to SDS is 2100 M -1 ~2500M -1 The range can be as follows:

[0168] kit In some embodiments, a kit for detecting a target nucleic acid sequence in a sample is provided. In some embodiments, the kit comprises a signal-generating oligonucleotide disclosed herein. The kit can include a lysis buffer containing one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence, and optionally, the one or more lysis agents include a detergent, and the detergent can include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit may include a reagent composition comprising one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions, the one or more components for amplification comprising: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophile polymerase activity capable of producing a nucleic acid amplification product. In some embodiments, the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer.

[0169] The kit can include at least one component that provides real-time detection activity for nucleic acid amplification products. The real-time detection activity can be provided by a molecular beacon. The reagent composition (e.g., a dried composition) can include a reverse transcriptase and / or a reverse transcription primer. The molar ratio of the one or more protectants to the one or more amplification reagents can be between about 10:1 and about 1:10 (e.g., about 2:1). In some embodiments, the one or more additives include Tween 20, Triton X-100, Tween 80, a non-ionic detergent (e.g., a non-ionic surfactant), or any combination thereof. In some embodiments, the one or more protectants include a cyclodextrin compound. In some embodiments, the one or more solubility reagents comprise about 0.001% (wt / vol) to about 1.0% (wt / vol) (e.g., about 0.2% (wt / vol)) of the processed sample. In some embodiments, the one or more solubility agents include a detergent. The detergent can include one or more of a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and an amphoteric surfactant. In some embodiments, it may be advantageous for one or more protecting agents to sequester one or more solubility agents, thereby preventing the one or more solubility agents from denaturing one or more amplification reagents.

[0170] The kit may include, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, as described herein. If one target is being amplified, a pair of primers (forward and reverse) may be included in the kit. If multiple target sequences are being amplified, multiple primer pairs may be included in the kit. The kit may include a control polynucleotide, and if multiple target sequences are being amplified, multiple control polynucleotides may be included in the kit. An enzyme with hyperthermophile polymerase activity can have an amino acid sequence that is at least about 90% or 95% identical to the amino acid sequence of SEQ ID NO: 7, or a functional fragment thereof. For example, an enzyme with hyperthermophile polymerase activity can include the amino acid sequence of SEQ ID NO: 7.

[0171] The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may include (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length. The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoan.

[0172] The kit may also contain one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates, and the like, or the components may be combined in various combinations in such containers. The components of the kit may, for example, be present in one or more containers. In some embodiments, all of the components are provided in a single container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a container separate from the primers. The components may, for example, be lyophilized, heat-dried, freeze-dried, or present in a stable buffer. In some embodiments, the polymerase and / or reverse transcriptase are present in a single container in lyophilized or heat-dried form, and the primers are either lyophilized, heat-dried, freeze-dried, or present in a buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase and primers are present in a single container in lyophilized or heat-dried form.

[0173] The kit may further include, for example, dNTPs or modified nucleotides used in the reaction, vessels, cuvettes, or other containers used for the reaction, or vials of water or buffer for rehydrating lyophilized or heat-dried components. The buffer used may be, for example, suitable for both polymerase activity and primer annealing activity. The kits may also include instructions for practicing one or more of the methods described herein and / or instructions for one or more of the components described herein. The instructions and / or instructions may be in printed form or may be included in a kit insert. The kits may also include a written description of an internet location that provides such instructions or instructions. The kit may further comprise reagents used in detection methods, such as reagents used in FRET, lateral flow devices, dipsticks, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. [Example]

[0174] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Example 1 FluB PB2 assay: nonspecific product formation (no 2'OM protection) This example demonstrates that nonspecific product formation between the reverse primer and molecular beacon can result in a false-positive signal in the absence of the assay target sequence. Figures 5A-5E depict data showing the concentration-dependent trend between FluB PB2 reverse primer and the occurrence of a false-positive signal under no-target conditions (NTC). Figures 5A-5E depict data showing FluB PB2 nonspecific product formation (no 2'OM protection). The primers and probes for the FluB PB2 assay are shown in Table 4. The FluB PB2 reverse primer was set at 600 nM (Figure 5A), 500 nM (Figure 5B), 400 nM (Figure 5C), 300 nM (Figure 5D), and 200 nM (Figure 5E). As the concentration of the reverse primer increased, the rate of false-positive NTCs increased, and the detection time for these NTCs occurred earlier.

[0175] [Table 4]

[0176] Example 2 FluB PB2 assay: Prevention of nonspecific product formation by 2'OM protection This example demonstrates that preventing unintended extension product formation reduces false-positive signals caused by primer readthrough. Figures 6B-6D depict results using three variations of FluB PB2 molecular beacons containing 2'OM modifications. These molecular beacons were observed to produce significantly fewer false-positive signals under no-target conditions (NTC) compared to the unmodified control beacon (Figure 6A). Figures 6A-6D depict data related to the prevention of nonspecific product formation in the FluB PB2 assay by 2'OM-modified beacons. Results are depicted using the unmodified control molecular beacon (Figure 6A; LNA3.13), 2'OM-modified version 1 (Figure 6B; LNA3.13m1), 2'OM-modified version 2 (Figure 6C; LNA3.13m2), and 2'OM-modified version 3 (Figure 6D; LNA3.13m3). In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 5' modification (e.g., 5HEX). In some embodiments, the probes (e.g., molecular beacons) provided herein comprise a 3' modification (e.g., 3IAbRQSp).

[0177] [Table 5]

[0178] Example 3 Neisseria gonorrhoeae ("NG", "GC") Assay & Chlamydia trachomatis ("CT") Assay False positive assessment This assay demonstrates the prevention of false positives in Neisseria gonorrhoeae and Chlamydia trachomatis assays by the protection probes provided herein.

[0179] First, false-positive evaluation was performed using 20 urine samples (10% in 1.11x GRBS) and 20 ProbeTec™ swabs (1.67% in 1x GRBS) using unmodified CT and GC ROX beacons. Figures 7A-7D depict data related to false-positive evaluation in the Neisseria gonorrhoeae and Chlamydia trachomatis assays performed on NTC 10% urine samples (Figures 7A-7B) and NTC vaginal swab samples (Figures 7C-7D) using 8 U 9dN polymerase (Figures 7A, 7C) and 12 U 9dN polymerase (Figures 7B, 7D). Tables 6 and 7 depict the results of testing NTC urine samples and NTC vaginal swab samples, respectively, with different amounts of 9dN polymerase.

[0180] [Table 6]

[0181] [Table 7]

[0182] Next, a false-positive comparison of the nominal probe versus the modified (protected) probe was performed on NTC urine samples (N = 20). Figures 8A–8D depict data related to false-positive evaluation in the Chlamydia trachomatis assay (Figures 8A–8B) and Neisseria gonorrhoeae assay (Figures 8C–8D) performed on NTC 15% urine samples using the nominal probe (Figures 8A, 8C) and the protected probe (Figures 8B, 8D). Use of the protected probe resulted in a reduction in false positives in the Chlamydia trachomatis assay from 7 / 20 (35%) to 1 / 20 (5%), with the only remaining false positive attributed to suspected target template contamination. Therefore, this higher false-positive frequency was mitigated by the O-methyl modification of the beacon. Identical performance (no false positives observed) was observed between the two probes in the Neisseria gonorrhoeae assay. A false-positive comparison of the nominal probe versus the modified (protected) probe was then performed on vaginal swabs (N = 20). Figures 9A–9D depict data related to false-positive evaluation in the Chlamydia trachomatis assay (Figures 9A–9B) and Neisseria gonorrhoeae assay (Figures 9C–9D) performed on vaginal swab samples using the nominal probe (Figures 9A, 9C) and the protected probe (Figures 9B, 9D). For both assays, a higher number of false-positives was observed for vaginal swab samples and was mitigated by O-methyl modification of the beacon. Use of the protected probe resulted in a reduction of false-positives in the Chlamydia trachomatis assay from 9 / 20 (45%) to 0 / 20 (0%). Use of the protected probe resulted in a reduction of false-positives in the Neisseria gonorrhoeae assay from 1 / 20 (5%) to 0 / 20 (0%).

[0183] Next, NTC testing with the modified probe was performed using NTC 10% urine samples (N = 40). The assay was switched to a HEX beacon (O-methyl modified) for C. trachomatis, and 0% FP for C. trachomatis and 2.5% for Neisseria gonorrhoeae (ROX) were observed. Figures 10A-10D depict data related to false-positive evaluation in the C. trachomatis assay (Figures 10A-10B) and Neisseria gonorrhoeae assay (Figures 10C-10D) performed with the protected probe on NTC urine samples. Next, NTC testing was performed with the modified probes using NTC vaginal swab samples (1.67% VM (N=20)). The 3-in-1 swabs were expressed in 1×GRBS (1.67%) and assayed using CT HEX (2'-O-methyl modified) beacons and GC ROX (2'-O-methyl modified) beacons. Figures 11A-11B depict data related to false-positive evaluation in the Chlamydia trachomatis assay (Figure 11A) and Neisseria gonorrhoeae assay (Figure 11B) performed with the protected probes on NTC vaginal swab samples. Zero percent FP was observed for both assays.

[0184] Example 4 FluA assay: RNA modified beacon This example demonstrates the use of RNA base incorporation for the protection of molecular beacons. Figures 13A-13C depict data related to RNA base incorporation for the protection of molecular beacons in a FluA assay. The assay was performed with the NTC only-Primer Drop (Figures 13A-13B), or the probe was screened with the target (Figure 13C). Under forward and reverse primer conditions, the RNA-modified probe (LP2(rna3)) produced the same rate of false positives as the nominal probe (LP2), no false positives were detected with the RNA-modified probe LP2(rna1), and one false positive out of four was detected with the RNA-modified probe LP2(rna2) (Figure 14A). Under reverse primer-only conditions, the RNA modification to the probe helped reduce false positive detection (Figure 13B). When the probes were screened with the target (Figure 13C), the RNA-modified probe LP2(rna3) produced the same FP rate as the nominal probe LP2 (3 out of 4 NTCs were detected). RNA modifications were shown to negatively affect target product binding to the probe (which may explain the lack of false positives observed with LP2(rna1) and LP2(rna2)). This example demonstrates that RNA base modifications of probes can confer protection from nonspecific product formation.

[0185] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims. With respect to the use of substantially any plural and / or singular terminology herein, those of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless stated otherwise.

[0186] Those skilled in the art will understand that the terms used in this specification, generally, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Furthermore, those skilled in the art will understand that where a specific number is intended in an introduced claim recitation, such intention will be expressly recited in the claim; otherwise, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means limiting any particular claim containing such an introduced claim recitation to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Additionally, even if a particular number is explicitly recited in an introduced claim recitation, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a base recitation of "two recitations" means at least two recitations, or two or more recitations, in the absence of other modifiers).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that a person of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, a system having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that a person of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, those skilled in the art will appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either of those terms, or both terms.

[0187] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is also thereby described in terms of every individual member or subgroup of members of the Markush group.

[0188] As one of ordinary skill in the art would understand, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as being capable of dividing that same range into at least two, three, four, five, ten, etc., as fully described. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Furthermore, as one of ordinary skill in the art would understand, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the recited numerical values ​​and that can be subsequently divided into subranges as discussed above. Finally, as one of ordinary skill in the art would understand, a range includes each individual member. Thus, for example, a group containing 1 to 3 items refers to groups containing 1, 2, or 3 items. Similarly, a group containing 1 to 5 items refers to groups containing 1, 2, 3, 4, or 5 items, etc.

[0189] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not limitation, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for detecting a target nucleic acid sequence in a sample, comprising: amplifying the target nucleic acid sequence in the amplification reaction mixture under isothermal amplification conditions, thereby producing a nucleic acid amplification product; and detecting said nucleic acid amplification products using signal-generating oligonucleotides, said signal-generating oligonucleotides being capable of hybridizing to said nucleic acid amplification products and comprising one or more polymerase stoppers; A method comprising:

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

3. the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain; the signal-generating oligonucleotide comprises a loop domain between the 5' subdomain and the 3' subdomain; intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain; the one or more polymerase stoppers are within the loop domain; The method of claim 1 or 2, wherein the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise the one or more polymerase stoppers.

4. The nucleic acid amplification product (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 being 1 to 10 bases in length. The method according to any one of claims 1 to 3, comprising:

5. the signal-generating oligonucleotide comprises a first region comprising at least a portion of the sequence of the reverse primer; the signal-generating oligonucleotide comprises a second region comprising a sequence complementary to at least a portion of the forward primer; and / or the signal-generating oligonucleotide comprises a spacer region comprising at least a portion of the sequence of the spacer sequence; 5. The method of any one of claims 1 to 4, wherein optionally the first region, the second region, and / or the spacer region comprises one or more polymerase stoppers.

6. 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 the two 3' terminal nucleotides of the forward primer and / or the reverse primer; and / or The method according to any one of claims 1 to 5, wherein the spacer region comprises a sequence complementary to at least two 3'-terminal nucleotides of the forward primer and / or the reverse primer.

7. the signal-generating oligonucleotide is from about 10 nucleotides to about 100 nucleotides in length; the forward primer and / or the reverse primer is from about 5 nucleotides to about 25 nucleotides in length; the second region, the spacer region, and / or the first region are from about 1 nucleotide to about 25 nucleotides in length; and / or 7. The method of any one of claims 1 to 6, wherein the 5' subdomain, the 3' subdomain, the loop domain, the 5' terminal domain, and / or the 3' terminal domain are from about 1 nucleotide to about 25 nucleotides in length.

8. the signal-generating oligonucleotide comprises a 5' terminal domain 5' of the 5' subdomain; and / or the signal-generating oligonucleotide comprises a 3' terminal domain 3' of the 5' subdomain; 8. The method of any one of claims 1 to 7, wherein optionally the 5'-terminal domain and / or the 3'-terminal domain does not comprise the one or more polymerase stoppers.

9. 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; The method according to any one of claims 1 to 8, wherein the second region comprises at least a portion of the loop domain and / or the 3' subdomain.

10. The method of any one of claims 1 to 9, wherein the one or more polymerase stoppers comprise one or more 2'-O-methyl (2'OM) RNA nucleotides.

11. 11. The method of any one of claims 1 to 10, wherein the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemical capture abasic site, or any combination thereof.

12. the stable abasic site comprises a 1',2'-dideoxy; The chemically trapped abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride; the abasic site comprises an apurinic base site, an apyrimidinic base site, or both; and / or 12. The method of claim 11, wherein the abasic site is generated by an alkylating agent or an oxidizing agent.

13. The one or more polymerase stoppers are one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2,6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3(OC 3 H 6 OPO 3 ) group, one or more photocleavable (PC) [OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(CH 3 ) OPO 3 ] group, one or more hexanediol groups, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, one or more spacer 18 (iSp18) [(OCH 2 CH 26 OPO 3 ] groups, or any combination thereof The method according to any one of claims 1 to 12, comprising:

14. The method of any one of claims 1 to 13, wherein the signal-generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids.

15. The method of any one of claims 1 to 14, wherein the signal-generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide.

16. 16. The method of any one of claims 1 to 15, wherein the signal-generating oligonucleotide comprises a label, optionally wherein the label comprises a quenchable label, and further optionally wherein the quenchable label is a fluorophore.

17. The signal-generating oligonucleotide comprises a quencher and, optionally, the label is in the 3'-terminal domain and the quencher is in the 5'-terminal domain; and / or The method of any one of claims 1 to 16, wherein the label is in the 5'-terminal domain and the quencher is in the 3'-terminal domain.

18. after the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide; 18. The method of any one of claims 1 to 17, wherein optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

19. after the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide; 19. The method of any one of claims 1 to 18, wherein optionally, one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second, undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

20. after an exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers can stop polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide; Optionally, said one or more polymerase stoppers are capable of terminating polymerase extension of said exogenous nucleic acid of said third undesired duplex beyond said one or more polymerase stoppers of said signal-generating oligonucleotide; 20. The method of any one of claims 1 to 19, wherein optionally the exogenous nucleic acid is selected from the group comprising a sample nucleic acid, a primer configured to hybridize to a second target nucleic acid sequence, a primer configured to hybridize to an internal standard, or any combination thereof.

21. The method according to any one of claims 1 to 20, wherein the sample nucleic acid comprises a nucleic acid comprising the target nucleic acid sequence.

22. The step of amplifying the target nucleic acid sequence comprises: The method includes a step of amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein the amplifying step comprises amplifying a nucleic acid comprising the target nucleic acid sequence by: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and ii) Enzymes with hyperthermophilic polymerase activity 22. The method of any one of claims 1 to 21, comprising contacting said nucleic acid amplification product with

23. 23. The method of claim 21 or 22, wherein the nucleic acid is double-stranded DNA.

24. 24. The method of any one of claims 21 to 23, wherein the nucleic acid is a product of a reverse transcription reaction, optionally the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid, and further optionally, the amplifying step comprises generating the nucleic acid by a reverse transcription reaction.

25. 25. The method of any one of claims 1 to 24, wherein the sample nucleic acid comprises sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA.

26. The step of amplifying the target nucleic acid sequence comprises: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying step comprises: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophilic polymerase activity thereby producing said nucleic acid amplification product. The method of any one of claims 1 to 25, comprising:

27. if the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity produces a first undesired extension product; the first undesired extension product can be amplified by the enzyme having hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product; the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex to generate a first extension stall product; 27. The method of any one of claims 1 to 26, wherein the first extension stalled product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the forward primer and the reverse primer to produce the first undesired amplification product.

28. 28. The method of any one of claims 1 to 27, wherein the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

29. if the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity produces a second undesired extension product; the second undesired extension product can be amplified by the enzyme having hyperthermophile polymerase activity in the presence of the reverse primer to form a second undesired amplification product; the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second undesired duplex to generate a second extension stall product; 29. The method of any one of claims 1 to 28, wherein the second extension stalled product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to produce the second undesired amplification product.

30. 30. The method of any one of claims 1 to 29, wherein the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second, undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

31. if the exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide by an enzyme having hyperthermophile polymerase activity produces a third undesired extension product; the third undesired extension product can be amplified by the enzyme having hyperthermophile polymerase activity in the presence of the reverse primer to form a third undesired amplification product; the one or more polymerase stoppers are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex to generate a third extension stall product; 31. The method of any one of claims 1 to 30, wherein the third extension stalled product cannot be amplified by the enzyme with hyperthermophile polymerase activity in the presence of the reverse primer to produce the third undesired amplification product.

32. 32. The method of any one of claims 1 to 31, wherein the one or more polymerase stoppers are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond the one or more polymerase stoppers of the signal-generating oligonucleotide.

33. 33. The method of any one of claims 1 to 32, wherein the detecting step comprises contacting the nucleic acid amplification product with the signal-generating oligonucleotide for hybridization.

34. The method of any one of claims 1 to 33, wherein the step of detecting the nucleic acid amplification product comprises the use of a real-time detection method.

35. the label is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; and / or the label generates a signal after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product; Optionally, the signal is fluorescence.

36. the detecting step includes detecting the signal of the label before the amplification reaction, after the amplification reaction, or both; detecting the nucleic acid amplification product comprises detecting a signal generated by a label of a signal-generating oligonucleotide, optionally wherein the label is a fluorophore and the signal is fluorescent; detecting the signal comprises detecting fluorescence emitted by the label; The method includes 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 comprising the target nucleic acid sequence in the sample; the presence, absence, and / or amount of the signal indicates the presence, absence, and / or amount of the target nucleic acid sequence in the sample; and / or 36. The method of any one of claims 1 to 35, wherein the presence, absence, and / or amount of the signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample.

37. the label is capable of generating a false positive signal after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product, and optionally the signal and the false positive signal are indistinguishable; and / or 37. The method of any one of claims 1 to 36, wherein after the signal-generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product, the label generates a false positive signal, and optionally the signal and the false positive signal are indistinguishable.

38. 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 38. The method of any one of claims 1 to 37, wherein detection of said false positive signal reduces the likelihood of an accurate determination of the presence, absence and / or amount of said target nucleic acid sequence in said sample.

39. 39. The method of any one of claims 1 to 38, wherein the presence of the one or more polymerase stoppers in the signal-generating oligonucleotide increases the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample by at least about 1.1 fold compared to the signal-generating oligonucleotide not comprising the one or more polymerase stoppers.

40. production of the first extension stall product, the second extension stall product, and / or the third extension stall product does not result in a false positive signal; and / or 40. The method of any one of claims 1 to 39, wherein the signal-generating oligonucleotide hybridized to the first extension stall product, the second extension stall product, and / or the third extension stall product does not generate a false positive signal.

41. the nucleic acid amplification product reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reach a detectable level; and / or 41. The method of any one of claims 1 to 40, wherein the signal reaches a detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before the false positive signal reaches a detectable level.

42. 42. The method of any one of claims 1 to 41, wherein the appearance of a detectable level of the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product is delayed by at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes compared to a comparable method in which the signal-generating oligonucleotide does not comprise the one or more polymerase stoppers.

43. 43. The method of any one of claims 1 to 42, wherein the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product do not reach detectable levels for at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the amplifying step begins.

44. 44. The method of any one of claims 1 to 43, wherein the occurrence 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 compared to a comparable method in which the signal-generating oligonucleotide does not comprise the one or more polymerase stoppers.

45. amplifying the target nucleic acid sequence comprises producing a detectable level of the nucleic acid amplification product within about 20 minutes, within about 15 minutes, or within about 10 minutes; and / or 45. The method of any one of claims 1 to 44, wherein the detecting step is performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the treated sample.

46. the lysis buffer comprises one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA; and / or 46. ​​The method of any one of claims 1 to 45, wherein the pH of the lysis buffer is from about 1.0 to about 10.0, and optionally, the pH of the lysis buffer is about 2.

2.

47. 47. The method of any one of claims 1 to 46, wherein the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid, optionally wherein the sample nucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof.

48. The one or more amplification reagents are reverse transcriptase; an enzyme having hyperthermophilic polymerase activity, optionally having reverse transcriptase activity; Forward primer; reverse primer; a reverse transcription primer; and / or dNTP 48. The method of any one of claims 1 to 47, comprising:

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

50. The one or more lytic reagents include: about 0.001% (wt / vol) to about 1.0% (wt / vol) of the treated sample, optionally about 0.2% (wt / vol) of the treated sample; and / or a detergent optionally comprising one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; 50. The method of any one of claims 1 to 49, comprising:

51. carried out in a single reaction vessel; does not include a step of using any enzyme other than the reverse transcriptase and the enzyme having hyperthermophile polymerase activity; does not include a step of using any enzyme other than the enzyme having hyperthermophilic polymerase activity; does not include a step of thermally and / or enzymatically denaturing the nucleic acid during the amplification step; and / or 51. The method of any one of claims 1 to 50, which does not include a step of contacting the nucleic acid with a single-stranded DNA binding protein.

52. the target nucleic acid sequence comprises a length of about 20 nucleotides or less to about 90 nucleotides or less, and optionally comprises a length of about 30 nucleotides; the forward primer, the reverse primer, and / or the reverse transcription primer are about 8 to 16 bases in length; the nucleic acid amplification product is about 20 to 40 bases in length; and / or 52. The method of any one of claims 1 to 51, wherein the spacer sequence comprises a portion of the target nucleic acid sequence and is optionally 1 to 10 bases in length.

53. Isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, further optionally about 55°C to about 75°C, optionally about 56°C to about 67°C; the amplifying step (a) is performed for about 5 minutes to about 60 minutes, optionally for about 15 minutes; and / or (b) is performed under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions; The amplifying step is carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA), and optionally, the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:7 or a functional fragment thereof, optionally the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO:7, further optionally the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:7, optionally the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity; and / or The method of any one of claims 1 to 52, wherein the sample ribonucleic acid is contacted with the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity simultaneously; optionally, the sample ribonucleic acid is contacted with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, the forward primer, and the reverse primer simultaneously; and further optionally, the sample ribonucleic acid is contacted with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer simultaneously.

54. The biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a viral particle, an exosome, a protoplast, and a microvesicle; The biological entity comprises a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof; and / or 54. The method of any one of claims 1 to 53, wherein the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a virus, bacterium, fungus, or protozoan.

55. The virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus; The bacteria include Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae pneumonia, S. agalactiae, and Listeria monocytogenes; the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or 55. The method of any one of claims 1 to 54, wherein the protozoan comprises one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.

56. the sample is a biological sample or an environmental sample; the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof; and / or 56. The method of any one of claims 1 to 55, wherein the biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, stool, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

57. wherein the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences, and wherein the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally wherein the two or more target nucleic acid sequences are specific to two or more different organisms, and further optionally wherein the two or more different organisms comprise one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C; The amplifying step does not include one or more of Archaeal Polymerase Amplification (APA), Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), Transcription-Mediated Amplification (TMA), Nicking Enzyme Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), Ramification (RAM), Circular Helicase-Dependent Amplification (cHDA), Single Primer Isothermal Amplification (SPIA), Signal-Mediated RNA Amplification Technology (SMART), Self-Sustained Sequence Replication (3SR), Genomic Exponential Amplification Reaction (GEAR), and Isothermal Multiple Displacement Amplification (IMDA), and optionally, the amplifying step does not include LAMP: the amplifying step comprises one or more of APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA, optionally, the amplifying step does not comprise LAMP; and / or 57. The method of any one of claims 1-56, wherein the method does not include one or more of: (i) diluting the processed sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the processed sample; (iv) sonicating the processed sample; (v) sonicating the amplification reaction mixture; (vi) adding an RNase inhibitor to the processed sample; (vii) adding an RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing the one or more lytic agents from the processed sample or the amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the processed sample or amplification reaction mixture.

58. a signal-generating oligonucleotide capable of hybridizing to a nucleic acid amplification product, a 5' subdomain, a 3' subdomain, intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain, and a loop domain between the 5' subdomain and the 3' subdomain; intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain; the loop domain comprises one or more polymerase stops; A signal-generating oligonucleotide, wherein the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not include the one or more polymerase stoppers.

59. 59. The signal-generating oligonucleotide of claim 58, wherein the 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.

60. The nucleic acid amplification product (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 being 1 to 10 bases in length.

60. The signal-generating oligonucleotide of claim 58 or 59, comprising:

61. the signal-generating oligonucleotide comprises a first region comprising at least a portion of the sequence of the reverse primer; the signal-generating oligonucleotide comprises a second region comprising a sequence complementary to at least a portion of the forward primer; and / or the signal-generating oligonucleotide comprises a spacer region comprising at least a portion of the spacer sequence; 61. The signaling oligonucleotide of any one of claims 58 to 60, wherein optionally the first region, the second region, and / or the spacer region comprises one or more polymerase stoppers.

62. 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 the two 3' terminal nucleotides of the forward primer and / or the reverse primer; and / or 62. The signaling oligonucleotide of any one of claims 58 to 61, wherein the spacer region comprises a sequence complementary to at least two 3'-terminal nucleotides of the forward primer and / or the reverse primer.

63. the forward primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence; the reverse primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; Optionally, the nucleic acid amplification product is generated by amplifying the target nucleic acid sequence using the forward primer and the reverse primer.

64. the signal-generating oligonucleotide is from about 10 nucleotides to about 100 nucleotides in length; the forward primer and / or the reverse primer is from about 5 nucleotides to about 25 nucleotides in length; the second region, the spacer region, and / or the first region are from about 1 nucleotide to about 25 nucleotides in length; and / or 64. The signaling oligonucleotide of any one of claims 58 to 63, wherein the 5' subdomain, the 3' subdomain, the loop domain, the 5' terminal domain, and / or the 3' terminal domain are from about 1 nucleotide to about 25 nucleotides in length.

65. the signal-generating oligonucleotide comprises a 5' terminal domain 5' of the 5' subdomain; and / or the signal-generating oligonucleotide comprises a 3' terminal domain 3' of the 5' subdomain; 65. The signaling oligonucleotide of any one of claims 58 to 64, wherein optionally the 5'-terminal domain and / or the 3'-terminal domain does not comprise the one or more polymerase stoppers.

66. 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; 66. The signaling oligonucleotide of any one of claims 58 to 65, wherein the second region comprises at least a portion of the loop domain and / or the 3' subdomain.

67. 67. The signaling oligonucleotide of any one of claims 58 to 66, wherein the one or more polymerase stoppers comprise one or more 2'-O-methyl (2'OM) RNA nucleotides.

68. 68. The signaling oligonucleotide of any one of claims 58 to 67, wherein the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemical capture abasic site, or any combination thereof.

69. the stable abasic site comprises a 1',2'-dideoxy; The chemically trapped abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride; the abasic site comprises an apurinic base site, an apyrimidinic base site, or both; and / or 69. The signal-generating oligonucleotide of claim 68, wherein the abasic site is generated by an alkylating agent or an oxidizing agent.

70. The one or more polymerase stoppers may be one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2,6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3(OC 3 H 6 OPO 3 ) group, one or more photocleavable (PC) [OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(CH 3 ) OPO 3 ] group, one or more hexanediol groups, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, one or more spacer 18 (iSp18) [(OCH 2 CH 26 OPO 3 ] groups, or any combination thereof 70. The signal-generating oligonucleotide of any one of claims 58 to 69, comprising:

71. the signal-generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids; The signal generating oligonucleotide comprises a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide; the signal-generating oligonucleotide comprises a label, optionally, the label comprises a quenchable label, and further optionally, the quenchable label is a fluorophore; the signal-generating oligonucleotide comprises a quencher, and optionally (a) the label is in the 3'-terminal domain and the quencher is in the 5'-terminal domain, and / or (b) the label is in the 5'-terminal domain and the quencher is in the 3'-terminal domain; and / or 71. The signal-generating oligonucleotide of any one of claims 58 to 70, wherein the label is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the nucleic acid amplification product, and optionally the signal is fluorescent.

72. after the forward primer binds to the signal-generating oligonucleotide to form a first undesired duplex, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal-generating oligonucleotide; 72. The signaling oligonucleotide of any one of claims 58 to 71, wherein optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signaling oligonucleotide.

73. after the reverse primer binds to the signal-generating oligonucleotide to form a second undesired duplex, the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal-generating oligonucleotide; 73. The signaling oligonucleotide of any one of claims 58 to 72, wherein optionally, one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second, undesired duplex beyond the one or more polymerase stoppers of the signaling oligonucleotide.

74. after an exogenous nucleic acid binds to the signal-generating oligonucleotide to form a third undesired duplex, the one or more polymerase stoppers can stop polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal-generating oligonucleotide; 74. The signaling oligonucleotide of any one of claims 58 to 73, wherein optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex beyond the one or more polymerase stoppers of the signaling oligonucleotide.

75. 1. A kit for detecting a target nucleic acid sequence in a sample, comprising: (a) a signal-generating oligonucleotide according to any one of claims 1 to 74; (b) a lysis buffer comprising one or more lysis agents capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence, optionally wherein the one or more lysis agents comprise a detergent, optionally wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; and / or (c) a reagent composition comprising one or more amplification reagents comprising one or more components for amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplification comprise: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the reverse primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, optionally having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:7 or a functional fragment thereof, optionally having an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO:7, and further optionally, the polymerase comprising the amino acid sequence of SEQ ID NO:

7. A reagent composition comprising Kit including:

76. 76. The kit of claim 75, wherein the reagent composition comprises a reverse transcriptase and / or a reverse transcription primer.

77. The nucleic acid amplification product is about 20 to 40 bases in length, and the nucleic acid amplification product is (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 being 1 to 10 bases in length.

77. The kit of claim 75 or 76, comprising:

78. The biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a viral particle, an exosome, a protoplast, and a microvesicle; The biological entity comprises a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof; and / or 78. The kit of any one of claims 75 to 77, wherein the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid is derived from a virus, bacterium, fungus, or protozoan.

79. The viruses are SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus; The bacteria include one or more of Mycobacterium tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma spp., Legionella pneumophila, Legionella dumophila, Mycoplasma fermentans, Ehrlichia spp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, S. agalactiae, and Listeria monocytogenes; the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or 79. The kit of any one of claims 75 to 78, wherein the protozoan comprises one or more of Trypanosoma cruzi, Leishmania spp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora spp., and Eimeria spp.

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

80. The kit of any one of claims 75 to 79, comprising:

81. 1. A method for detecting Neisseria gonorrhoeae in a sample, comprising: contacting the sample with at least one primer pair, the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, and each primer in the at least one primer pair comprises any one of the sequences of SEQ ID NOs: 2-3 and 16-17, or a sequence that exhibits at least about 85% identity to any one of the sequences of SEQ ID NOs: 2-3 and 16-17; If the sample contains Neisseria gonorrhoeae, generating an amplicon of the target nucleic acid sequence; and determining the presence or amount of said amplicon as an indication of the presence of Neisseria gonorrhoeae in said sample. A method comprising:

82. 82. The method of claim 81 , wherein the at least one primer pair comprises a first primer comprising the sequence of SEQ ID NO: 2 or 17, and a second primer comprising the sequence of SEQ ID NO: 3 or 16.

83. 83. The method of claim 81 or 82, wherein the at least one primer pair capable of hybridizing to the target nucleic acid sequence of Neisseria gonorrhoeae is SEQ ID NO: 2 and 3, SEQ ID NO: 2 and 16, SEQ ID NO: 17 and 3, or SEQ ID NO: 17 and 16.

84. determining the presence or amount of the amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14; Optionally, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14; and / or Optionally, each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

85. 1. A method for detecting Chlamydia trachomatis in a sample, comprising: contacting the sample with at least one primer pair, the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, and each primer in the at least one primer pair comprises any one of the sequences of SEQ ID NOs: 20-23, 25-26, 28-29, and 31, or a sequence that exhibits at least about 85% identity to any one of the sequences of SEQ ID NOs: 20-23, 25-26, 28-29, and 31; If the sample contains Chlamydia trachomatis, generating an amplicon of the target nucleic acid sequence; and determining the presence or amount of said amplicon as an indication of the presence of Chlamydia trachomatis in said sample. A method comprising:

86. 86. The method of claim 85, wherein the at least one primer pair comprises a first primer comprising the sequence of SEQ ID NO: 20, 22, 25, or 28, and a second primer comprising the sequence of SEQ ID NO: 21, 23, 26, 29, or 31.

87. 87. The method of claim 85 or 86, wherein the at least one primer pair capable of hybridizing to the target nucleic acid sequence of Chlamydia trachomatis is SEQ ID NOs:20 and 21, SEQ ID NOs:20 and 23, SEQ ID NOs:20 and 26, SEQ ID NOs:20 and 29, SEQ ID NOs:20 and 31, SEQ ID NOs:22 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:22 and 26, SEQ ID NOs:22 and 29, SEQ ID NOs:22 and 31, SEQ ID NOs:25 and 21, SEQ ID NOs:25 and 23, SEQ ID NOs:25 and 26, SEQ ID NOs:25 and 29, SEQ ID NOs:25 and 31, SEQ ID NOs:28 and 21, SEQ ID NOs:28 and 23, SEQ ID NOs:28 and 26, SEQ ID NOs:28 and 29, or SEQ ID NOs:28 and 31.

88. determining the presence or amount of the amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32; Optionally, each of the one or more signaling oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32; and / or 88. The method of any one of claims 85-87, optionally wherein each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

89. 1. A method for detecting influenza B virus in a sample, comprising: contacting the sample with at least one primer pair, the at least one primer pair is capable of hybridizing to a target nucleic acid sequence of influenza B virus, and each primer in the at least one primer pair comprises any one of the sequences of SEQ ID NOs: 36-37 and 41, or a sequence that exhibits at least about 85% identity to any one of the sequences of SEQ ID NOs: 36-37 and 41; If the sample contains influenza B virus, generating an amplicon of the target nucleic acid sequence; and determining the presence or amount of said amplicon as an indication of the presence of influenza B virus in said sample. A method comprising:

90. 90. The method of claim 89, wherein the at least one primer pair comprises a first primer comprising the sequence of SEQ ID NO: 36 and a second primer comprising the sequence of SEQ ID NO: 37 or 41.

91. 91. The method of claim 89 or 90, wherein the at least one primer pair capable of hybridizing to the target nucleic acid sequence of influenza B virus is SEQ ID NO: 36 and 37, or SEQ ID NO: 36 and 41.

92. determining the presence or amount of the amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45; Optionally, each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45; and / or 92. The method of any one of claims 89-91, optionally wherein each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

93. 93. The method of any one of claims 89 to 92, comprising contacting sample ribonucleic acid of said sample with a reverse transcriptase and a primer that exhibits at least about 85% identity to SEQ ID NO:

38.

94. the sample is a biological sample or an environmental sample; the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof; and / or 94. The method of any one of claims 81 to 93, wherein the biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, stool, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

95. 95. The method of any one of claims 81 to 94, wherein the sample is contacted with a reagent composition comprising the at least one primer pair to produce the amplification reaction mixture.

96. contacting a sample containing biological entities with a lysis buffer to produce a processed sample, the lysis buffer comprising one or more lysis agents capable of lysing the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence; and contacting a reagent composition comprising the at least one primer pair with the processed sample to produce the amplification reaction mixture, the reagent composition comprising one or more amplification reagents. The method of any one of claims 81 to 95, comprising:

97. The one or more amplification reagents are reverse transcriptase; an enzyme having hyperthermophilic polymerase activity, optionally having reverse transcriptase activity; a reverse transcription primer; and / or dNTP 97. The method of any one of claims 81 to 96, comprising:

98. The step of generating an amplicon of the target nucleic acid sequence comprises: amplifying said target nucleic acid sequence in said amplification reaction mixture under amplification conditions, thereby producing an amplicon of said target nucleic acid sequence.

98. The method of any one of claims 81 to 97, comprising:

99. The amplifying step is carried out using a method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA), and optionally, the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); the amplifying step does not include one or more of Archaeal Polymerase Amplification (APA), Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), Transcription-Mediated Amplification (TMA), Nicking Enzyme Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), Ramification (RAM), Circular Helicase-Dependent Amplification (cHDA), Single Primer Isothermal Amplification (SPIA), Signal-Mediated RNA Amplification Technology (SMART), Self-Sustained Sequence Replication (3SR), Genomic Exponential Amplification Reaction (GEAR), and Isothermal Multiple Displacement Amplification (IMDA), and optionally, the amplifying step does not include LAMP; the amplifying step comprises one or more of APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA, optionally, the amplifying step does not comprise LAMP; and / or 99. The method of claim 98, wherein the method does not include one or more of: (i) diluting the processed sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the processed sample; (iv) sonicating the processed sample; (v) sonicating the amplification reaction mixture; (vi) adding an RNase inhibitor to the processed sample; (vii) adding an RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing the one or more lytic agents from the processed sample or the amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the processed sample or amplification reaction mixture.

100. 100. The method of any one of claims 81 to 99, wherein said determining step comprises contacting an amplicon of said target nucleic acid sequence with said signal-generating oligonucleotide for hybridization, and optionally said determining step comprises the use of a real-time detection method.

101. the label of the signal-generating oligonucleotide is capable of generating a signal after the signal-generating oligonucleotide hybridizes to the amplicon of the target nucleic acid sequence; and / or 101. The method of any one of claims 81 to 100, wherein the label generates a signal after the signal-generating oligonucleotide hybridizes to an amplicon of the target nucleic acid sequence, and optionally the signal is fluorescent.

102. 1. A composition for the detection of Neisseria gonorrhoeae in a sample, comprising: At least one primer pair capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, wherein each primer in said at least one primer pair comprises any one of the sequences of SEQ ID NOs: 2-3 and 16-17, or a sequence exhibiting at least about 85% identity with any one of the sequences of SEQ ID NOs: 2-3 and 16-17. A composition comprising:

103. The composition of claim 102, wherein at least one primer pair capable of hybridizing to the target nucleic acid sequence of Neisseria gonorrhoeae comprises a primer comprising the sequence of SEQ ID NO: 2 or 17 and a primer comprising the sequence of SEQ ID NO: 3 or 16.

104. 104. The composition of claim 102 or 103, further comprising one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

105. 105. The composition of any one of claims 102 to 104, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

106. 106. The composition of any one of claims 102 to 105, wherein each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 1 and 12-14.

107. 1. A composition for the detection of Chlamydia trachomatis in a sample, comprising: At least one primer pair capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in said at least one primer pair comprises any one of the sequences of SEQ ID NOs: 20-23, 25-26, 28-29, and 31, or a sequence that exhibits at least about 85% identity with any one of the sequences of SEQ ID NOs: 20-23, 25-26, 28-29, and 31. A composition comprising:

108. The composition of claim 107, wherein at least one primer pair capable of hybridizing to the target nucleic acid sequence of Chlamydia trachomatis comprises a primer comprising the sequence of SEQ ID NO: 20, 22, 25, or 28, and a primer comprising the sequence of SEQ ID NO: 21, 23, 26, 29, or 31.

109. 109. The composition of claim 107 or 108, further comprising one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

110. 110. The composition of any one of claims 107 to 109, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

111. 111. The composition of any one of claims 107-110, wherein each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32.

112. 1. A composition for the detection of influenza B virus in a sample, comprising: At least one primer pair capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in said at least one primer pair comprises any one of the sequences of SEQ ID NOs: 36-37 and 41, or a sequence that exhibits at least about 85% identity to any one of the sequences of SEQ ID NOs: 36-37 and 41. A composition comprising:

113. The composition of claim 112, wherein at least one primer pair capable of hybridizing to the target nucleic acid sequence of influenza B virus comprises a primer comprising the sequence of SEQ ID NO: 36 and a primer comprising the sequence of SEQ ID NO: 37 or 41.

114. 114. The composition of claim 112 or 113, further comprising a primer exhibiting at least about 85% identity to SEQ ID NO:

38.

115. 115. The composition of any one of claims 112-114, further comprising one or more signal-generating oligonucleotides, each of the one or more signal-generating oligonucleotides comprising a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45, or a sequence that exhibits at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

116. 116. The composition of any one of claims 112-115, wherein each of the one or more signal-generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

117. 117. The composition of any one of claims 112-116, wherein each of the one or more signal-generating oligonucleotides consists of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

118. the signal-generating oligonucleotide comprises a 5' subdomain and a 3' subdomain; the signal-generating oligonucleotide comprises a loop domain between the 5' subdomain and the 3' subdomain; intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain; the loop domain comprises one or more polymerase stoppers; 118. The method or composition of any one of claims 81 to 117, wherein the 5' subdomain, the paired stem domain, and / or the 3' subdomain do not comprise the one or more polymerase stoppers.

119. the signal-generating oligonucleotide comprises a 5' terminal domain 5' of the 5' subdomain; and / or the signal-generating oligonucleotide comprises a 3' terminal domain 3' of the 5' subdomain; 119. The method or composition of claim 118, wherein optionally, the 5'-terminal domain and / or the 3'-terminal domain does not comprise the one or more polymerase stoppers.

120. 120. The method or composition of claim 118 or 119, wherein the one or more polymerase stoppers comprise one or more 2'-O-methyl (2'OM) RNA nucleotides.

121. 121. The method of any one of claims 118-120, wherein the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemical capture abasic site, or any combination thereof.

122. the stable abasic site comprises a 1',2'-dideoxy; The chemically trapped abasic site comprises an abasic site reacted with an alkoxyamine or sodium borohydride; the abasic site comprises an apurinic base site, an apyrimidinic base site, or both; and / or 122. The method or composition of any one of claims 118 to 121, wherein the free base site is generated by an alkylating agent or an oxidizing agent.

123. The one or more polymerase stoppers may be one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2,6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more iso-deoxycytidines (iso-dC), one or more iso-deoxyguanosines (iso-dG), one or more C3 (OC 3 H 6 OPO 3 ) group, one or more photocleavable (PC) [OC 3 H 6 -C(O)NHCH 2 -C 6 H 3 NO 2 -CH(CH 3 ) OPO 3 ] group, one or more hexanediol groups, one or more spacers 9 (iSp9) [(OCH 2 CH 2 ) 3 OPO 3 ] group, one or more spacer 18 (iSp18) [(OCH 2 CH 26 OPO 3 ] groups, or any combination thereof; the signal-generating oligonucleotide comprises one or more phosphorothioate linkages and / or one or more locked nucleic acids; the signal-generating oligonucleotide comprises a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide; and / or 123. The method or composition of any one of claims 118 to 122, wherein the signal-generating oligonucleotide comprises a label, optionally wherein the label comprises a quenchable label, and further optionally wherein the quenchable label is a fluorophore.

124. A signal-generating oligonucleotide or primer up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, said signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, and optionally the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17; the signal-generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NOs: 1-3, 12-14, and 16-17; and / or The signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 12 to 14, and 16 to 17.

125. A signal-generating oligonucleotide or primer up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, said signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, and optionally the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32; the signal-generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32; and / or The signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32.

126. A signal-generating oligonucleotide or primer up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of influenza B virus, said signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 36-45, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 36-45, and optionally the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 36-45, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 36-45; the signal-generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NOs: 36-45; and / or The signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 36 to 45.

127. A signal-generating oligonucleotide or primer up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of influenza A virus, said signal-generating oligonucleotide or primer comprising a sequence selected from the group consisting of SEQ ID NOs: 33-35, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 33-35, and optionally the signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 33-35, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 33-35; the signal-generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NOs: 33-35; and / or The signal-generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 33 to 35.

128. 128. A composition comprising two or more signal-generating oligonucleotides or primers according to any one of claims 124 to 127.

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

129. The composition of claim 128, comprising:

130. The one or more components for amplification include:

1. An enzyme having hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, wherein the enzyme optionally has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:7 or a functional fragment thereof, optionally has an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO:7, and further optionally is a polymerase comprising the amino acid sequence of SEQ ID NO:

7.

130. The composition of claim 128 or 129, comprising:

Images