Modified molecular beacons for improved detection specificity

Abstract

The disclosure herein includes methods, compositions, and kits for detecting a target nucleic acid sequence in a sample. The method can include generating an oligonucleotide using a signal capable of hybridizing to a nucleic acid amplification product and comprising one or more polymerase terminators. In some embodiments, assays employing the protected signal generating oligonucleotides provided herein exhibit reduced non-specific product formation and less false positives.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 374,772, filed on September 7, 2022; and U.S. Provisional Application No. 63 / 374,774, filed on September 7, 2022. The entire contents of these applications are hereby expressly incorporated by reference in their entirety.

[0003] Reference to a sequence listing

[0004] This application is submitted together with a sequence listing in electronic format. The sequence listing is provided as a file entitled 68EB-317332-WO, created on 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.

[0005] background

[0006] field

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

[0008] Description of the Prior Art

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

[0010] Overview

[0011] The disclosure herein includes methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: amplifying a target nucleic acid sequence in an amplification reaction mixture under isothermal amplification conditions, thereby producing a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing with the nucleic acid amplification product and comprises one or more polymerase terminators (stopper). The method may include: contacting a sample comprising a biological entity with a lysis buffer to produce a treated sample, wherein the lysis buffer comprises one or more cleavage agents capable of cleaving the biological entity to release the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence; the method may include: contacting a reagent composition with a treated sample to produce an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0012] The disclosure herein includes signal generation oligonucleotide. In some embodiments, the signal generation oligonucleotide can hybridize with the nucleic acid amplification product. In some embodiments, the signal generation oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal generation oligonucleotide comprises a loop domain between the 5' subdomain and the 3' subdomain. In some embodiments, the 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 terminators. In some embodiments, the 5' subdomain, the paired stem domain and / or the 3' subdomain do not comprise one or more polymerase terminators. In some embodiments, the nucleic acid amplification product is produced by amplifying a target nucleic acid sequence comprising a first chain and a second chain complementary to each other. In some embodiments, the nucleic acid amplification product is produced by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0013] The signal generating oligonucleotide may comprise a 5' subdomain and a 3' subdomain. In some embodiments, the signal generating oligonucleotide comprises a loop domain between the 5' subdomain and the 3' subdomain. In some embodiments, the 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 terminators are located in 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 terminators. In some embodiments, the nucleic acid amplification product comprises: (1) the sequence of the forward primer and its reverse complement, (2) the sequence of the reverse primer and its reverse complement, and (3) a spacer sequence, which is 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 long. In some embodiments, the signal generating oligonucleotide comprises a first district, and the first district comprises the sequence of at least a portion of the reverse primer. In some embodiments, the signal generating oligonucleotide comprises a second district, and the second district comprises a sequence complementary to at least a portion of the forward primer. In some embodiments, the signal generating oligonucleotide comprises a spacer, and the spacer comprises a sequence of at least a portion of the spacer sequence. One or more of the first district, the second district and / or the spacer may comprise one or more polymerase terminators. The first district may comprise a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. The second district may comprise a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. In some embodiments, the spacer comprises a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer.

[0014] The length of the signal generating oligonucleotide can be from about 10 nucleotides to about 100 nucleotides. In some embodiments, the length of the forward primer and / or the reverse primer is from about 5 nucleotides to about 25 nucleotides; in some embodiments, the length of the second district, the spacer and / or the first district is from about 1 nucleotide to about 25 nucleotides. In some embodiments, the length of the 5' subdomain, the 3' subdomain, the ring domain, the 5' terminal domain and / or the 3' terminal domain is from about 1 nucleotide to about 25 nucleotides. In some embodiments, the signal generating oligonucleotide comprises a 5' terminal domain located at 5' of the 5' subdomain. In some embodiments, the signal generating oligonucleotide comprises a 3' terminal domain located at 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 terminators.

[0015] In some embodiments, the first region comprises at least a portion of the 5' subdomain and / or the ring domain. In some embodiments, the spacer comprises at least a portion of the ring domain. In some embodiments, the second region comprises at least a portion of the ring 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. In some embodiments, the ring domain comprises at least a portion of the spacer, 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.

[0016] One or more polymerase terminators may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. In some embodiments, one or more polymerase terminators include one or more of abasic sites, stable abasic sites, chemically captured abasic sites, or any combination thereof. In some embodiments, stable abasic sites include 1', 2'-dideoxy. In some embodiments, chemically captured abasic sites include abasic sites reacted with alkoxyamines or sodium borohydride. In some embodiments, abasic sites include apurinic sites, apyrimidinic sites, or both. In some embodiments, abasic sites are produced by alkylating agents or oxidizing agents. In some embodiments, the one or more polymerase terminators include: one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromodeoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more isodeoxycytidine (Iso-dC), one or more isodeoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(o)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (spacer 9, iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups, or any combination thereof.

[0017] In some embodiments, the signal generation oligonucleotide comprises one or more phosphorothioate bonds and / or one or more locked nucleic acids. In some embodiments, the signal generation oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon (molecular beacon) detection probe oligonucleotide or a molecular torch (molecular torch) detection probe oligonucleotide. In some embodiments, the signal generation oligonucleotide comprises a label, such as a quenchable label (such as a fluorophore). In some embodiments, the signal generation oligonucleotide comprises a quencher. In some embodiments, the label is located in the 3' terminal domain and the quencher is located in the 5' terminal domain, and / or the label is located in the 5' terminal domain and the quencher is located in the 3' terminal domain.

[0018] In some embodiments, when the forward primer binds to the signal generating oligonucleotide to form a first undesired duplex, one or more polymerase terminators are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal generating oligonucleotide, and in some embodiments, one or more polymerase terminators are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide. In some embodiments, when the reverse primer binds to the signal generating oligonucleotide to form a second undesired duplex, one or more polymerase terminators are capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal generating oligonucleotide, and in some embodiments, one or more polymerase terminators are capable of terminating polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide. In some embodiments, when the exogenous nucleic acid binds to the signal generating oligonucleotide to form a third undesirable duplex, one or more polymerase terminators are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesirable duplex to the 5' end of the signal generating oligonucleotide, and in some embodiments, one or more polymerase terminators are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesirable duplex beyond one or more polymerase terminators of the signal generating oligonucleotide. In some embodiments, the exogenous nucleic acid is selected from the group consisting of: a sample nucleic acid, a primer configured to hybridize with a second target nucleic acid sequence, a primer configured to hybridize with an internal control, or any combination thereof.

[0019] In some embodiments, the sample nucleic acid includes a nucleic acid comprising a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence includes: amplifying a target nucleic acid sequence comprising a first chain and a second chain complementary to each other under isothermal amplification conditions, wherein the amplification includes contacting the nucleic acid comprising the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer can hybridize with the sequence of the first chain of the target nucleic acid sequence, and the reverse primer can hybridize with the sequence of the second chain of the target nucleic acid sequence; and ii) an enzyme with hyperthermophilic biopolymerase activity, thereby producing a nucleic acid amplification product. In some embodiments, the nucleic acid is a double-stranded DNA. In some embodiments, the nucleic acid is the product of a reverse transcription reaction. In some embodiments, the nucleic acid is the product of a reverse transcription reaction produced from a sample ribonucleic acid. In some embodiments, amplification includes producing nucleic acid by a reverse transcription reaction. In some embodiments, the sample nucleic acid includes a sample ribonucleic acid, and wherein the method includes contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA. In some embodiments, amplifying the target nucleic acid sequence includes: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having a hyperthermophilic biopolymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises the target nucleic acid sequence, and wherein the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplification includes 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 a hyperthermophilic biopolymerase activity, thereby producing a nucleic acid amplification product.

[0020] In some embodiments, if the forward primer binds to the signal generating oligonucleotide to form a first undesired duplex, the forward primer of the first undesired duplex is extended to the 5' end of the signal generating oligonucleotide by an enzyme having a hyperthermophilic polymerase activity to generate a first undesired extension product, and in some embodiments, the first undesired extension product can be amplified by an enzyme having a hyperthermophilic polymerase activity in the presence of a forward primer and a reverse primer to form a first undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesired duplex to generate a first stalled extension product. In some embodiments, the first stalled extension product cannot be amplified by an enzyme having a hyperthermophilic polymerase activity in the presence of a forward primer and a reverse primer to generate a first undesired amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide. In some embodiments, if the reverse primer is combined with the signal generating oligonucleotide to form a second undesirable duplex, the reverse primer of the second undesirable duplex generates a second undesirable extension product by extension of the enzyme with hyperthermophilic polymerase activity to the 5' end of the signal generating oligonucleotide. In some embodiments, the second undesirable extension product can be amplified by the enzyme with hyperthermophilic polymerase activity in the presence of the reverse primer to form a second undesirable amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesirable duplex to produce a second stagnant extension product. In some embodiments, the second stagnant extension product cannot be amplified by the enzyme with hyperthermophilic polymerase activity in the presence of the reverse primer to produce a second undesirable amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesirable duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide. In some embodiments, if the exogenous nucleic acid is combined with the signal generating oligonucleotide to form a third undesirable duplex, the exogenous nucleic acid of the third undesirable duplex is extended to the 5' end of the signal generating oligonucleotide by an enzyme having a hyperthermophilic polymerase activity to generate a third undesirable extension product. In some embodiments, the third undesirable extension product can be amplified by an enzyme having a hyperthermophilic polymerase activity in the presence of a reverse primer to form a third undesirable amplification product. In some embodiments, one or more polymerase terminators can terminate the polymerase extension of the exogenous nucleic acid of the third undesirable duplex to produce a third stalled extension product. In some embodiments, the third stalled extension product cannot be amplified by an enzyme having a hyperthermophilic polymerase activity in the presence of a reverse primer to produce a third undesirable amplification product.In some embodiments, 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.

[0021] In some embodiments, the detection step includes contacting the nucleic acid amplification product with a signal generating oligonucleotide for hybridization. In some embodiments, detecting the nucleic acid amplification product includes using a real-time detection method. In some embodiments, the label can generate a signal when the signal generating oligonucleotide hybridizes with the nucleic acid amplification product. In some embodiments, when the signal generating oligonucleotide hybridizes with the nucleic acid amplification product, the label generates a signal (e.g., fluorescence). In some embodiments, the detection step includes 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 includes detecting the signal generated by the label of the signal generating oligonucleotide. In some embodiments, the label is a fluorophore and the signal is fluorescence. In some embodiments, detecting the signal includes detecting the fluorescence emitted by the label. In some embodiments, the method includes determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample. In some embodiments, determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample includes determining the presence, absence, and / or amount of the dsDNA and / or nucleic acid containing the target nucleic acid sequence in the sample. The presence, absence, and / or amount of the signal can 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 a signal indicates the presence, absence, and / or amount of dsDNA and / or nucleic acids comprising the target nucleic acid sequence in the sample.

[0022] In some embodiments, mark can produce false positive signal when signal produces oligonucleotide and the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product hybridization.In some embodiments, when signal produces oligonucleotide and the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product hybridization, mark produces false positive signal.In some embodiments, signal and false positive signal are indistinguishable.In some embodiments, the generation of the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product reduces the possibility of accurately determining the existence, absence and / or amount of target nucleic acid sequence in sample.In some embodiments, the detection of false positive signal reduces the possibility of accurately determining the existence, absence and / or amount of target nucleic acid sequence in sample.

[0023] Compared to signal generating oligonucleotides that do not contain one or more polymerase terminators, the presence of one or more polymerase terminators in the signal generating oligonucleotide can increase the probability of accurately determining the presence, absence and / or amount of the target nucleic acid sequence in the sample to at least about 1.1 times. In some embodiments, the generation of the first stagnant extension product, the second stagnant extension product and / or the third stagnant extension product does not generate a false positive signal. In some embodiments, the signal generating oligonucleotide hybridized with the first stagnant extension product, the second stagnant extension product and / or the third stagnant extension product does not generate a false positive signal. In some embodiments, the nucleic acid amplification product reaches a detectable level before or at least about 1, 2, 5, 10, 15 or 20 minutes before the first undesirable amplification product, the second undesirable amplification product and / or the third undesirable amplification product reaches a detectable level. In some embodiments, the signal reaches a detectable level before or at least about 1, 2, 5, 10, 15 or 20 minutes before the false positive signal reaches a detectable level. In some embodiments, compared with the appropriate method in which the signal generation oligonucleotide does not include one or more polymerase terminators, the occurrence of the false positive signal, the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product of the detectable level is delayed or delayed at least about 1, 2, 5, 10, 15 or 20 minutes. In some embodiments, the false positive signal, the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product continue or continue for at least about 5, 10, 15 or 20 minutes after the amplification step begins and do not reach the detectable level. In some embodiments, wherein, compared with the appropriate method in which the signal generation oligonucleotide does not include one or more polymerase terminators, the generation of the false positive signal, the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product is reduced by at least about 1.1 times.

[0024] In some embodiments, amplifying the target nucleic acid sequence comprises producing a nucleic acid amplification product at a detectable level within or about within 20, 15, or 10 minutes. In some embodiments, the detection is carried out within less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the treated sample.

[0025] 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 cell RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, one or more amplification reagents include: reverse transcriptase; an enzyme with hyperthermophilic biopolymerase activity; and / or dNTP. In some embodiments, the enzyme with hyperthermophilic biopolymerase activity has reverse transcriptase activity: forward primer; reverse primer; reverse transcription primer.

[0026] The reagent composition can be freeze-dried, heat-dried and / or include one or more additives. In some embodiments, one or more additives include: Tween 20, Triton X-100 and / or tween 80; amino acids; sugars or sugar alcohols; and / or polymers. Sugars or sugar alcohols can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof. In some embodiments, polymers include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides or any combination thereof. In some embodiments, contacting the reagent composition with the treated sample includes dissolving the reagent composition in the treated sample. In some embodiments, the one or more lysis reagents include: about 0.001% (w / v) to about 1.0 (w / v) of the treated sample (e.g., about 0.2% (w / v) of the treated sample); and / or 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 the use of any enzyme other than a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity; does not include the use of any enzyme other than an enzyme having hyperthermophilic polymerase activity; does not include thermal and / or enzymatic denaturation of the nucleic acid during the amplification step; and / or does not include contacting the nucleic acid with a single-stranded DNA binding protein.

[0027] The target nucleic acid sequence may include a length of no longer than about 20 nucleotides to no longer than about 90 nucleotides (e.g., about 30 nucleotides). In some embodiments, the forward primer, reverse primer and / or reverse transcription primer are about 8 to 16 bases long. In some embodiments, the nucleic acid amplification product is about 20 to 40 bases long. In some embodiments, the spacer sequence includes a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1 to 10 bases long. In some embodiments, isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, such as about 55°C to about 75°C or about 56°C to about 67°C. In some embodiments, amplification is performed: a time period of about 5 minutes to about 60 minutes (e.g., a time period of about 15 minutes). In some embodiments, amplification is performed under isothermal amplification conditions without helicase, single-stranded binding protein, cleavage agent, and recombinase. In some embodiments, amplification is performed 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, immune amplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). In some embodiments, PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0028] In some embodiments, the enzyme having hyperthermophilic polymerase activity has an amino acid sequence or a functional fragment thereof that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the enzyme having hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the enzyme having hyperthermophilic polymerase activity has low exonuclease activity or no exonuclease activity. In some embodiments, the sample ribonucleic acid is contacted with a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity at the same time. In some embodiments, the sample ribonucleic acid is contacted with a reverse transcriptase, an enzyme having hyperthermophilic polymerase activity, and a forward primer and a reverse primer at the same time. In some embodiments, the sample ribonucleic acid is contacted with a reverse transcriptase, an enzyme having hyperthermophilic polymerase activity, a forward primer, a reverse primer, and a reverse transcription primer at the same time.

[0029] Biological entities can include one or more of prokaryotic cells, eukaryotic cells, viral particles, exosomes, protoplasts and microvesicles. In some embodiments, biological entities include viruses, bacteria, fungi, protozoa, parts thereof or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, bacteria, fungi or protozoa. In some embodiments, the sample nucleic acid is derived from a virus, bacteria, fungi or protozoa. The virus can be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV) or hantavirus. In some embodiments, the bacteria include Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes.In some embodiments, fungi include Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum. In some embodiments, protozoa include Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., or Eimeria sp. One or more. The sample may be a biological sample or an environmental sample. In some embodiments, the environmental sample is, or is obtained from, a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is, or is obtained from, a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof.

[0030] The amplification step may comprise multiplex amplification of two or more target nucleic acid sequences, and wherein the detection step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally wherein the two or more target nucleic acid sequences are specific for two or more different organisms, and optionally wherein the two or more different organisms comprise one or more of SARS-CoV-2, influenza A, influenza B and / or influenza C. In some embodiments, amplification includes and / or does not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (Ramification, RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA). In some embodiments, amplification does not include LAMP. In some embodiments, the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) addition of a ribonuclease inhibitor to the treated sample; (vii) addition of a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal and / or enzymatic denaturation of the sample nucleic acid prior to and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0031] The disclosure herein includes a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises: a signal generating oligonucleotide disclosed herein. The kit may comprise: a lysis buffer, comprising one or more lysis agents capable of lysing a biological entity to release a sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of containing a target nucleic acid sequence, optionally one or more lysis agents comprising a detergent, and wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit may include: a reagent composition, comprising one or more amplification reagents, the amplification reagents comprising one or more components for amplification for amplifying a target nucleic acid sequence under isothermal amplification conditions, wherein the one or more components for amplification include: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing with a sequence of a first strand of the target nucleic acid sequence, and the reverse primer is capable of hybridizing with a sequence of a second strand of the target nucleic acid sequence; and / or (ii) an enzyme having a hyperthermophilic biopolymerase activity capable of producing a nucleic acid amplification product, optionally the enzyme having a hyperthermophilic biopolymerase 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 having a hyperthermophilic biopolymerase activity has an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO: 7, and optionally the enzyme having a hyperthermophilic biopolymerase activity 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.

[0032] The disclosure herein includes methods for detecting Neisseria gonorrhea (N.Gonorrhea) in a sample. In some embodiments, the method includes: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of Neisseria gonorrhea, wherein each primer in the at least one pair of primers comprises a sequence of any one of SEQ ID NO: 2-3 and 16-17, or a sequence that exhibits at least about 85% identity with any one of the sequences SEQ ID NO: 2-3 and 16-17. The method may include: if the sample contains Neisseria gonorrhea, an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence is generated. The method may include: determining the presence or amount of the amplicon as an indication of the presence of Neisseria gonorrhea in the sample.

[0033] In some embodiments, at least one pair of primers includes a first primer comprising a sequence of SEQ ID NO: 2 or 17 and a second primer comprising a sequence of SEQ ID NO: 3 or 16. In some embodiments, at least one pair of primers 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 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 NO: 1 and 12-14, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NO: 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 NO: 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 NO: 1 and 12-14.

[0034] The disclosure herein includes methods for detecting Chlamydia trachomatis (C. trachomatis) in a sample. In some embodiments, the method includes: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 20-23, 25-26, 28-29 and 31, or a sequence that exhibits at least about 85% identity to any one of the sequences SEQ ID NO: 20-23, 25-26, 28-29 and 31. The method may include: if the sample contains Chlamydia trachomatis, an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence is generated. The method may include: determining the presence or amount of the amplicon as an indication of the presence of Chlamydia trachomatis in the sample.

[0035] In some embodiments, at least one pair of primers includes 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 pair of primers 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 23, SEQ ID NOs: 22 and 26, SEQ ID NOs: 22 and 29, SEQ ID NOs: 25 and 23, SEQ ID NOs: 25 and 29, SEQ ID NOs: 25 and 31, SEQ ID NOs: 28 and 23, SEQ ID NOs: 28 and 26, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 28 and 26, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 28 and 29, or SEQ ID NOs: 28 and 26. 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 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. 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] The disclosure herein includes methods for detecting influenza B virus in a sample. In some embodiments, the method includes: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one pair of primers comprises a sequence of any one of SEQ ID NOs: 36-37 and 41, or a sequence that exhibits at least about 85% identity to any one of SEQ ID NOs: 36-37 and 41. The method may include: if the sample contains influenza B virus, then generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. 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.

[0037] In some embodiments, at least one pair of primers includes a first primer comprising a sequence of SEQ ID NO: 36 and a second primer comprising a sequence of SEQ ID NO: 37 or 41. In some embodiments, at least one pair of primers 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. In some embodiments, determining the presence or amount of an amplicon of a target nucleic acid sequence includes 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 NO: 39-40 and 42-45, or a sequence exhibiting at least about 85% identity with a sequence selected from the group consisting of SEQ ID NO: 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 NO: 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 NO: 39-40 and 42-45. The method may include contacting the 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.

[0038] In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the environmental sample is the following, or is obtained from: a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is the following, or is obtained from: a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof. In some embodiments, the sample is contacted with a reagent composition comprising at least one pair of primers to produce an amplification reaction mixture. The method may include: contacting a sample containing a biological entity with a lysis buffer to produce a treated sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entity to release sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence; and contacting a reagent composition comprising at least one pair of primers with the treated sample to produce an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents. In some embodiments, the one or more amplification reagents include: a reverse transcriptase; an enzyme having hyperthermophilic biopolymerase activity, optionally the enzyme having hyperthermophilic biopolymerase activity has reverse transcriptase activity; a reverse transcription primer; and / or dNTPs.

[0039] In some embodiments, generating an amplicon of a 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, amplification is performed 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, immune amplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA), optionally wherein the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR). In some embodiments, amplification does not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA), optionally amplification does not include LAMP. In some embodiments, amplification includes one or more of the following: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR and IMDA, optionally amplification does not include LAMP.

[0040] In some embodiments, the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) addition of a ribonuclease inhibitor to the treated sample; (vii) addition of a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal and / or enzymatic denaturation of the sample nucleic acid prior to and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0041] The determining step can include contacting the amplicon of the target nucleic acid sequence with a signal generating oligonucleotide to hybridize, optionally, determining includes using a real-time detection method. In some embodiments, the labeling of the signal generating oligonucleotide can generate a signal when the signal generates an oligonucleotide and the amplicon of the target nucleic acid sequence hybridizes. In some embodiments, when the signal generates an oligonucleotide and the amplicon of the target nucleic acid sequence hybridizes, the labeling generates a signal, and optionally the signal is fluorescence.

[0042] The disclosure herein includes compositions for detecting Neisseria gonorrhoeae in a sample. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, wherein each primer in the at least one pair of primers comprises any one of the sequences SEQ ID NOs: 2-3 and 16-17 or a sequence exhibiting at least about 85% identity to any one of the sequences SEQ ID NOs: 2-3 and 16-17. In some embodiments, at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae comprises a primer comprising the sequence SEQ ID NOs: 2 or 17 and a primer comprising the sequence SEQ ID NOs: 3 or 16. 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: 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.

[0043] The disclosure herein includes compositions for detecting Chlamydia trachomatis in a sample. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises any one of the sequences SEQ ID NO: 20-23, 25-26, 28-29 and 31 or a sequence exhibiting at least about 85% identity to any one of the sequences SEQ ID NO: 20-23, 25-26, 28-29 and 31. In some embodiments, at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis comprises a primer comprising the sequence SEQ ID NO: 20, 22, 25 or 28 and a primer comprising the sequence 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% 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.

[0044] The disclosure herein includes compositions for detecting influenza B virus in a sample. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in at least one pair of primers comprises any one of the sequences SEQ ID NO:36-37 and 41 or a sequence exhibiting at least about 85% identity to any one of the sequences SEQ ID NO:36-37 and 41. In some embodiments, at least one pair of primers capable of hybridizing to a target nucleic acid sequence of influenza B virus comprises a primer comprising the sequence SEQ ID NO:36 and a primer comprising the sequence 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, wherein each of the one or more signal generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NO: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 NO: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.

[0045] In some embodiments, the signal generation oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal generation oligonucleotide comprises a loop domain between the 5' subdomain and the 3' subdomain. In some embodiments, the 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 terminators. In some embodiments, the 5' subdomain, the paired stem domain and / or the 3' subdomain do not comprise one or more polymerase terminators. In some embodiments, the signal generation oligonucleotide comprises a 5' terminal domain located at 5' subdomain 5'. In some embodiments, the signal generation oligonucleotide comprises a 3' terminal domain located at 5' subdomain 3'. In some embodiments, the 5' terminal domain and / or the 3' terminal domain do not comprise one or more polymerase terminators.

[0046] One or more polymerase terminators may comprise one or more 2'-O-methyl (2'OM) RNA nucleotides. In some embodiments, one or more polymerase terminators include one or more of abasic sites, stable abasic sites, chemically captured abasic sites, or any combination thereof. In some embodiments, stable abasic sites include 1', 2'-dideoxy. In some embodiments, chemically captured abasic sites include abasic sites reacted with alkoxyamines or sodium borohydride. In some embodiments, abasic sites include apurinic sites, apyrimidinic sites, or both. In some embodiments, abasic sites are produced by alkylating agents or oxidizing agents. In some embodiments, the one or more polymerase terminators include: one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromodeoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases. group, one or more isodeoxycytidine (Iso-dC), one or more isodeoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(o)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups, or any combination thereof.

[0047] In some embodiments, the signal generating oligonucleotide comprises one or more phosphorothioate bonds and / or one or more locked nucleic acids. In some embodiments, the signal generating oligonucleotide is a TaqMan detection probe oligonucleotide, a molecular beacon detection probe oligonucleotide, or a molecular torch detection probe oligonucleotide. In some embodiments, the signal generating oligonucleotide comprises a label, such as a quenchable label (e.g., a fluorophore). In some embodiments, the signal generating oligonucleotide comprises a quencher. In some embodiments, the label is located in the 3' terminal domain and the quencher is located in the 5' terminal domain, and / or the label is located in the 5' terminal domain and the quencher is located in the 3' terminal domain.

[0048] In some embodiments, a signal generating oligonucleotide or primer is provided, up to about 100 nucleotides in length, capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, wherein 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, or a sequence exhibiting at least about 85% identity with 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 exhibiting at least about 85% identity with 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 comprises 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.

[0049] In some embodiments, a signal generating oligonucleotide or primer is provided, up to about 100 nucleotides in length, capable of hybridizing with a target nucleic acid sequence of Chlamydia trachomatis, wherein the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-15 and 18-32, or a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-15 and 18-32 that exhibits at least about 85% identity. In some embodiments, the signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NO: 15 and 18-32, or a sequence selected from the group consisting of SEQ ID NO: 15 and 18-32 that exhibits at least about 85% identity. In some embodiments, the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 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 NO: 15 and 18-32.

[0050] In some embodiments, a signal generating oligonucleotide or primer is provided, up to about 100 nucleotides in length, capable of hybridizing with a target nucleic acid sequence of influenza B virus, wherein the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 36-45, or a sequence selected from the group consisting of SEQ ID NO: 36-45 that exhibits at least about 85% identity. In some embodiments, the signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NO: 36-45, or a sequence selected from the group consisting of SEQ ID NO: 36-45 that exhibits at least about 85% identity. In some embodiments, the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 36-45. In some embodiments, the signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NO: 36-45.

[0051] In some embodiments, a signal generating oligonucleotide or primer is provided, up to about 100 nucleotides in length, capable of hybridizing with a target nucleic acid sequence of influenza A virus, wherein the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 33-35, or a sequence exhibiting at least about 85% identity with a sequence selected from the group consisting of SEQ ID NO: 33-35. In some embodiments, the signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NO: 33-35, or a sequence exhibiting at least about 85% identity with a sequence selected from the group consisting of SEQ ID NO: 33-35. In some embodiments, the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 33-35. In some embodiments, the signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NO: 33-35.

[0052] In some embodiments, a composition comprising two or more signal generating oligonucleotides and / or primers provided herein is provided. The composition may include: a lysis buffer comprising one or more lysing agents capable of lysing a biological entity to release a sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of containing a target nucleic acid sequence, optionally, one or more lysing agents comprising a detergent, and wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and a zwitterionic surfactant; and / or a reagent composition comprising one or more amplification reagents comprising one or more components for amplification of a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the one or more components for amplification include: an enzyme with hyperthermophilic polymerase activity that is capable of producing a nucleic acid amplification product, optionally the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:7 or a functional fragment thereof, optionally the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:7, and optionally, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO:7. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1A-1B A non-limiting exemplary schematic diagram of an isothermal amplification reaction provided herein is shown.

[0055] Figure 2A-2C Non-limiting exemplary primers and probes (molecular beacons) for an archaeal polymerase amplification (APA)-based assay for Neisseria Gonorrhea are depicted. Figure 2A The positions of primers and molecular beacons relative to the template are depicted. Figure 2B Depicts the unbound hairpin formation Figure 2A of molecular beacons. Figure 2C A non-limiting exemplary protected molecular beacon is depicted in an unbound hairpin formation (where "M" indicates a polymerase terminator, such as a 2' OM-modified base).

[0056] Figure 3 Describes the ability to accompany Figure 2A-2B Non-limiting exemplary non-specific product formation produced by the APA-based Neisseria gonorrhoeae assay depicted in FIG.

[0057] Figure 4 Depicts the use of protected probes (e.g. Figure 2C The protected probe shown in Figure 2A-2BNon-limiting exemplary prevention of non-specific product formation in an APA-based Neisseria gonorrhoeae assay is depicted in FIG.

[0058] Figure 5A-5E Data showing nonspecific product formation (without 2'OM protection) in the FluB PB2 assay are depicted. The FluB PB2 reverse primer was titrated to 600 nM ( Figure 5A )、500nM( Figure 5B )、400nM( Figure 5C )、300nM( Figure 5D ) and 200nM( Figure 5E ).

[0059] Figure 6A-6D Depicted are data related to the prevention of nonspecific product formation in the FluB PB2 assay by 2'OM-modified beacons. Depicted are data related to the prevention of nonspecific product formation in the FluB PB2 assay by 2'OM-modified beacons. Fig. 6A ; LNA3.13), 2'OM modified form 1 ( Figure 6B ; LNA3.13m1), 2'Om modified form 2 ( Figure 6C ; LNA3.13m2) and 2'OM modified version 3 ( Fig.6D ; LNA3.13m3) produced results.

[0060] Figure 7A-7D Depicted is the use of 8U 9dN polymerase ( Fig. 7A , Figure 7C ) and 12U 9dN polymerase ( Figure 7B , Fig.7D ) with NTC 10% urine sample ( Figure 7A-7B ) and NTC vaginal swab samples ( Figure 7C-D ) for the evaluation of false positives in assays for Neisseria gonorrhoeae and Chlamydia trachomatis.

[0061] Figure 8A-8D Plotted with the nominal probe ( Fig. 8A , Figure 8C ) and protected probes ( Figure 8B , Fig.8D ) Chlamydia trachomatis assay using NTC 15% urine samples ( Figure 8A-8B ) and Neisseria gonorrhoeae assay ( Figure 8C-Figure 8D ) in the data related to false positive assessment. Figure 8B Contains 1 erroneous amplification associated with a suspected Chlamydia trachomatis contamination incident.

[0062] Figure 9A-9D Plotted with the nominal probe ( Fig.9A , Fig. 9C ) and protected probes ( Fig. 9B , Fig.9D ) Chlamydia trachomatis assay using vaginal swab samples ( Figure 9A-9B ) and Neisseria gonorrhoeae assay ( Figure 9C-D ) in the data related to false positive assessment.

[0063] Figures 10A-10D Depicted is a Chlamydia trachomatis assay performed with NTC urine samples and using protected probes ( Figure 10A-10B ) and Neisseria gonorrhoeae assay ( Figure 10C-10D ) in the data related to false positive assessment. Fig. 10D Contains 1 erroneous amplification associated with a suspected N. gonorrhoeae contamination event.

[0064] Figure 11A-11B Depicts the Chlamydia trachomatis assay performed with NTC vaginal swab samples and using protected probes ( Fig.11A ) and Neisseria gonorrhoeae assay ( Fig. 11B ) in the data related to false positive assessment.

[0065] Fig.12 Depicted are non-limiting exemplary protected molecular beacons in an unbound hairpin formation for use in a Chlamydia trachomatis assay (where "M" indicates a polymerase terminator, such as a 2' OM-modified base).

[0066] Figure 13A-13C Depicted are data related to RNA base incorporation used to protect molecular beacons in a FluA assay. The FluA assay was performed using NTC primer-only titers ( Figure 13A-13B ) or probes are used to screen targets ( Fig. 13C ). DETAILED DESCRIPTION

[0067] Reference is made to the accompanying drawings forming a part of this document in the following detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context otherwise indicates. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the accompanying drawings can be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form a part of the present disclosure.

[0068] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

[0069] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of the present disclosure, the following terms are defined below.

[0070] The disclosure herein includes methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: amplifying a target nucleic acid sequence in an amplification reaction mixture under isothermal amplification conditions, thereby producing a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal-generating oligonucleotide, wherein the signal-generating oligonucleotide is capable of hybridizing with the nucleic acid amplification product and comprises one or more polymerase terminators. The method may include: contacting a sample comprising a biological entity with a lysis buffer to produce a treated sample, wherein the lysis buffer comprises one or more lysis agents capable of cleaving the biological entity to release the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence; the method may include: contacting a reagent composition with a treated sample to produce an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0071] The disclosure herein includes signal generation oligonucleotides, such as signal generation oligonucleotides that can hybridize with nucleic acid amplification products. In some embodiments, the signal generation oligonucleotide comprises a 5' subdomain and a 3' subdomain, and optionally a ring domain between the 5' subdomain and the 3' subdomain. In some embodiments, the intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the ring domain comprises one or more polymerase terminators. In some embodiments, the 5' subdomain, the paired stem domain and / or the 3' subdomain do not comprise one or more polymerase terminators. In some embodiments, the nucleic acid amplification product is produced by amplifying a target nucleic acid sequence comprising a first chain and a second chain that are complementary to each other. In some embodiments, the nucleic acid amplification product is produced by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0072] The disclosure herein includes a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit comprises: a signal generating oligonucleotide disclosed herein. The kit may comprise: a lysis buffer comprising one or more lysis agents capable of lysing a biological entity to release sample nucleic acids contained therein, wherein the sample nucleic acid is suspected of containing a target nucleic acid sequence, and optionally one or more lysis agents comprising a detergent. The kit may comprise: a reagent composition comprising one or more amplification reagents, the amplification reagents comprising one or more components for amplification for amplifying a target nucleic acid sequence under isothermal amplification conditions.

[0073] The disclosure herein includes methods for detecting Neisseria gonorrhoeae in a sample. In some embodiments, the method includes contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of Neisseria gonorrhoeae, wherein each primer in the at least one pair of primers comprises a sequence of any one of SEQ ID NOs: 2-3 and 16-17, or a sequence that exhibits at least about 85% identity to any one of SEQ ID NOs: 2-3 and 16-17. The method may include generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence if the sample contains Neisseria gonorrhoeae. The method may include determining the presence or amount of the amplicon as an indication of the presence of Neisseria gonorrhoeae in the sample.

[0074] The disclosure herein includes methods for detecting Chlamydia trachomatis in a sample. In some embodiments, the method includes contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises a sequence of any one 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 SEQ ID NOs: 20-23, 25-26, 28-29, and 31. The method may include generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence if the sample contains Chlamydia trachomatis. The method may include determining the presence or amount of the amplicon as an indication of the presence of Chlamydia trachomatis in the sample.

[0075] The disclosure herein includes methods for detecting influenza B virus in a sample. In some embodiments, the method includes: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one pair of primers comprises a sequence of any one of SEQ ID NOs: 36-37 and 41, or a sequence that exhibits at least about 85% identity to any one of SEQ ID NOs: 36-37 and 41. The method may include: if the sample contains influenza B virus, then generating an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence. 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.

[0076] The disclosure herein includes compositions for detecting Neisseria gonorrhoeae in a sample. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, wherein each primer in the at least one pair of primers comprises a sequence of any one of SEQ ID NOs: 2-3 and 16-17 or a sequence exhibiting at least about 85% identity to any one of SEQ ID NOs: 2-3 and 16-17.

[0077] The disclosure herein includes compositions for detecting Chlamydia trachomatis in a sample. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises any one of the sequences 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 SEQ ID NOs: 20-23, 25-26, 28-29 and 31.

[0078] The disclosure herein includes compositions for detecting influenza B virus in a sample. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one pair of primers comprises any one of the sequences SEQ ID NOs: 36-37 and 41 or a sequence exhibiting at least about 85% identity to any one of the sequences SEQ ID NOs: 36-37 and 41.

[0079] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, wherein 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, 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.

[0080] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-15 and 18-32, or a sequence exhibiting at least about 85% identity with a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-15 and 18-32.

[0081] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein the signal generating oligonucleotide or primer comprises 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.

[0082] In some embodiments, a signal generating oligonucleotide or primer is provided, up to about 100 nucleotides in length, capable of hybridizing to a target nucleic acid sequence of influenza A virus, wherein the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 33-35, or a sequence exhibiting at least about 85% identity to a sequence selected from the group consisting of SEQ ID NO: 33-35. In some embodiments, a composition comprising two or more signal generating oligonucleotides and / or primers provided herein is provided.

[0083] Methods and compositions for pathogen detection

[0084] As described herein, nucleic acid amplification and detection reactions can be performed to determine the presence, absence, type, and / or level of a pathogen in a sample, such as, for example, Neisseria gonorrhoeae, Chlamydia trachomatis, and / or influenza virus (e.g., influenza B virus). In some embodiments, the presence, absence, and / or level of one or more of Neisseria gonorrhoeae, Chlamydia trachomatis, and 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, multiple reactions can be performed to detect the presence, absence, or level of two or more of Neisseria gonorrhoeae, Chlamydia trachomatis, and influenza virus.

[0085] In some embodiments, a method for detecting Neisseria gonorrhoeae in a sample is provided. In some embodiments, the method includes: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of Neisseria gonorrhoeae, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 2-3 and any one of 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) with any one of the sequences SEQ ID NO: 2-3 and 16-17. The method may include: if the sample contains Neisseria gonorrhoeae, an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence is generated. The method may include: determining the presence or amount of the amplicon as an indication of the presence of Neisseria gonorrhoeae in the sample. The at least one pair of primers may 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 pair of primers 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. 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, wherein each of the one or more signal generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14, or 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) with a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14. Each of the one or more signal generating oligonucleotides can include or consist of the following: a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14.

[0086] In some embodiments, a method for detecting Chlamydia trachomatis in a sample is provided. In some embodiments, the method includes: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 20-23, 25-26, 28-29 and 31, 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) with any one of the sequences SEQ ID NO: 20-23, 25-26, 28-29 and 31. The method may include: if the sample contains Chlamydia trachomatis, an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence is generated. The method may include determining the presence or amount of the amplicon as an indication of the presence of Chlamydia trachomatis in the sample. The at least one pair of primers may include a first primer comprising the sequence SEQ ID NO: 20, 22, 25 or 28 and a second primer comprising the sequence SEQ ID NO: 21, 23, 26, 29 or 31. In some embodiments, at least one pair of primers 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 23, SEQ ID NOs: 22 and 26, SEQ ID NOs: 22 and 29, SEQ ID NOs: 25 and 23, SEQ ID NOs: 25 and 29, SEQ ID NOs: 25 and 31, SEQ ID NOs: 28 and 23, SEQ ID NOs: 28 and 26, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 28 and 26, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 28 and 29, or SEQ ID NOs: 28 and 26.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, wherein each of the one or more signal generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NO: 15, 18-19, 24, 27, 30 and 32, or a sequence selected from the group consisting of SEQ ID NO: 15, 18-19, 24, 27, 30 and 32 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). Each of the one or more signal generating oligonucleotides can include or consist of a sequence selected from the group consisting of SEQ ID NO: 15, 18-19, 24, 27, 30 and 32.

[0087] 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 pair of primers, wherein the at least one pair of primers is capable of hybridizing with a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 36-37 and any one of 41, 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) with any one of the sequences SEQ ID NO: 36-37 and 41. The method may include: if the sample contains influenza B virus, an amplicon (e.g., a nucleic acid amplification product) of the target nucleic acid sequence is generated. 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 pair of primers may include a first primer comprising the sequence SEQ ID NO: 36 and a second primer comprising the sequence SEQ ID NO: 37 or 41. In some embodiments, at least one pair of primers capable of hybridizing to a target nucleic acid sequence of influenza B virus is SEQ ID NOs: 36 and 37 or SEQ ID NOs: 36 and 41. Determining the presence or amount of an amplicon of a target nucleic acid sequence may include 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 (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 may comprise or consist of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45. The method may comprise contacting a sample ribonucleic acid of the sample with a reverse transcriptase and a primer having 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 SEQ ID NO: 38.

[0088] In some embodiments, the sample is contacted with a reagent composition comprising at least one pair of primers to produce an amplification reaction mixture. The method may include: contacting a sample containing a biological entity with a lysis buffer to produce a treated sample, wherein the lysis buffer comprises one or more lysis agents capable of lysing the biological entity to release the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of containing a target nucleic acid sequence; and contacting a reagent composition comprising at least one pair of primers with the treated sample to produce an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents. Producing an amplicon of a 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, and optionally, determining includes using a real-time detection method. The label of the signal generating oligonucleotide may be capable of generating a signal when the signal generating oligonucleotide hybridizes with the amplicon of the target nucleic acid sequence. In some embodiments, when the signal generating oligonucleotide hybridizes with the amplicon of the target nucleic acid sequence, the label generates a signal, and optionally the signal is fluorescence.

[0089] In some embodiments, a composition for detecting Neisseria gonorrhoeae in a sample is provided. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing with a target nucleic acid sequence of Neisseria gonorrhoeae, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 2-3 and any one of 16-17, 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) with any one of the sequences SEQ ID NO: 2-3 and 16-17. In some embodiments, at least one pair of primers capable of hybridizing with a target nucleic acid sequence of Neisseria gonorrhoeae comprises a primer comprising a sequence of SEQ ID NO: 2 or 17 and a primer comprising a sequence of SEQ ID NO: 3 or 16. The composition may include: 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 NO: 1 and 12-14, or a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14 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). Each of the one or more signal generating oligonucleotides may include or consist of a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14.

[0090] In some embodiments, a composition for detecting Chlamydia trachomatis in a sample is provided. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 20-23, 25-26, 28-29 and 31, 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) with any one of SEQ ID NO: 20-23, 25-26, 28-29 and 31. In some embodiments, at least one pair of primers capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis comprises a primer comprising a sequence of SEQ ID NO: 20, 22, 25 or 28 and a primer comprising a 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 NO: 15, 18-19, 24, 27, 30 and 32, 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 NO: 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.

[0091] In some embodiments, a composition for detecting influenza B virus in a sample is provided. In some embodiments, the composition comprises: at least one pair of primers capable of hybridizing with a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one pair of primers comprises a sequence of SEQ ID NO: 36-37 and any one of 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) with any one of the sequence SEQ ID NO: 36-37 and 41. In some embodiments, at least one pair of primers capable of hybridizing with a target nucleic acid sequence of influenza B virus comprises a primer comprising the sequence SEQ ID NO: 36 and a primer comprising the sequence 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). Compositions may include: 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 selected from the group consisting of SEQ ID NOs: 39-40 and 42-45 showing 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). Each of the one or more signal generating oligonucleotides may include or consist of a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

[0092] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of Neisseria gonorrhoeae, wherein 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, 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 is composed of a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-14 and 16-17, or a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-14 and 16-17 showing 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). The signal generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 1-3, 12-14 and 16-17.

[0093] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein the signal generating oligonucleotide or primer comprises a sequence selected from the group consisting of SEQ ID NO: 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 NO: 15 and 18-32. In some embodiments, the signal generating oligonucleotide or primer is composed of a sequence selected from the group consisting of SEQ ID NO: 15 and 18-32, or a sequence selected from the group consisting of SEQ ID NO: 15 and 18-32 showing 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). The signal generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 15 and 18-32.

[0094] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein the signal generating oligonucleotide or primer comprises 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 is composed of a sequence selected from the group consisting of SEQ ID NO: 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 NO: 36-45. The signal generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 36-45.

[0095] In some embodiments, a signal generating oligonucleotide or primer is provided, having a length of up to about 100 nucleotides, capable of hybridizing to a target nucleic acid sequence of influenza A virus, wherein the signal generating oligonucleotide or primer comprises 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 is composed of a sequence selected from the group consisting of SEQ ID NO: 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 NO: 33-35. The signal generating oligonucleotide or primer may comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 33-35.

[0096] In some embodiments, provide and comprise two or more signal generation oligonucleotides and / or primer compositions provided herein.Also provided herein is oligonucleotide (for example amplification primer or signal generation oligonucleotide) comprising 1,2,3,4 or more mismatches or universal nucleotides relative to SEQ ID NO:1-45 or its complement, including with SEQ ID NO:1-45 or its complement at least 80% identical (for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values ​​​​between numbers or scopes) oligonucleotides.

[0097] Protected probe

[0098] The disclosure herein includes methods and compositions comprising modified molecular beacons (e.g., "protected probes") that can improve assay specificity in some embodiments. The protected probes disclosed herein can be used in assays that employ archaeal polymerase amplification ("APA") to isothermally amplify regions of interest within a target DNA (or cDNA) template for the purpose of real-time analyte detection. In some embodiments, the protected probes provided herein comprise a polymerase terminator (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). Without being 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 have not yet used 2'OM bases specifically for this purpose. Spacer modifications (such as C3 spacers) can be used for this same effect, but C3 spacers have been shown to be incompatible with the molecular beacons employed in the APA assays described herein. Without being bound by any particular theory, in some embodiments, the compositions and methods provided herein utilize the ability of 9dN (DNA-dependent DNA polymerase) to read RNA templates. In particular, when encountering 2'OM bases (i.e., methylated RNA bases) in a given template, enzymatic continuous synthesis (e.g., the enzyme cannot successfully "read" the position in the DNA template) is theoretically prevented. 2'-O-methyl RNA can be present in small RNAs (e.g., tRNA), and is a post-transcriptional modification, which is a natural modification of RNA. Oligonucleotides comprising 2'-O-methyl RNA can be directly synthesized. This modification can increase the melting temperature of RNA: RNA duplexes, while also only causing a moderate change in RNA: DNA stability. Additionally, this modification can show stability for single-stranded ribonuclease attacks, and the sensitivity to DNA enzymes is generally 5 to 10 times lower than DNA. 2'OM modification can be used in antisense oligonucleotides for improving stability and binding affinity with targets. In addition to the 2'OM modification described herein, the following alternative base modifications were tested in the context of molecular beacons for APA: C3 spacer modification; abasic site modification; and unmethylated RNA bases. Based on the data generated, the 2'OM modification provided the greatest protection (without rendering the molecular beacon incompatible with APA).

[0099] Figure 2A-2C Depicted are non-limiting exemplary primers and probes (molecular beacons) for use in the rapid nucleic acid amplification and detection assays provided herein, including the Archaeal Polymerase Amplification (APA)-based Neisseria gonorrhoeae assay. Figure 2A The positions of primers and molecular beacons relative to the template are depicted. Figure 2B Depicts the unbound hairpin formation Figure 2AThe spacer is indicated by an arrow and corresponds to the template region located between the binding sites of the forward primer and the reverse primer. In some embodiments, one or more bases of the probes provided herein are LNA bases ( Figure 2B ). Figure 2C Depicted are protected molecular beacons in an unbound hairpin formation (where "M" indicates a polymerase terminator, such as a 2'OM-modified base). Figure 3 Describes the ability to accompany Figure 2A-2B Non-limiting exemplary non-specific product formation produced by an APA-based Neisseria gonorrhoeae assay depicted in (and not using the 2'OM protected probes described herein). Depicted are undesired, non-specific interactions between a reverse primer and a molecular beacon that can theoretically occur. This is a unique challenge for some embodiments of the APA assay design provided herein due to the expected overlap of primer / probe footprints. In this example, the assay employs a molecular beacon that completely overlaps the reverse primer. Polymerase extension of the reverse primer bound to the molecular beacon can produce a product that can be exponentially amplified via the reverse primer. Figure 4 Described is the use of protected probes comprising polymerase terminators as described herein (e.g., Figure 2C The protected probe shown in Figure 2A-2B A non-limiting exemplary prevention of non-specific product formation in an APA-based Neisseria gonorrhoeae assay depicted in . Depicted therein are unexpected, non-specific interactions that can occur between a reverse primer and a molecular beacon. By preventing read-through of a primer footprint embedded within a molecular beacon, the methods and compositions provided herein using protected probes can prevent the formation of unexpected extension products. 2'OM can be used as a control point to achieve this read-through prevention. Without being bound by any particular theory, the position of the modification determines the level of prevention of non-specific product formation - it is located at the very beginning of the primer overlap region to prevent read-through at the beginning (i.e., so that none of the overlapping bases are copied). In some embodiments, one or more polymerase terminators are located near or at the 3' end of the first region (immediately adjacent to the spacer) and are located within the loop domain.

[0100] Due to the expected overlap of primer / probe footprints (a signal generating oligonucleotide (e.g., a molecular beacon) may include a first region of a sequence comprising at least a portion of a reverse primer and / or a second region comprising a sequence complementary to at least a portion of a forward primer), undesired interactions between a signal generating oligonucleotide (e.g., a molecular beacon) and a forward primer and / or a reverse primer result in nonspecific product formation, which may be a unique and significant challenge for some embodiments of the amplification / detection assays provided herein. Additionally, due to the hairpin nature of the signal generating oligonucleotides provided herein, the repetition of the two 3' terminal nucleotides of the reverse primer in the stem loop may produce undesired reverse primer / probe interactions. Therefore, these inherent elements of some signal generating oligonucleotide-based amplification / detection assays provided herein may produce nonspecific product formation (and thereby produce false positive signals). However, the methods and compositions provided herein address these problems in the art and produce assays with reduced nonspecific product formation, reduced false positive signals, and / or increased likelihood of accurately determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample. The sequences of the Neisseria gonorrhoeae assay primers and probes described above are shown in Table 1 below. Additionally, Fig.12 Table 2 and Table 3 provide sequences of Chlamydia trachomatis assay primers and protected probes. Table 3 provides non-limiting exemplary FluA and FluB assay primers and probes. In some embodiments, the probes (e.g., molecular beacons) provided herein include 5' modifications (e.g., 5TEX615, FAM). In some embodiments, the probes (e.g., molecular beacons) provided herein include 3' modifications (e.g., 3IAbRQSp, IBFQ).

[0101] Table 1: Primers and probes for Neisseria gonorrhoeae ("NG") assay

[0102]

[0103] Table 2: Primers and probes for Chlamydia trachomatis ("CT") assay

[0104]

[0105]

[0106] Table 3: Flu assay primers and probes

[0107]

[0108] In some embodiments, a method for detecting a target nucleic acid sequence in a sample is provided. In some embodiments, the method includes: amplifying a target nucleic acid sequence in an amplification reaction mixture under isothermal amplification conditions, thereby producing a nucleic acid amplification product; and detecting the nucleic acid amplification product with a signal generating oligonucleotide, wherein the signal generating oligonucleotide is capable of hybridizing with the nucleic acid amplification product and comprises one or more polymerase terminators. The method may include: contacting a sample comprising a biological entity with a lysis buffer to produce a treated sample, wherein the lysis buffer comprises one or more cleaving agents capable of cleaving the biological entity to release the sample nucleic acid contained therein, and wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence. The method may include: contacting a reagent composition with a treated sample to produce an amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

[0109] In some embodiments, a signal generation oligonucleotide is provided. In some embodiments, the signal generation oligonucleotide can hybridize with a nucleic acid amplification product. In some embodiments, the signal generation oligonucleotide comprises a 5' subdomain and a 3' subdomain. In some embodiments, the signal generation oligonucleotide comprises a ring domain between the 5' subdomain and the 3' subdomain. In some embodiments, the intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain. In some embodiments, the ring domain comprises one or more polymerase terminators. In some embodiments, the 5' subdomain, the paired stem domain and / or the 3' subdomain do not comprise one or more polymerase terminators. In some embodiments, the nucleic acid amplification product is produced by amplifying a target nucleic acid sequence comprising a first chain and a second chain complementary to each other. In some embodiments, the nucleic acid amplification product is produced by amplifying the target nucleic acid sequence with a forward primer and a reverse primer.

[0110] The signal generating oligonucleotide may include a 5' subdomain and a 3' subdomain. The signal generating oligonucleotide may include a loop domain between the 5' subdomain and the 3' subdomain. The intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain may be able to form a paired stem domain. One or more polymerase terminators may be located in the loop domain. In some embodiments, the 5' subdomain, the paired stem domain and / or the 3' subdomain do not include one or more polymerase terminators.

[0111] The nucleic acid amplification product may 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 may be 1 to 10 bases in length.

[0112] The signal generating oligonucleotide may include a first region, wherein the first region includes a sequence of at least a portion of the reverse primer. The signal generating oligonucleotide may include a second region, wherein the second region includes a sequence complementary to at least a portion of the forward primer. The signal generating oligonucleotide may include a spacer region, wherein the spacer region includes a sequence of at least a portion of the spacer sequence. The first region, the second region, and / or the spacer region may include one or more polymerase terminators. The first region may include a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. The second region may include a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. The spacer region may include a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. The first region may include a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. The second region may include a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer. The spacer region may include a sequence complementary to at least two 3' terminal nucleotides of the forward primer and / or the reverse primer.

[0113] The length of the signal generating oligonucleotide can be from about 10 nucleotides to about 100 nucleotides. The length of the forward primer and / or the reverse primer can be from about 5 nucleotides to about 25 nucleotides. The length of the second district, the spacer and / or the first district can be from about 1 nucleotide to about 25 nucleotides. The length of the 5' subdomain, the 3' subdomain, the ring domain, the 5' terminal domain and / or the 3' terminal domain can be from about 1 nucleotide to about 25 nucleotides. The signal generating oligonucleotide can include a 5' terminal domain located at 5' subdomain 5'. The signal generating oligonucleotide can include a 3' terminal domain located at 3' of the 5' subdomain. In some embodiments, the 5' terminal domain and / or the 3' terminal domain do not include one or more polymerase terminators.

[0114] The first region may include at least a portion of a 5' subdomain and / or a ring domain. The spacer may include at least a portion of a ring domain. The second region may include at least a portion of a ring domain and / or a 3' subdomain. The 5' subdomain may include at least a portion of the first region and / or the spacer. The ring domain may include at least a portion of the spacer, the first region, and / or the second region. The 3' subdomain may include at least a portion of the second region and / or the spacer. In some embodiments, one or more polymerase terminators are located near the 3' end of the first region or at the 3' end (immediately adjacent to the spacer) and are located within the ring domain.

[0115] As used herein, "polymerase terminator" is a molecule (e.g., modified nucleotide) that can terminate or inhibit polymerization. In some embodiments, at least one of one or more polymerase terminators is a 2'-O-methylated nucleotide. Non-limiting examples of 2'-O-methylated nucleotides include 2'-O-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine and 2'-O-methylguanosine. One or more polymerase terminators may include one or more 2'-O-methyl (2'OM) RNA nucleotides. One or more polymerase terminators may include abasic sites, stable abasic sites, chemically captured abasic sites, or any combination thereof. The stable abasic sites may include 1', 2'-dideoxy. The chemically captured abasic sites may include abasic sites reacted with alkoxyamines or sodium borohydride. Abasic sites may include apurinic sites, apyrimidinic sites, or both. Abasic sites may be produced by alkylating agents or oxidants. In some embodiments, the one or more polymerase terminators include: one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromodeoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA Base, one or more isodeoxycytidine (Iso-dC), one or more isodeoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(o)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups or any combination thereof. The protected probes provided herein can contain one or more 2'-O-methyl ribonucleosides, such as, for example, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylpseudouridine, 2'-O-methylguanosine, 2'-O-methyl-5-methyluridine and / or 2'-O-methyluridine.

[0116] The signal generating oligonucleotide may comprise one or more phosphorothioate bonds 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 comprise a label. The label may comprise a quenchable label (e.g., a fluorophore). The signal generating oligonucleotide may comprise a quencher. The label may be located in the 3' terminal domain, and the quencher may be located in the 5' terminal domain. The label may be located in the 5' terminal domain, and the quencher may be located in the 3' terminal domain.

[0117] When the forward primer combines with the signal to produce an oligonucleotide to form the first undesirable duplex, one or more polymerase terminators may be able to terminate the forward primer of the first undesirable duplex to the signal to produce the polymerase extension of the 5' end of the oligonucleotide. One or more polymerase terminators may be able to terminate the forward primer of the first undesirable duplex to exceed the polymerase extension of the one or more polymerase terminators of the signal to produce the oligonucleotide. When the reverse primer combines with the signal to produce an oligonucleotide to form the second undesirable duplex, one or more polymerase terminators may be able to terminate the reverse primer of the second undesirable duplex to produce the polymerase extension of the 5' end of the oligonucleotide. One or more polymerase terminators may be able to terminate the reverse primer of the second undesirable duplex to exceed the polymerase extension of the one or more polymerase terminators of the signal to produce the oligonucleotide. When the exogenous nucleic acid combines with the signal to produce an oligonucleotide to form the third undesirable duplex, one or more polymerase terminators may be able to terminate the exogenous nucleic acid of the third undesirable duplex to produce the polymerase extension of the 5' end of the oligonucleotide. The one or more polymerase stoppers may be capable of terminating polymerase extension of the third undesired duplex of the exogenous nucleic acid beyond the one or more polymerase stoppers of the signal generating oligonucleotide.

[0118] The sample nucleic acid may include a nucleic acid containing a target nucleic acid sequence. In some embodiments, amplifying the target nucleic acid sequence includes: amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other under isothermal amplification conditions, wherein amplification includes contacting the nucleic acid comprising the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer can hybridize with the sequence of the first strand of the target nucleic acid sequence, and the reverse primer can hybridize with the sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme with hyperthermophilic biopolymerase activity, thereby producing a nucleic acid amplification product. The nucleic acid may be a double-stranded DNA. The nucleic acid may be the product of a reverse transcription reaction. The nucleic acid may be the product of a reverse transcription reaction produced from a sample ribonucleic acid. Amplification may include producing 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 produce cDNA. In some embodiments, amplifying the target nucleic acid sequence includes: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having a hyperthermophilic biopolymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises the target nucleic acid sequence, and wherein the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplification includes 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 a hyperthermophilic biopolymerase activity, thereby producing a nucleic acid amplification product.

[0119] In some embodiments, if the forward primer binds to the signal generating oligonucleotide to form a first undesirable duplex, the forward primer of the first undesirable duplex generates a first undesirable extension product by extension of the enzyme with hyperthermophilic polymerase activity to the 5' end of the signal generating oligonucleotide, and the first undesirable extension product can be amplified by the enzyme with hyperthermophilic polymerase activity in the presence of the forward primer and the reverse primer to form the first undesirable amplification product. One or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesirable duplex to produce a first stalled extension product. In some embodiments, the first stalled extension product cannot be amplified by the enzyme with hyperthermophilic polymerase activity in the presence of the forward primer and the reverse primer to produce the first undesirable amplification product. One or more polymerase terminators can terminate the polymerase extension of the forward primer of the first undesirable duplex beyond the one or more polymerase terminators of the signal generating oligonucleotide. In some embodiments, if the reverse primer combines with the signal generation oligonucleotide to form a second undesirable duplex, the reverse primer of the second undesirable duplex generates a second undesirable extension product by an enzyme with a hyperthermophilic polymerase activity to the 5' end of the signal generation oligonucleotide. The second undesirable extension product can be amplified by an enzyme with a hyperthermophilic polymerase activity to form a second undesirable amplification product in the presence of a reverse primer. One or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesirable duplex to produce a second stagnant extension product. In some embodiments, the second stagnant extension product cannot be amplified by an enzyme with a hyperthermophilic polymerase activity to produce a second undesirable amplification product in the presence of a reverse primer. One or more polymerase terminators can terminate the polymerase extension of the reverse primer of the second undesirable duplex beyond one or more polymerase terminators of the signal generation oligonucleotide. In some embodiments, if the exogenous nucleic acid combines with the signal generation oligonucleotide to form a third undesirable duplex, the exogenous nucleic acid of the third undesirable duplex is extended to the 5' end of the signal generation oligonucleotide by an enzyme with a hyperthermophilic polymerase activity to generate a third undesirable extension product. The third undesirable extension product can be amplified by an enzyme with a hyperthermophilic polymerase activity in the presence of a reverse primer to form a third undesirable amplification product. One or more polymerase terminators can terminate the polymerase extension of the exogenous nucleic acid of the third undesirable duplex to produce a third stagnant extension product. In some embodiments, the third stagnant extension product cannot be amplified by an enzyme with a hyperthermophilic polymerase activity in the presence of a reverse primer to produce a third undesirable amplification product. One or more polymerase terminators can terminate the polymerase extension of the exogenous nucleic acid of the third undesirable duplex beyond the one or more polymerase terminators of the signal generation oligonucleotide.

[0120] The detection 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 is capable of generating a signal when the signal generating oligonucleotide hybridizes with the nucleic acid amplification product. In some embodiments, the label generates a signal (e.g., fluorescence) when the signal generating oligonucleotide hybridizes with the nucleic acid amplification product. The detection 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 the 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 the fluorescence emitted by the label. The method may include 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 may include determining the presence, absence, and / or amount of dsDNA and / or nucleic acids containing 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 the dsDNA and / or nucleic acids containing the target nucleic acid sequence in the sample.

[0121] Mark can produce false positive signal when signal produces oligonucleotide and the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product hybridization.In some embodiments, when signal produces oligonucleotide and the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product hybridization, mark produces false positive signal.Signal and false positive signal can be indistinguishable.In some embodiments, the generation of the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product reduces the possibility of accurately determining the existence, absence and / or amount of target nucleic acid sequence in sample.In some embodiments, the detection of false positive signal reduces the possibility of accurately determining the existence, absence and / or amount of target nucleic acid sequence in sample.

[0122] In some embodiments, the presence of one or more polymerase terminators in the signal generating oligonucleotide increases the likelihood of accurately determining the presence, absence, and / or amount of a target nucleic acid sequence in a sample by at least about 1.1 times (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or a number or range between any of these values) compared to a signal generating oligonucleotide that does not include one or more polymerase terminators. In some embodiments, the generation of the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate a false positive signal. In some embodiments, the signal generating oligonucleotide that hybridizes to the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate a false positive signal. In some embodiments, nucleic acid amplification product reaches detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes or about 20 minutes or any one of these values ​​before the first undesirable amplification product, the second undesirable amplification product and / or the third undesirable amplification product reach detectable level.In some embodiments, signal reaches detectable level at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes or about 20 minutes or any one of these values ​​before the false positive signal reaches detectable level or a numeral or scope between them.Compared with the appropriate method that wherein signal produces oligonucleotide and does not comprise one or more polymerase terminators, the false positive signal of detectable level, the first undesirable amplification product, the second undesirable amplification product and / or the third undesirable amplification product can be delayed at least about 1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes or about 20 minutes or any one of these values ​​or a numeral or scope between them. In some embodiments, false positive signal, the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product do not reach detectable levels within at least about 5 minutes, about 10 minutes, about 15 minutes or about 20 minutes after the amplification step begins. Compared with a suitable method in which the signal generation oligonucleotide does not include one or more polymerase terminators, the generation of false positive signal, the first unexpected amplification product, the second unexpected amplification product and / or the third unexpected amplification product can be reduced by at least about 1.1 times (e.g., 1.1 times, 1.3 times, 1.5 times, 1.7 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or any one of these values ​​between the number or scope).

[0123] Amplifying the target nucleic acid sequence can include producing nucleic acid amplification products at a detectable level within about 20 minutes, about 15 minutes, or about 10 minutes. Detection is performed in less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the treated sample.

[0124] The lysis buffer may include one or more of magnesium sulfate, ammonium sulfate, EDTA, and EGTA. The pH of the lysis buffer may be from about 1.0 to about 10.0 (e.g., about 2.2). The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample nucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or a combination thereof. In some embodiments, one or more amplification reagents include: reverse transcriptase; an enzyme with hyperthermophilic biopolymerase activity; and / or dNTPs. In some embodiments, the enzyme with hyperthermophilic biopolymerase activity has reverse transcriptase activity: forward primer; reverse primer; reverse transcription primer. The reagent composition may be freeze-dried, heat-dried, and / or include one or more additives. In some embodiments, one or more additives include: tween 20, Triton X-100, and / or tween 80; amino acids; sugars or sugar alcohols; and / or polymers. Sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof. Polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptide, collagen peptide or any combination thereof. Contacting the reagent composition with the treated sample can include dissolving the reagent composition in the treated sample. In some embodiments, one or more lysis reagents include: about 0.001% (w / v) to about 1.0 (w / v) of treated sample (e.g., about 0.2% (w / v) of treated sample); and / or detergent (e.g., one or more of cationic surfactants, anionic surfactants, nonionic surfactants and amphoteric surfactants).

[0125] In some embodiments, the method: is performed in a single reaction vessel; does not include the use of any enzyme other than reverse transcriptase and an enzyme with thermophilic polymerase activity; does not include the use of any enzyme other than an enzyme with thermophilic polymerase activity; does not include thermal and / or enzymatic denaturation of the nucleic acid during the amplification step; and / or does not include contacting the nucleic acid with a single-stranded DNA binding protein.

[0126] The target nucleic acid sequence may comprise a length of no longer than about 20 nucleotides to no longer than about 90 nucleotides (e.g., about 30 nucleotides). The forward primer, reverse primer and / or reverse transcription primer may be about 8 to 16 bases long. The nucleic acid amplification product may be about 20 to 40 bases long. The spacer sequence may comprise a portion of the target nucleic acid sequence. The spacer sequence may be 1 to 10 bases long. Isothermal amplification conditions may include a constant temperature of about 30°C to about 72°C, optionally about 55°C to about 75°C, optionally about 56°C to about 67°C. Amplification may be performed for a period of about 5 minutes to about 60 minutes (e.g., a period of about 15 minutes). Amplification may be performed under isothermal amplification conditions without helicase, single-stranded binding protein, cleavage agent, and recombinase. Amplification can be performed 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, immune amplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification, and transcription-mediated amplification (TMA). PCR can be real-time PCR and / or quantitative real-time PCR (QRT-PCR).

[0127] The enzyme with hyperthermophilic biopolymerase activity has an amino acid sequence or a functional fragment thereof that is at least about 90% identical to the amino acid sequence of SEQ ID NO:7. The enzyme with hyperthermophilic biopolymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:7. The enzyme with hyperthermophilic biopolymerase activity can be a polymerase comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the enzyme with hyperthermophilic biopolymerase activity has low exonuclease activity or no exonuclease activity. The sample ribonucleic acid can be contacted with a reverse transcriptase and an enzyme with hyperthermophilic biopolymerase activity at the same time. The sample ribonucleic acid can be contacted with a reverse transcriptase, an enzyme with hyperthermophilic biopolymerase activity, and a forward primer and a reverse primer at the same time. The sample ribonucleic acid can be contacted with a reverse transcriptase, an enzyme with hyperthermophilic biopolymerase activity, a forward primer, a reverse primer, and a reverse transcription primer at the same time.

[0128] In some embodiments, amplification includes and / or does not include one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, amplification does not include LAMP.

[0129] In some embodiments, the method does not include one or more of the following: (i) dilution of the treated sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) addition of a ribonuclease inhibitor to the treated sample; (vii) addition of a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal and / or enzymatic denaturation of the sample nucleic acid prior to and / or during amplification; and (xiii) addition of ribonuclease H to the treated sample or the amplification reaction mixture.

[0130] The term "isothermal amplification reaction" should be given its usual meaning, and should also include reactions in which the temperature does not change significantly during the reaction. In some embodiments, during the main enzymatic reaction step where amplification occurs, the temperature deviation of the isothermal amplification reaction does not exceed 10°C, for example, does not exceed 5°C or does not exceed 2°C. According to the method for isothermal amplification of nucleic acids, different enzymes can be used for amplification. Isothermal amplification compositions and methods are described in the PCT application published as WO2017176404, the contents of which are incorporated herein by reference in their entirety.

[0131] In some embodiments, the methods and components described herein include storage-stable lysis buffer. In some embodiments, the lysis buffer resists the formation of precipitation for a period of time under storage conditions (e.g., storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer resists precipitation are described in International Application No. PCT / US23 / 61980, entitled "NON-OPAQUELYTIC BUFFER COMPOSITION FORMULATIONS" and filed on February 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0132] In addition to acid and / or low pH conditions, some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote chain separation and / or promote chain melting). Compositions, kits, and methods for nucleic acid detection in which nucleic acid chains dissociate under low pH conditions (e.g., via contact with an acidic lysis buffer) to promote subsequent rapid amplification and detection are described in International Application No. PCT / US23 / 61978, entitled "METHOD FOR SEPARATING GENOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID Targets," and filed on February 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0133] In some embodiments, the methods and compositions described herein may include a lysis buffer and / or a reagent composition. A lysis buffer comprising a lysing agent and a reducing agent and a reagent composition comprising an amplifying agent and one or more protective agents (e.g., cyclodextrin compounds) capable of chelating the lysing agent are described in International Application No. PCT / US22 / 21015, entitled "ISOTHERMAL AMPLIFICATION OFPATHOGENS" and filed on March 18, 2022, the contents of which are incorporated herein by reference in their entirety.

[0134] In some embodiments, some embodiments of the methods and compositions described herein can be used in conjunction with the systems, methods, compositions, and kits for monitoring amplification reactions described in U.S. Provisional Patent Application No. 63 / 374,835, entitled “HAIRPIN INTERNAL CONTROL FOR ISOTHERMAL NUCLEIC ACID AMPLIFICATION,” filed on September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0135] 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 enable multiplexing of targets and / or one or more internal controls. Compositions, kits, and methods for multiplex nucleic acid detection are described in U.S. Provisional Patent Application No. 63 / 374,831, entitled “ARCHEAL POLYMERASE AMPLIFICATION” and filed on September 7, 2022, the contents of which are incorporated herein by reference in their entirety.

[0136] Nucleic Acids, Subjects, Samples, and Nucleic Acid Processing

[0137] Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" may be used interchangeably herein. The term refers to nucleic acids of any composition, such as DNA (e.g., complementary DNA (cDNA), genomic DNA (gDNA), etc.), RNA (e.g., messenger RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs and / or non-natural backbones, etc.), RNA / DNA hybrids and polyamide nucleic acids (polyamide nucleic acids). Acids, PNAs), all of which may be in single-stranded or double-stranded form, and unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be or may be derived from a plasmid, a bacteriophage, an autonomously replicating sequence (ARS), a centromere, an artificial chromosome, a chromosome, or other nucleic acids that can replicate or be replicated in vitro or in a host cell, a cell, a nucleus, a mitochondria, or the cytoplasm of a cell. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, interspecies homologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as explicitly indicated sequences. In particular, degenerate codon substitutions may be obtained by generating a third position in which one or more selected (or all) codons are mixed. The term nucleic acid can be used interchangeably with locus, gene, cDNA and mRNA encoded by a gene. As an equivalent, the term can also include derivatives, variants and analogs of RNA or DNA synthesized from nucleotide analogs, single-stranded ("sense" or "antisense", "positive" strand or "negative" strand, "forward" reading frame or "reverse" reading frame, "forward" strand or "reverse" strand) and double-stranded polynucleotides. The term "gene" means a DNA segment involved in producing a polypeptide chain. ; and generally include regions before and after the coding region (leader and trailer) that participate in the transcription / translation of the gene product and the regulation of transcription / translation, as well as intermediate sequences (introns) between the various coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). For RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed in terms of the number of bases.

[0138] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid can be referred to as a target sequence, a target polynucleotide and / or a target polynucleotide sequence, and can include a double-stranded nucleic acid molecule and a single-stranded nucleic acid molecule. The target nucleic acid can be, for example, DNA or RNA. When the target nucleic acid is an RNA molecule, the molecule can be, for example, double-stranded, single-stranded, or the RNA molecule can include a single-stranded target sequence. When the target nucleic acid is double-stranded, the target nucleic acid generally includes a first strand and a second strand. The first strand and the second strand can be referred to as a forward strand and a reverse strand, and are generally complementary to each other. When the target nucleic acid is single-stranded, a complementary strand can be produced, for example, by polymerization and / or reverse transcription, so that the target nucleic acid is double-stranded and has a first / forward strand and a second / reverse strand.

[0139] The target sequence can refer to the sense strand or antisense strand of the nucleic acid sequence, and can also refer to the sequence present in the target nucleic acid, amplified copy or amplified product of the original target sequence. The target sequence can be a subsequence in a larger polynucleotide. For example, the target sequence can be a short sequence (for example, 20 to 50 bases) in a nucleic acid fragment, a chromosome, a plasmid, which is targeted for amplification. In some embodiments, the target sequence can refer to a sequence complementary to an oligonucleotide (for example, a primer) for amplifying nucleic acid in a target nucleic acid. Therefore, the target sequence can refer to the entire sequence targeted for amplification, or can refer to a subsequence of oligonucleotide binding in a target nucleic acid. Amplified product can be a larger molecule comprising the target sequence and at least one other sequence or other nucleotides. The amplified product can be a length approximately the same as the target sequence, for example, a length identical to the target sequence. The amplified product can include the target sequence or be composed of the target sequence.

[0140] The length of the target sequence and / or guanosine cytosine (GC) concentration (percentage) can depend in part on the temperature at which the amplification reaction is run, and the temperature can depend in part on the stability of the polymerase used in the reaction. Sample assays can be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, when the polymerase is stable at up to 60°C to 65°C, the length of the target sequence can be, for example, from 19 to 50 nucleotides, or the length can be, for example, from about 40 to 50, 20 to 45, 20 to 40 or 20 to 30 nucleotides. The GC concentration under these conditions can be, for example, less than 60%, less than 55%, less than 50% or less than 45%.

[0141] Target nucleic acid can include, for example, genomic nucleic acid, plasmid nucleic acid, mitochondrial nucleic acid, cellular nucleic acid, extracellular nucleic acid, bacterial nucleic acid and viral nucleic acid. In some embodiments, target nucleic acid can include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, gene, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA or synthetic oligonucleotide. Genomic nucleic acid can include any nucleic acid from any genome, for example, animal, plant, insect, virus and bacterial genome (for example, the genome present in spore). In some embodiments, genomic target nucleic acid is in a specific genomic locus or more than one genomic locus. Genomic locus can include any one or combination of open reading frame DNA, non-transcribed DNA, intron sequence, exon sequence, promoter sequence, enhancer sequence, flanking sequence or any sequence that is considered to be related to a given genomic locus.

[0142] The target sequence may comprise one or more repeat elements (e.g., multiple repeats, inverted repeats, palindromes, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence is present in the sample nucleic acid (e.g., in a nucleic acid fragment, chromosome, genome, plasmid) as a repeat element (e.g., multiple repeats, inverted repeats, palindromes, tandem repeats, microsatellites, minisatellites, etc.). For example, the target sequence may appear multiple times as a repeat element, and one, some, or all occurrences of the target sequence in the repeat element may be amplified using the methods described herein (e.g., using a single pair of primers). In some embodiments, the target sequence is present in the sample nucleic acid (e.g., in a nucleic acid fragment, chromosome, genome, plasmid) as a repeat and / or homolog within the species.

[0143] The target nucleic acid may include microRNA. MicroRNA, miRNA or small temporal RNAs (stRNA) are short (e.g., about 21 to 23 nucleotides long) and single-stranded RNA sequences involved in gene regulation. MicroRNA can interfere with the translation of messenger RNA and is partially complementary to messenger RNA. The target nucleic acid may include microRNA precursors, such as primary transcripts (pri-miRNA) and pre-miRNA stem-loop structure RNAs that are further processed into miRNA. The target nucleic acid may include short interfering RNA (siRNA), which is a short (e.g., about 20 to 25 nucleotides long) and at least partially double-stranded RNA molecule involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0144] The nucleic acid used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, from the sample separation obtained from the subject. Subject can be any living or non-living organism, including but not limited to humans, non-human animals, plants, bacteria, fungi, viruses or protozoa. Any humans or non-human animals can be selected, including but not limited to mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (for example, cattle), equines (for example, horses), caprines and ovines (for example, sheep, goats), swine (for example, pigs), camelids (for example, camels, llamas, alpacas), monkeys, apes (for example, gorillas, chimpanzees), ursids (for example, bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales and sharks. Subject can be male or female, and subject can be any age (for example, embryo, fetus, infant, child, adult).

[0145] Sample or test sample can be any sample separated or obtained from a subject or part thereof. The non-limiting examples of sample include fluid or tissue from a subject, including but not limited to blood or blood products (e.g., serum, plasma, etc.), cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, stomach, peritoneum, catheter, ear, arthroscopy), serum, plasma, urine, aspirate (aspirate), biopsy sample, abdominal puncture sample, cell (e.g., blood cell) or its part (e.g., mitochondria, nucleus, extract, etc.), female reproductive tract washings, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, hard tissue (e.g., liver, spleen, kidney, lung or ovary), etc. or its combination. The term blood includes any fraction of whole blood, blood products or blood, such as serum, plasma, buffy coat or the analog of conventional definition. Plasma refers to the whole blood fraction produced by centrifugation of blood treated with an anticoagulant. Serum refers to the aqueous portion of the fluid remaining after the blood sample has coagulated. Fluid or tissue samples are usually collected according to the standard protocols usually followed in hospitals or clinics. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) is usually collected and can be stored according to standard procedures before or after preparation.

[0146] Samples can include samples containing spores, viruses, cells, nucleic acids from prokaryotes or eukaryotes, and / or any free nucleic acids. For example, the methods described herein can be used to detect nucleic acids outside of spores (e.g., without the need for lysis). Samples can be isolated from any material suspected of containing the target sequence, such as from a subject described above. In some embodiments, the target sequence is present in air, plants, soil, or other materials suspected of containing biological organisms.

[0147] Nucleic acids can be obtained (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method can be used to isolate, extract and / or purify nucleic acids from a biological sample, including DNA preparation methods known in the art, 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 TM Blood DNA Isolation Kit (Promega, Madison, Wis.), GFX TM Genomic blood DNA purification kit (Amersham, Piscataway, NJ), etc. or a combination thereof. US Patent No. 7,888,006 provides a DNA purification method and does not disclose the compositions (eg, lysis buffer, protective agent) and methods provided herein.

[0148] In some embodiments, a cell lysis procedure is performed. Cell lysis can be performed before starting 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 (e.g., detergent, hypotonic solution, enzymatic procedure, etc., or a combination thereof), physical (e.g., French press, ultrasonic treatment, etc.) or electrolytic lysis methods. For example, chemical methods typically use lysing agents to destroy cells and extract nucleic acids from cells, and then treat with chaotropic salts. In some embodiments, cell lysis includes the use of detergents (e.g., ions, non-ions, anions, zwitterions). In some embodiments, cell lysis includes the use of ionic detergents (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods such as grinding after freezing / thawing, using cell crushing, etc. may also be useful. High salt lysis procedures may also be used. For example, an alkaline lysis procedure may be used. The latter procedure traditionally involves the use of a phenol-chloroform solution, and an alternative phenol-chloroform-free procedure involving three solutions may be used. In the latter procedure, one solution may contain 15 mM Tris, pH 8.0; 10 mM EDTA and 100 μg / ml RNase; a second solution may contain 0.2 N NaOH and 1% SDS; and a third solution may contain, for example, 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and compositions described herein.

[0149] Nucleic acid can be provided for carrying out the methods described herein without processing samples containing nucleic acid. For example, nucleic acid can be provided for carrying out the amplification methods described herein without prior nucleic acid purification. In some embodiments, the target sequence is amplified directly from the sample (for example, without performing any nucleic acid extraction, separation, purification and / or partial purification steps). In some embodiments, after processing a sample containing nucleic acid, nucleic acid is provided for carrying out the methods described herein. For example, nucleic acid can be extracted, separated, purified or partially purified from a sample. The term "isolated" generally refers to nucleic acid taken out from its original environment (for example, if it is naturally present, it is a natural environment, if it is exogenously expressed, it is a host cell), and thus changed from its original environment by human intervention (for example, "by human hands"). The term "isolated nucleic acid" can refer to nucleic acid taken out from a subject (for example, a human subject). Isolated nucleic acid can provide non-nucleic acid components (for example, proteins, lipids, carbohydrates) less than the amount of components present in the source sample. The composition comprising isolated nucleic acid can be free of about 50% to greater than 99% of non-nucleic acid components. The composition comprising the nucleic acid of separation can be free of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% non-nucleic acid components. The term "purified" generally refers to that the non-nucleic acid components (e.g., protein, lipid, carbohydrate) provided by the nucleic acid are less than the amount of the non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. The composition comprising the nucleic acid of purification can be free of other non-nucleic acid components by about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%.

[0150] Nucleic acids can be provided for performing the methods described herein without modifying the nucleic acids. Modifications can include, for example, denaturation, digestion, nicking, melting, incorporation and / or ligation of heterologous sequences, addition of epigenetic modifications, addition of labels (e.g., radioactive labels such as 32 P. 33 P. 125 I or 35 S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, fluorescent dyes), etc. Therefore, in some embodiments, unmodified nucleic acids are amplified.

[0151] 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 a high degree of sensitivity. In some embodiments, the methods can be used to detect target DNA / RNA present in a sample containing more than one RNA / DNA (including target RNA / DNA and more than one non-target RNA / DNA), wherein the target RNA / DNA is present in a sample at a concentration of 10, 20, 25, 50, 100, 500, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 15 ... 3 pcs, 5×10 3 10 4 pcs, 5×10 4 10 5 pcs, 5×10 5 10 6 or 10 7 One or more copies of each non-target DNA / RNA are present. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" should be given their ordinary meanings, and should also refer to DNA, or RNA, or a combination of DNA and RNA.

[0152] For methods of detecting target RNA / DNA in a sample, the detection threshold can be, for example, 10 nM or less. The term "detection threshold" should be given its ordinary meaning, and should also describe the minimum amount of target RNA / DNA that must be present in a sample in order to detect. As an illustrative example, when the detection threshold is 10 nM, a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the disclosed methods have a detection threshold of 5nM or less, 1nM or less, 0.5nM or less, 0.1nM or less, 0.05nM or less, 0.01nM or less, 0.005nM or less, 0.001nM or less, 0.0005nM or less, 0.0001nM or less, 0.00005nM or less, 0.00001nM or less, 10pM or less, 1pM or less, 500fM or less, 250fM or less, 100fM or less, 50fM or less, 500aM (attomolar) or less, 250aM or less, 100aM or less, 50aM or less, 10aM or less, or 1aM or less. In some embodiments, the disclosed compositions or methods exhibit detection sensitivity of attomole / liter (aM), femtomolar / liter (fM), picomolar / liter (pM), and / or nanomolar / liter (nM).

[0153] The sample can include sample nucleic acids (e.g., more than one sample nucleic acid). The term "more than one" is used herein to mean two or more. Thus, in some embodiments, the sample includes two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1,000 or more, or 5,000 or more) sample nucleic acids (e.g., DNA / RNA). The disclosed methods can be used as a very sensitive means of detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, the sample includes 5, 10, 20, 25, 50, 100, 500, 1000 or more) sample nucleic acids that are different in sequence from each other. 3 5×10 3 10 4 5×10 4 10 5 5×10 5 10 6 Species or 10 7 In some embodiments, the sample comprises DNA / RNA from cells (e.g., eukaryotic cells, mammalian cells, or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0154] The term "sample" as used herein should be given its ordinary meaning and should include any sample containing RNA and / or DNA (e.g., in order to determine whether a target DNA and / or target RNA is present in a RNA and / or DNA population). The sample can be a biological sample or an environmental sample. The sample can be derived from any source, for example, the sample can be a synthetic combination of purified DNA and / or RNA; the sample can be a cell lysate, a cell lysate enriched in DNA / RNA, or DNA / RNA isolated and / or purified from a cell lysate. The sample can be from a patient (e.g., for diagnostic purposes). The sample can be from permeabilized cells, cross-linked cells, tissue sections, or a combination thereof. The sample can be from a tissue prepared by cross-linking followed by delipidation and adjustment to form a uniform refractive index. The sample can contain a target nucleic acid (e.g., a target DNA / RNA) and more than one non-target DNA / RNA. In some embodiments, the target DNA / RNA is present in 10, 20, 25, 50, 100, 500, 10 3 pcs, 5×10 3 10 4 pcs, 5×10 4 10 5 pcs, 5×105 10 6 or 10 7 One copy of each non-target DNA / RNA is present in the sample.

[0155] Samples about patients include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, as well as samples that have been manipulated in any way after their acquisition (such as by treatment with reagents), washed or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA). Samples may include the following or are the following: biological samples, including but not limited to clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), and also tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, etc. Biological samples may include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), for example, samples containing RNA obtained from such cells (e.g., cell lysates or other cell extracts containing RNA). In some embodiments, the environmental sample is, or is obtained from, a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof.

[0156] The source of the sample can be (or suspected to be) pathological cells, fluids, tissues or organs; or normal (non-lesion) cells, fluids, tissues or organs. In some embodiments, the source of the sample is (or suspected to be) pathogen-infected cells, tissues or organs. For example, the source of the sample can be an individual who may be infected or may not be infected, and the sample can be any biological sample collected from the individual (for example, blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, stool sample, cerebrospinal fluid, fine needle aspiration, swab sample (for example, oral swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal mucus, tears, buffy coat, mucosal sample, epithelial cell sample (for example, epithelial cell scraping) etc.) and its culture. The sample can be a cell-free liquid sample or a liquid sample comprising cells. Pathogens can be viruses, fungi, worms, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites, etc. "Herms" include roundworms, heartworms, and phytophagous nematodes (Nematoda), flukes (Trematoda), acanthocephala, and tapeworms (Cestoda). Protozoan infections include Giardia spp., Trichomonas spp. infections, African trypanosomiasis, amoebic dysentery, babesiosis, balantidium dysentery, Chaga's disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens such as parasitic / protozoan pathogens include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include, for example, immunodeficiency viruses (e.g., HIV); influenza virus; dengue fever; West Nile virus; herpes virus; yellow fever virus; hepatitis C virus; hepatitis A virus; hepatitis B virus; papillomavirus, etc. Pathogenic viruses can include DNA viruses such as: papovaviruses (e.g., HPV, polyomavirus); hepadnavirus; herpesviruses (e.g., HSV (e.g., HSV I, HSV II), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, Pityriasis rosea (PityriasisRosea, Kaposi's sarcoma-associated herpesvirus); adenovirus (e.g., thymovirus, avian adenovirus, ichtadenovirus, mammalian adenovirus, sialidase adenovirus); poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, capripox virus, pseudovaccinia, bovine papular stomatitis virus; tanapox virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvovirus (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parvovirus 4G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like. Non-limiting examples of pathogens include Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, leprae), Brucella abortus, rabies virus, human serum parvovirus-like virus, respiratory syncytial virus, measles virus, adenovirus, human T-cell leukemia virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovisbovis), Eimeria species (e.g., tenella), Onchocerca volvulus, Leishmania species (e.g., tropica), Streptococcus pneumonia, 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 virus, herpes virus 6, herpes virus 7, JC virus, influenza virus A, influenza virus B, influenza virus C, rotavirus, human adenovirus, human enterovirus, hantavirus, Legionella dumov dumoffii, Mycoplasma fermentans, Haemophilus influenzae, Rickettsia rickettsii, Ehrlichia sp. (e.g., chaffeensis), Borrelia burgdorferi, Yersinia pestis, Chlamydia pneumoniae, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma sp.) (e.g., arthritidis), M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae.

[0157] Amplification

[0158] Provided herein is a method for amplifying nucleic acid. In some embodiments, nucleic acid is amplified using a suitable amplification method. Nucleic acid amplification generally relates to the enzymatic synthesis of nucleic acid amplicon (copy), which comprises a sequence complementary to the nucleotide sequence amplified. In some embodiments, amplification method is carried out in a single container, a single chamber and / or a single volume (i.e., a continuous volume). In some embodiments, amplification method and detection method (e.g., detection method described herein) are carried out in a single container, a single chamber and / or a single volume (i.e., a continuous volume).

[0159] The terms "amplify," "amplification," "amplification reaction," or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification sometimes refers to an "exponential" increase in a target nucleic acid. "Amplification" may also refer to a linear increase in the number of target nucleic acids, but is distinct from a single, single primer extension step. In some embodiments, a limited amplification reaction, also referred to as pre-amplification, may be performed. Pre-amplification is a method in which a limited amount of amplification occurs because a small number of cycles, such as 10 cycles, are performed. Pre-amplification may allow some amplification, but stops amplification before the exponential phase and typically produces about 500 copies of the desired nucleotide sequence. The use of pre-amplification may limit inaccuracies associated with depleted reactants in certain amplification reactions, and may also reduce amplification biases due to nucleotide sequence or species abundance of the target. In some embodiments, a primer extension may be performed as a prelude to linear or exponential amplification.

[0160] A general description of amplification process is presented herein. Primers (e.g., oligonucleotides described herein) contact with target nucleic acid, and for example, complementary sequences anneal or hybridize to each other. Primers can anneal with target nucleic acid at or near the sequence of interest (e.g., adjacent, contiguous, etc.). Primers annealed with target can be referred to as primer-target hybrids, hybridized primer-targets or primer-target duplexes. When referring to a nucleotide sequence of interest, the term near or adjacent to the distance (e.g., base number) or region between one or more nucleotides (e.g., nucleotide sequence) of the primer end and the target. Typically, proximity is a range of about 1 nucleotide to about 50 nucleotides from a nucleotide or nucleotide sequence of interest (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 nucleotides). In some embodiments, a group of primers (e.g., a pair of primers, forward and reverse primers, a first oligonucleotide and a second oligonucleotide) anneal within about 1 to 20 nucleotides from a nucleotide or nucleotide sequence of interest, and produce an amplified product. In some embodiments, the primers are annealed within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended by a polymerase along the target (i.e., template strand) to produce a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplified product is produced. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step.

[0161] The components of the amplification reaction (e.g., one or more amplification reagents) may include, for example, one or more primers (e.g., single primers, primer pairs, primer sets, oligonucleotides, multiple primer sets for multiple amplification, etc.), nucleic acid targets (e.g., target nucleic acids from samples), one or more polymerases, nucleotides (e.g., dNTPs, etc.), and suitable buffers (e.g., buffers containing detergents, reducing agents, monovalent ions, and divalent ions). The amplification reaction may also include one or more of the following: reverse transcriptase, reverse transcription primers, and one or more detection agents.

[0162] Nucleic acid amplification can be carried out in the presence of natural nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derived nucleotides. Natural nucleotides generally refer to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid or uridylic acid. Derived nucleotides are generally nucleotides different from natural nucleotides. Ribonucleotide triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T or U. Monomeric nucleotide subunits may be represented herein as A, G, C, T or U, without particular reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs may be used, such as analogs containing detectable labels (e.g., fluorescent or colorimetric labels). For example, nucleic acid amplification can be carried out in the presence of labeled dNTPs, such as radioactive labels, such as 32 P. 33 P. 125 I or 35 S; enzyme labels, such as alkaline phosphatase; fluorescent labels, such as fluorescein isothiocyanate (FITC); or other labels, such as biotin, avidin, digoxin, antigens, haptens, or fluorescent dyes. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, for example, heat-activated dNTPs (e.g., CleanAmp from TriLink TM dNTP).

[0163] One or more amplification reagents can include non-enzyme components and enzyme components.Non-enzyme components can include, for example, primers, nucleotides, buffer, salt, reducing agent, detergent and ion.In some embodiments, non-enzyme components do not include protein (for example, nucleic acid binding protein), enzyme or protein with enzymatic activity, for example, polymerase, reverse transcriptase, helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nickase, recombinase etc.In some embodiments, enzyme component is made up of polymerase or is made up of polymerase and reverse transcriptase.Therefore, such enzyme component will exclude other proteins (for example, nucleic acid binding protein and / or protein with enzymatic activity), for example, helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction endonuclease, nickase, recombinase etc.

[0164] In some embodiments, the amplification conditions include enzymatic activity (e.g., enzymatic activity provided by a polymerase or enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include enzymatic activity provided by enzymes other than polymerases and / or reverse transcriptases, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, etc. The polymerase activity and reverse transcriptase activity can be provided by separate enzymes or separate enzyme types (e.g., polymerase and reverse transcriptase), or by a single enzyme or enzyme type (e.g., polymerase).

[0165] The amplification of nucleic acid can include the PCR of non-thermal cycle type. In some embodiments, the amplification of nucleic acid includes isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process carried out at a constant temperature. Terms such as isothermal conditions, isothermally and constant temperature generally refer to reaction conditions in which the reaction temperature remains constant during the amplification reaction process. Isothermal amplification conditions do not generally include thermal cycling (i.e., circulating between higher temperatures and lower temperatures) components in the amplification process. When amplified under isothermal conditions, the reaction can be maintained at a substantially constant temperature, which means that the temperature can be maintained at a temperature inaccurately. For example, in the isothermal amplification process, due to, for example, environment or variables based on equipment, small fluctuations of temperature (such as ± 1 ° C to 5 ° C) may occur. Generally, the whole reaction volume is maintained at a substantially constant temperature, and the isothermal reaction herein does not generally include the amplification conditions that depend on the temperature gradient generated in the reaction vessel and / or the temperature cycle based on convection.

[0166] Isothermal amplification reaction herein can be carried out at a substantially constant temperature. In some embodiments, isothermal amplification reaction herein is carried out at a temperature of about 55 ° C to a temperature of about 75 ° C, for example, at the following temperature or at about the following temperature: 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 67 ° C, 68 ° C, 69 ° C, 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C or about 75 ° C or in any two of these values ​​between the number or scope. In some embodiments, temperature element (for example, heat source) is maintained at a substantially constant temperature, for example, at or below about 75 ° C, at or below about 70 ° C, at or below about 65 ° C, or at or below about 60 ° C of substantially constant temperature.

[0167] Amplification process herein can be carried out for a certain time length, for example until detectable nucleic acid amplification product is produced. Nucleic acid amplification product can be detected by any suitable detection process and / or detection process described herein. Amplification process can be carried out for a time length of about 20 minutes or less time or about 10 minutes or less time. For example, amplification process can be carried out in about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 minutes or in any two of these values ​​numerals or scopes.

[0168] In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, during the amplification step (and / or other steps), the nucleic acid target can be amplified without exposure to agents or conditions that promote strand separation. In some embodiments, during the amplification step (and / or other steps), the nucleic acid target can be amplified without exposure to agents or conditions that promote unwinding. Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote unwinding can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemicals, proteins (e.g., enzymatic agents), etc.

[0169] In some embodiments, the methods disclosed herein do not include thermal denaturation of nucleic acids (e.g., heating a solution containing nucleic acids to a high temperature, such as, for example, a temperature higher than 75°C, 80°C, 90°C, or 95°C or higher) or protein-based (e.g., enzymatic) denaturation. Protein-based (e.g., enzymatic) denaturation can include contacting nucleic acids with one or more of helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, RNA replicases, and nucleic acid binding proteins (e.g., single-stranded binding proteins). In some embodiments, compositions provided herein do not include helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, RNA replicases, and / or nucleic acid binding proteins (e.g., single-stranded binding proteins). In some embodiments, compositions and methods provided herein do not include intercalating agents, alkylating agents, and / or chemicals, such as formamide, glycerol, urea, dimethyl sulfoxide (DMSO) or N,N,N-trimethylglycine (betaine). In some embodiments, the disclosed method does not include contacting the nucleic acid with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions for denaturing the nucleic acid (e.g., promoting chain separation and / or promoting unwinding). In some embodiments, in addition to a polymerase (e.g., a hyperthermophilic biopolymerase), the amplification step (e.g., step (c)) does not include agents and / or conditions for denaturing the nucleic acid (e.g., promoting chain separation and / or promoting unwinding). In some embodiments, in addition to a polymerase (e.g., a hyperthermophilic biopolymerase) and / or low pH conditions (e.g., contacting with one or more acids), the methods and compositions provided herein do not include agents and / or conditions for denaturing the nucleic acid (e.g., promoting chain separation and / or promoting unwinding).

[0170] The nucleic acid target can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction endonucleases, nickases, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-stranded binding proteins) or any combination thereof. For example, the nucleic acid target can be amplified without exposure to a helicase, which includes but is not limited to DNA helicases and RNA helicases. Amplification conditions that do not include the use of a helicase are amplification conditions without a helicase.

[0171] The nucleic acid target can be amplified without being exposed to a recombinase, and the recombinase includes but is not limited to Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, T4 uvsX. In some embodiments, the nucleic acid target is amplified without being exposed to a recombinase auxiliary protein, such as a recombinase loading factor (e.g., T4 uvsY). The nucleic acid target can be amplified without being exposed to a nucleic acid binding protein (e.g., a single-stranded binding protein or a single-stranded DNA binding protein (SSB)), such as T4gp32. In some embodiments, the nucleic acid target is amplified without being exposed to a topoisomerase. The nucleic acid target can be amplified under exposure to or without being exposed to an agent or condition that destabilizes the nucleic acid. As used herein, the term "destabilization" should be given its ordinary meaning, and should also refer to the destruction of the overall organization and geometric orientation of nucleic acid molecules (e.g., double helix structures) by one or more of the tilting, rolling, twisting, sliding and flipping effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, 17 pages). Destabilization does not generally refer to the unwinding or separation (e.g., denaturation) of nucleic acid chains. Nucleic acid destabilization can be achieved by, for example, exposure to agents such as intercalating agents or alkylating agents and / or chemicals such as formamide, urea, dimethyl sulfoxide (DMSO) or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, nucleic acid targets are amplified without exposure to ligases and / or RNA replicases.

[0172] In some embodiments, the nucleic acid target can be amplified without cleavage or digestion. For example, the nucleic acid target can be amplified without being pre-exposed to one or more cleavage agents, and the complete nucleic acid is amplified. In some embodiments, the nucleic acid target is amplified without being exposed to one or more cleavage agents during amplification. In some embodiments, the nucleic acid target is amplified without being exposed to one or more cleavage agents after amplification. Amplification conditions that do not include the use of cleavage agents may be referred to as cleavage-free amplification conditions herein. The term "cleavage agent" generally refers to an agent that can cleave nucleic acids at one or more specific or non-specific sites, sometimes a chemical substance or enzyme. Specific cleavage agents are generally cleaved according to specific nucleotide sequences at specific sites. Cleavage agents may include endonucleases (e.g., restriction endonucleases, nickases, etc.); exonucleases (DNA enzymes, RNA enzymes (e.g., RNAse H), 5' to 3' exonucleases (e.g., exonucleases II), 3' to 5' exonucleases (e.g., exonucleases I) and poly (A) specific 3' to 5' exonucleases); and chemical cleavage agents.

[0173] Nucleic acid target can be amplified without restriction endonuclease and / or nickase. In some embodiments, nucleic acid is amplified without being exposed to restriction endonuclease and / or nickase in advance. In some embodiments, nucleic acid is amplified without being exposed to restriction endonuclease and / or nickase during amplification. In some embodiments, nucleic acid is amplified without being exposed to restriction endonuclease and / or nickase after amplification. Nucleic acid target can be amplified without exonuclease treatment. Exonuclease includes, for example, DNA enzyme, RNA enzyme (for example, RNA enzyme H), 5' to 3' exonuclease (for example, exonuclease II), 3' to 5' exonuclease (for example, exonuclease I) and multi (A) specific 3' to 5' exonuclease. In some embodiments, nucleic acid is amplified without exonuclease treatment before, during and / or after amplification. Exonuclease-free amplification conditions are not included. In some embodiments, nucleic acid is amplified without DNA enzyme treatment and / or RNA enzyme treatment. In some embodiments, the nucleic acid is amplified in the absence of RNase H treatment.

[0174] The nucleic acid of amplification can be referred to as nucleic acid amplification product or amplicon in this article.In some embodiments, amplification product comprises naturally occurring nucleotide, non-naturally occurring nucleotide, nucleotide analog etc. and aforementioned combination.Amplification product usually has the nucleotide sequence identical or substantially identical with the sequence (for example, target sequence) or its complement in sample nucleic acid.The nucleotide sequence of "substantially identical" in amplification product usually will have the sequence identity (for example, about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity) of height with the nucleotide sequence amplified or its complement, and variation is the result of polymerase infidelity (infidelity) or other variables sometimes.

[0175] The nucleic acid amplification product may include polynucleotides that are continuously complementary or substantially identical to the target sequence in the sample nucleic acid. Continuous complementation generally refers to a nucleotide sequence in the first chain, for example, wherein each ordered base (e.g., reading 5' to 3') is paired with a corresponding ordered base in the second chain, and there are no gaps, additional sequences, or unpaired bases in the sequence considered to be continuously complementary. In other words, continuous complementation generally refers to that all continuous bases of the nucleotide sequence in the first chain are complementary to the corresponding continuous bases of the nucleotide sequence in the second chain. For example, a first chain with a sequence of 5'-ATGCATGCATGC-3' (SEQ ID NO: 9) will be considered to be continuously complementary to a second chain with a sequence of 5'-GCATGCATGCAT-3' (SEQ ID NO: 10), wherein all continuous bases in the first chain are complementary to all corresponding continuous bases in the second chain. However, a first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO: 11) would not be considered to be continuously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 10) because the six adenine (6 A) sequence in the middle of the first strand would not pair with bases in the second strand. A continuous complementary sequence is sometimes about 5 to about 25 continuous bases in length, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 continuous bases in length, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of polynucleotides that are continuously complementary to or substantially identical to a target sequence in a sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequences that are not continuously complementary or substantially identical to the target sequence (e.g., at the 5' and / or 3' ends, or within the product), e.g., additional sequences incorporated into the amplification product by tailing primers or ligation, and / or additional sequences that provide a cleavage agent recognition site (e.g., a nickase recognition site). Typically, unless the target sequence includes tandem repeats, the amplification product does not include products in the form of tandem repeats.

[0176] The nucleic acid amplification product may comprise a sequence complementary to or substantially identical to one or more primers used in the amplification reaction. In some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is continuously complementary to or identical to a first primer sequence, and a second nucleotide sequence that is continuously complementary to or identical to a second primer sequence.

[0177] The nucleic acid amplification product may include a spacer sequence. As described herein, the spacer sequence in the amplification product is a sequence (1 or more bases) that is continuously complementary or substantially identical to a portion of the target sequence in the sample nucleic acid, and the flank is a sequence that is complementary or substantially identical to one or more primers used in the amplification reaction in the amplification product. The spacer sequence flanking the sequence in the amplification product is generally located between the first sequence (complementary or substantially identical to the first primer) and the second sequence (complementary or substantially identical to the second primer). Therefore, the amplification product generally includes the first sequence, followed by the spacer sequence, followed by the second sequence. The spacer sequence is generally not complementary or substantially identical to the sequence in the primer. The spacer sequence can be the following or can include the following: about 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases. In some embodiments, the nucleic acid amplification product is composed of or substantially composed of: a first nucleotide sequence that is continuously complementary or identical to the first primer sequence, a second nucleotide sequence that is continuously complementary or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence (e.g., at the 5' end and / or 3' end; or within the product), which is not continuously complementary or identical to the first primer sequence and the second primer sequence, and is not part of the spacer sequence, for example, additional sequences incorporated into the amplification product by tailing or circularizing primers, ligation or other mechanisms. In some embodiments, the nucleic acid amplification product generally does not include additional sequences (e.g., at the 5' end and / or 3' end; or within the product), which is not continuously complementary or identical to the first primer sequence and the second primer sequence, and is not part of the spacer sequence, for example, additional sequences incorporated into the amplification product by tailing or circularizing primers, ligation or other mechanisms. However, in such embodiments, the nucleic acid amplification product may include some mismatched (i.e., non-complementary) bases or one or more additional bases (e.g., at the 5' end and / or 3' end; or within the product), which are introduced into the product, for example, by errors or confounding during the amplification process.

[0178] The length of the nucleic acid amplification product can be up to 50 bases, including 10, 15, 20, 25, 30, 35, 40, 45, 50 bases, or a number or range of bases between any two of these values. In some embodiments, the nucleic acid amplification products of a given target sequence have the same length or substantially the same length (e.g., within 1 to 10 bases). Therefore, the nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel), and generally do not produce multiple signals indicating multiple lengths (e.g., a ladder or tail on an electrophoresis gel). For multiple reactions, the nucleic acid amplification products of different target sequences can have different lengths.

[0179] The methods and components described herein can be used for multiple amplification, which generally refers to the amplification of more than one nucleic acid of interest (for example, the amplification of more than one target sequence). For example, multiple amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in the sample. For example, the amplification step can include the multiple amplification of two or more target nucleic acid sequences, and the detection step can include the multiple detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. Two or more target nucleic acid sequences can be specific to two or more different organisms (for example, one or more of SARS-CoV-2, influenza A, influenza B and / or influenza C). Multiple amplification can also refer to amplifying one or more sequences present in multiple samples simultaneously or in a stepwise manner. For example, multiple amplification can be used to amplify at least two target sequences that can be amplified (for example, the amplification reaction includes suitable primers and enzymes to amplify at least two target sequences). In some embodiments, an amplification reaction is prepared to detect at least two target sequences, but only one target sequence exists in the sample being tested, so that although both sequences can be amplified, only one sequence is amplified. In some embodiments, when there are two target sequences, the amplification reaction results in the amplification of the two target sequences. Multiple amplification reactions can result in the amplification of one, some or all target sequences, wherein appropriate primers and enzymes are included. In some embodiments, an amplification reaction is prepared to detect two sequences with a pair of primers, wherein one sequence is a target sequence, and one sequence is a control sequence (e.g., a synthetic sequence that can be amplified by the same primer as the target sequence and has a spacer base or sequence different from the target). In some embodiments, an amplification reaction is prepared to detect multiple groups of sequences with corresponding primer pairs, wherein each group of sequences includes a target sequence and a control sequence.

[0180] Primers

[0181] Nucleic acid amplification is usually carried out in the presence of one or more primers. Primers are usually characterized as oligonucleotides, which include nucleotide sequences that can hybridize or anneal with target nucleic acids at or near (e.g., adjacent to) a specific region of interest (i.e., a target sequence). For example, a primer can allow the specific determination of a target nucleic acid nucleotide sequence or the detection of a target nucleic acid or its characteristics (e.g., the presence or absence of a sequence). Primers can be naturally occurring or synthetic. The term specific, or specificity, usually refers to the combination or hybridization of a molecule with another molecule, such as a primer for a target polynucleotide. That is, specific or specific refers to the recognition, contact and formation of a stable complex between two molecules, in contrast, any one of the two molecules with the recognition, contact or complex formation of other molecules is much less. The term annealing or hybridization usually refers to the formation of a stable complex between two molecules. When referring to primers, the terms primer, oligomer or oligonucleotide can be used interchangeably herein.

[0182] Primers can be designed and synthesized using a suitable method, and can have any length suitable for hybridizing with the target sequence and performing amplification processes described herein. Primers are generally designed according to the sequence in the target nucleic acid. Primer length in some embodiments can be from about 5 to about 30 bases, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length. Primers can include naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides) or mixtures thereof. Modified and modified bases can include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite TM , Adenylation, Azide (NHS ester), Digoxigenin (NHS ester), Cholesteryl-TEG, I-Linker TM , amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, Uni-Link TM amino modifications), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylations (e.g., biotin, biotin(azide), biotin-dT, biotin-TEG, dibiotin, PC-biotin, desthiobiotin-TEG), thiol modifications (e.g., thiol modifier C3 SS, dithiol, thiol modifier C6 SS)); fluorophores (e.g., Freedom TM Dye, Alexa Dye, LI-COR ATTO TM Dyes, Rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas -X (NHS ester), 640 (NHS ester), Dy 750 (NHS ester), Iowa Dark quencher modification (e.g., Iowa FQ, Iowa RQ), dark quencher modification (e.g., Black Hole -1. Black Hole -2, Dabcyl); spacers (C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, reverse dT, reverse dideoxy-T, dideoxy-C, 5-methyl dC, deoxyinosine, Super Super Locked nucleic acids (LNA's), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), iso-dC, iso-dG, fluorine C, fluorine U, fluorine A, fluorine G; phosphorothioate (PS) bond modifications (e.g., phosphorothioate DNA bases, phosphorothioate RNA bases, phosphorothioate 2'-O-methyl bases, phosphorothioate LNA bases); and click chemistry modifications. In some embodiments, the modified and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS connections, 3' phosphate groups, spacer bases (such as, C3 spacer bases or other spacer bases). For example, a primer may contain one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is typically a post-transcriptional modification of RNA found in tRNA and other small RNAs. Primers comprising 2'-O-methyl RNA bases can be directly synthesized. For example, this modification can increase the Tm of RNA: RNA duplexes and provide stability in the presence of single-stranded ribonucleases and DNA enzymes. 2'-O-methyl RNA bases can be included in primers, for example, to increase stability and binding affinity to target sequences. In some embodiments, primers can include one or more phosphorothioate (PS) connections (for example, PS key modifications). PS keys replace non-bridging oxygen in the primer phosphate backbone with sulfur atoms. This modification usually makes internucleotide bonds resist nuclease degradation. For example, PS keys can be introduced between about the last 3 to 5 nucleotides of the 5'-end or 3'-end of the primer to inhibit exonuclease degradation. In some embodiments, PS keys included in the entire primer can help reduce the attack of endonucleases. Primers can, for example, include 3' phosphate groups. In some cases, 3' phosphorylation can inhibit the degradation of certain 3'-exonucleases and can be used to block the extension of DNA polymerase. In some embodiments, the primer comprises one or more spacer bases (e.g., one or more C3 spacers). The C3 spacer phosphoramidite can be incorporated into the interior or 5' end of the primer. For example, multiple C3 spacers can be added to either end of the primer to introduce a long hydrophilic spacer arm for attaching a fluorophore or other pendant group.

[0183] Primer can comprise DNA base, RNA base or both, wherein one or more DNA base and RNA base are modified or unmodified.For example, primer can be a mixture of DNA base and RNA base.Primer can be made up of DNA base (for example, modified DNA base and / or unmodified DNA base).In some embodiments, primer is made up of unmodified DNA base.In some embodiments, primer is made up of modified DNA base.Primer can be made up of RNA base (for example, modified RNA base and / or unmodified RNA base).In some embodiments, primer is made up of unmodified RNA base.In some embodiments, primer is made up of modified RNA base.In some embodiments, primer does not comprise RNA base.In some embodiments, primer does not comprise DNA base.In some embodiments, primer does not comprise cleavage agent recognition site (for example, does not comprise nickase recognition site).In some embodiments, primer does not comprise tail (for example, does not comprise the tail of nickase recognition site).

[0184] In some embodiments, all or part of the primer sequence can be complementary or substantially complementary to the target nucleic acid.Substantially complementary about sequence generally refers to nucleotide sequences that will hybridize to each other.The stringency of hybridization conditions can be changed to tolerate different numbers of sequence mispairings.The target sequence and primer sequence can be, for example, at least 75% complementary to each other, including 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to each other.The primer substantially complementary to the target nucleic acid sequence is also generally identical with the complement of the target nucleic acid sequence (that is, the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid can be at least 75% identical in sequence, e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to each other.

[0185] In some embodiments, primer includes a pair of primers. A pair of primers can include a forward primer and a reverse primer (for example, a primer combined with the sense and antisense strands of the target nucleic acid). In some embodiments, primers are composed of a pair of primers (that is, a forward primer and a reverse primer). Therefore, in some embodiments, a pair of primers is used to amplify the target sequence, and other primers or oligonucleotides are not included in the amplification of the target sequence (for example, the amplification reaction component does not include other primer pairs of a given target sequence, does not include nested primers, does not include a buffer primer (bumper primer), does not include oligonucleotides other than primers, does not include probes, etc.). In some embodiments, primers are composed of a pair of primers. In some embodiments, amplification reaction can include other primer pairs for amplifying different target sequences, such as in multiple amplification. In some embodiments, primers are composed of a pair of primers, however, in some embodiments, amplification reaction can include other primers, oligonucleotides or probes for detection process, and this detection process is not considered to be a part of amplification. In some embodiments, primers are used in groups. An amplification primer set may include a pair of forward and reverse primers for a given target sequence. For multiplex amplification, primers that amplify a first target sequence are considered a primer set, and primers that amplify a second target sequence are considered a different primer set.

[0186] Nucleic acid described herein (e.g., amplification product, sample nucleic acid, target nucleic acid sequence) can include a first strand and a second strand that are complementary to each other. Amplification reaction components can include or consist of a first primer (first oligonucleotide) complementary to a target sequence in a first strand (e.g., sense strand, forward strand) of a sample nucleic acid and a second primer (second oligonucleotide) complementary to a target sequence in a second strand (e.g., antisense strand, reverse strand) of a sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) includes a first polynucleotide that is continuously complementary to a target sequence in a first strand of a sample nucleic acid, and the second primer (second oligonucleotide) includes a second polynucleotide that is continuously complementary to a target sequence in a second strand of a sample nucleic acid. The continuous complementarity of primer-target generally refers to a nucleotide sequence in a primer, wherein each base is paired with a corresponding ordered base in a target sequence in order, and a gap in the sequence, an additional sequence, or an unpaired base is not considered to be continuously complementary. In some embodiments, the primer does not include any additional sequence that is not continuously complementary to the target sequence (e.g., at the 5' and / or 3' end, or within the primer), for example, an additional sequence present in a tailing primer or a circularizing primer, and / or an additional sequence that provides a cleavage agent recognition site (e.g., a nickase recognition site). In some embodiments, the amplification reaction components do not include primers containing additional sequences (i.e., sequences other than the sequence that is continuously complementary to the target sequence), for example, tailing primers, circularizing primers, primers that can form a stem-loop structure, a hairpin structure, and / or additional sequences that provide a cleavage agent recognition site (e.g., a nickase recognition site), etc.

[0187] In some embodiments, the primer may include modifications such as one or more inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers, or any modifiers that change primer binding properties. In some embodiments, the primer may include detectable molecules or entities (e.g., fluorophores, radioisotopes, colorimetric agents, particles, enzymes, etc.).

[0188] Polymerase

[0189] Amplification reaction components (e.g., one or more amplification reagents) may include one or more polymerases. Polymerases are proteins that can specifically incorporate nucleotides to extend primer molecules (e.g., amplification primers described herein) for nucleic acid target sequences (e.g., primers annealing thereto) to extend the 3' hydroxyl termini of primer molecules. Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that are active at elevated reaction temperatures (e.g., higher than 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C). Hyperthermophilic polymerases may be referred to as hyperthermophilic biopolymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, polymerases may incorporate about 1 to about 50 nucleotides in a single synthesis, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in a single synthesis, or numbers or ranges between any two of these values.

[0190] The amplification reaction components may include one or more DNA polymerases selected from the group consisting of: 9°N DNA polymerase; 9°Nm TM DNA polymerase; Therminator TM DNA polymerase; Therminator TM IIDNA polymerase; Therminator TM IIIDNA polymerase; Therminator TM gamma DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exosome); T4 DNA polymerase; T7 DNA polymerase; Deep VentR TM (Exo)DNA polymerase; Deep VentR TM DNA polymerase; DyNAzyme TM EXT DNA; DyNAzyme TMII hot start DNA polymerase; Phusion TM High-fidelity DNA polymerase; DNA polymerase; (Exo)DNA polymerase; RepliPHI TM Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm TM DNA polymerase); MasterAmp TM AmpliTherm TM DNA polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase β; and ThermoPhi DNA polymerase.

[0191] In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermally stable at high temperatures). The hyperthermophilic DNA polymerase may have a half-life of about 5 to 10 hours at 95°C and a half-life of about 1 to 3 hours at 100°C. For example, the amplification reaction components may include one or more hyperthermophilic DNA polymerases from archaea (e.g., hyperthermophilic DNA polymerases from Thermococcus or hyperthermophilic DNA polymerases from Thermococcaceae archaea). In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components comprise one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0192] In some embodiments, the amplification reaction components include hyperthermophilic organism DNA polymerase or its functional fragment. Functional fragments usually retain one or more functions of full-length polymerase, such as the ability of polymerized DNA (for example, in amplification reaction). In some cases, functional fragments perform functions (for example, polymerized DNA in amplification reaction) at least about 50%, at least about 75%, at least about 90%, at least about 95% of the level of full-length polymerase functional level. The level of polymerase activity can be, for example, assessed using a detectable nucleic acid amplification method, such as described herein. In some embodiments, the amplification reaction components include hyperthermophilic organism DNA polymerase, which includes the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8 or the functional fragment of SEQID NO:7 or SEQ ID NO:8.

[0193] In some embodiments, the amplification reaction components (e.g., one or more amplification reagents) comprise a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophilic biopolymerase or a functional fragment thereof. In some embodiments, the amplification reaction components comprise a polymerase comprising an amino acid sequence at least about 90%, 95%, or 99% identical to an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or a functional fragment thereof.

[0194] The polymerase may have reverse transcription capability. In such an embodiment, the amplification reaction may amplify the RNA target in a single step without using a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase capability include Bst (large fragment), 9°N DNA polymerase, 9°Nm TM DNA polymerase, Therminator TM 、Therminator TM II, etc. The amplification reaction components may include one or more independent reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction may include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity.

[0195] In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases without or with low exonuclease activity are used during amplification. In some embodiments, the polymerase without or with low exonuclease activity comprises one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, compared with unmodified polymerase, the modified polymerase with low exonuclease activity can have 10% or less exonuclease activity, for example, compared with unmodified polymerase, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% exonuclease activity. In some embodiments, the polymerase does not have 5' to 3' exonuclease activity or has low 5' to 3' exonuclease activity, and / or does not have 3' to 5' exonuclease activity or has low 3' to 5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand-dependent exonuclease activity, and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of the polymerase include one or more amino acid substitutions at positions 141 and / or 143 and / or 458 of SEQ ID NO:7 (e.g., D141A, E143A, E143D, and A485L) or at positions corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:7.

[0196] Detection and quantification

[0197] The methods described herein may include detecting and / or quantifying nucleic acid amplification products. For example, the amplification products may be detected and / or quantified by any suitable detection and / or quantification method described herein. Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, endpoint), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5' to 3' exonuclease hydrolysis probes (e.g., TAQMAN), embedding / binding dyes, absorbance (e.g., colorimetry, turbidimetry), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, quantitative amplification, primer extension methods (e.g., iPLEX TM), molecular inversion probe (MIP) technology from Affymetrix, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, gene chip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, genetic bit analysis (GBA), multiplex microsequencing, SNaPshot, GOOD assay, microarray microsequencing, array primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed incorporation (TDI), colorimetric oligonucleotide ligation assay (OLA), sequence-encoded OLA, microarray ligation, ligase chain reaction, padlock probes, invader assay (invader In some embodiments, the detection of nucleic acid amplification products includes the use of real-time detection methods (i.e., detection and / or continuous monitoring of products during the amplification process). In some embodiments, the detection of nucleic acid amplification products includes the use of endpoint detection methods (i.e., detection of products after completion or cessation of the amplification process). Nucleic acid detection methods can also be used using labeled nucleotides directly incorporated into the target sequence or in a probe containing a target complementary sequence. Such labels can be radioactive and / or fluorescent in nature and can be distinguished in any manner discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, the detection method can be used in conjunction with the measurement of signal intensity and / or the generation (or reference) of a standard curve and / or a lookup table for quantitative nucleic acid amplification products.

[0198] Detection of nucleic acid amplification products can include the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule, wherein the detectable properties of the molecule are detectable under certain conditions, so that the molecule can function as a specific and informative signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or speed of passing through a known size opening. Molecular beacons for detecting nucleic acid molecules can be, for example, hairpin-shaped oligonucleotides, one end of which contains a fluorophore, and the other end contains a quenching dye. The loop of the hairpin can contain a probe sequence complementary to the target sequence, and the stem is formed by annealing of the complementary arm sequence on either side of the probe sequence. Fluorophores and quenching molecules can be covalently linked at the opposite ends of each arm. Under conditions that prevent oligonucleotides from hybridizing with their complementary targets, or when molecular beacons are free in solution, fluorescent molecules and quenching molecules approach each other, thereby preventing FRET. When molecular beacons encounter target molecules (e.g., nucleic acid amplification products), hybridization can occur, and the ring structure is converted into a stable, more rigid conformation, resulting in the separation of fluorophores and quencher molecules, thereby generating fluorescence. Due to the specificity of the probe, the generation of fluorescence is usually entirely due to the synthesis of the expected amplified product. In some cases, the molecular beacon probe sequence hybridizes with the sequence in the amplified product, and the sequence in the amplified product is identical or complementary to the sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes with the sequence in the amplified product, and the sequence in the amplified product is not identical or complementary to the sequence in the target nucleic acid (for example, hybridizing with a sequence added to the amplified product by tailing amplification primers or connection). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with fluorophores of different colors and different target sequences, so that several products can be detected simultaneously in the same reaction (for example, in multiple reactions). For quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each amplification cycle, and because the unhybridized molecular beacons are dark, it is not necessary to separate the probe-target hybrid to quantitatively determine the amount of the amplified product. The signal generated is proportional to the amount of the amplified product. Detection using molecular beacons can be completed in real time or as an end point detection method.

[0199] Detection of nucleic acid amplification products may include the use of lateral flow. The use of lateral flow generally includes the use of lateral flow devices, including but not limited to test strip assays and thin layer chromatography plates with various suitable coatings. Fixed in the flow path are various binding reagents for the sample, binding partners, or conjugates involving binding partners for the sample and a signal generating system.

[0200] Detection of nucleic acid amplification products can include the use of FRET, which is an energy transfer mechanism between two chromophores (donor and acceptor molecules). In short, the donor fluorophore molecule is excited at a specific excitation wavelength. When the donor molecule returns to its ground state, the subsequent emission of the donor molecule can transfer the excitation energy to the acceptor molecule through a long-range dipole-dipole interaction. The emission intensity of the acceptor molecule can be monitored and changes with the distance between the donor and the acceptor, the overlap of the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be used for quantitative molecular dynamics, for example, in the DNA-DNA interactions described for molecular beacons. In order to monitor the generation of a specific product, the probe can be labeled with a donor molecule on one end and an acceptor molecule on the other end. Probe-target hybridization causes the distance or orientation of the donor and the acceptor to change, and FRET changes are observed.

[0201] Detecting nucleic acid amplification products can include the use of fluorescence polarization (FP). FP technology is based on the principle that when excited by linearly polarized light, a fluorescently labeled compound will emit fluorescence, and its degree of polarization is inversely proportional to its rotation rate. Therefore, when a molecule with a fluorescent label such as a tracer-nucleic acid conjugate is excited by linearly polarized light, the emitted light remains highly polarized because the fluorophore is constrained and cannot rotate between the time when the light is absorbed and emitted. When a free tracer compound (i.e., not bound to a nucleic acid) is excited by linearly polarized light, it rotates much faster than the corresponding tracer-nucleic acid conjugate, and the molecule is more randomly oriented, so the emitted light is depolarized. Therefore, fluorescence polarization provides a quantitative method for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0202] Detection of nucleic acid amplification products can include the use of surface capture, such as by fixing specific oligonucleotides to the surface to achieve, thereby producing a biosensor that is both highly sensitive and selective. Example surfaces that can be used to attach probes include gold and carbon. Detection of nucleic acid amplification products can include the use of 5' to 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 TAQMAN probe principle relies on 1) the 5' to 3' exonuclease activity of Taq polymerase to cleave dual-labeled probes during hybridization with complementary target sequences and 2) fluorophore-based detection. The fluorescent signal generated allows the accumulation of amplified products to be quantitatively measured during the exponential phase of amplification, and the TAQMAN probe can significantly increase the specificity of detection.

[0203] Detection of nucleic acid amplification products may include the use of intercalating and / or binding dyes, including dyes that specifically stain nucleic acids (e.g., intercalating dyes that exhibit enhanced fluorescence when bound to DNA or RNA). Dyes may include DNA or RNA intercalating fluorophores, including but not limited to 82. Acridine orange, ethidium bromide, Hoechst dye, Propidium iodide, I (asymmetric cyanine dyes), II, TOTO (thiazole orange dimer) and YOYO (oxazole yellow dimer). Detection of nucleic acid amplification products can include the use of absorbance methods (e.g., colorimetry, turbidimetry). In some embodiments, the detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260nm) to concentration. The direct measurement of nucleic acids can be converted to concentration using the Beer Lambert law, which uses the measured path length and extinction coefficient to associate absorbance with concentration. Detection of nucleic acid amplification products can include the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or the use of mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and amount of nucleic acids, and can be used to provide rapid analysis of complex mixtures. After amplification, the sample can be ionized, and the ions produced are separated in an electric field and / or magnetic field according to their mass-to-charge ratio, and the detector measures the mass-to-charge ratio of the ions. Mass spectrometry 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 (eg, matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can have high throughput due to high-speed signal acquisition and automated analysis off solid surfaces.

[0204] Detecting nucleic acid amplification products can include using nucleic acid sequencing. The entire sequence or a partial sequence of the amplified product can be determined, and the determined nucleotide sequence can be referred to as a read. For example, a linear amplification product can be directly analyzed without further amplification (e.g., by using a single molecule sequencing method). In some embodiments, the linear amplification product is further amplified and then analyzed (e.g., using a ligation sequencing or pyrophosphate sequencing method). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, sequencing based on reversible terminators, ligation sequencing, pyrophosphate sequencing, synthetic sequencing, single molecule sequencing, multiple sequencing, solid phase single nucleotide sequencing, and nanopore sequencing. Detecting nucleic acid amplification products can include using digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., company).

[0205] Lysis buffer

[0206] Lysis Agent

[0207] As disclosed herein, the lysing agent may include a detergent. The detergent may include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The anionic surfactant may include NH4 + , K + 、Na + or Li + As a counter ion. Cationic surfactants may include I - Br - or Cl - as a counter ion.

[0208] The lysing agent provided herein can serve as a denaturant. "Denaturing agent" or "denaturant" used herein should be given its common meaning, and include any compound or material that will cause the reversible unfolding of the protein. The intensity of the denaturing agent or denaturant will be determined by the characteristics and concentration of the specific denaturing agent or denaturant. Suitable denaturing agents or denaturants include chaotropes, detergents, organic solvents, water-miscible solvents, phospholipids or a combination of two or more such agents. Suitable chaotropes include but are not limited to urea, guanidine and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), sodium lauryl creatine (sarkosyl), mild nonionic detergents (e.g., digitonin), mild cationic detergents (e.g., N->2,3-(dioleyloxy)-propyl-N,N,N-trimethylammonium), mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including but not limited to sulfobetaines (Zwittergent), 3-(3-chloroamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO). Organic, water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols, such as ethanol or isopropanol), or lower alkane diols (especially C2-C4 alkanols such as ethylene glycol) can be used as denaturants. The phospholipids can be naturally occurring phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol, or synthetic phospholipid derivatives or variants, such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0209] Suitable surfactant levels can be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, the use of anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof) can be advantageous.

[0210] Anionic surfactants useful herein include water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl group, and water-soluble salts of sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant.

[0211] Suitable cationic surfactants can be broadly defined as derivatives of fatty quaternary ammonium compounds having a long alkyl chain containing about 8 to 18 carbon atoms, such as lauryl trimethyl ammonium chloride; cetyl pyridinium chloride; benzalkonium chloride; cetyl trimethyl ammonium bromide; diisobutyl phenoxyethyl dimethyl benzyl ammonium chloride; coconut alkyl trimethyl ammonium nitrite; cetyl pyridinium fluoride, etc. In the compositions disclosed herein, certain cationic surfactants may also act as bactericides.

[0212] Suitable nonionic surfactants that can be used in the compositions, methods and kits of the present disclosure can be broadly defined as compounds produced by the condensation of alkylene oxide groups (hydrophilic in nature) with organic hydrophobic compounds that can be aliphatic and / or aromatic in nature. Examples of suitable nonionic surfactants include poloxamers; sorbitan derivatives, such as sorbitan diisostearate; ethylene oxide condensates of hydrogenated castor oil, such as PEG-30 hydrogenated castor oil; ethylene oxide condensates of fatty alcohols or alkylphenols; products produced by the condensation of ethylene oxide with the reaction products of propylene oxide and ethylenediamine; long chain tertiary amine oxides; long chain tertiary phosphine oxides; long chain dialkyl sulfoxides and mixtures of these materials. These materials can be used to stabilize foam without causing excessive viscosity to the consumer product composition.

[0213] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphine, and sulfone compounds in which the aliphatic radical can be straight or branched chain and one of the aliphatic substituents contains from about 8 to 18 carbon atoms and one contains an anionic water-solubilizing group such as carboxy, sulfonate, sulfate, phosphate, or phosphonate.

[0214] Exemplary anionic, single-chain surfactants include alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates and saturated or unsaturated fatty acids and salts thereof. The portion containing the polar head group in the cationic surfactant may include, for example, quaternary ammonium, pyridine, sulfonium and / or phosphonium groups. For example, the polar head group may include trimethylammonium. Exemplary cationic, single-chain surfactants include alkyl trimethyl ammonium halides, alkyl trimethyl toluene sulfonate ammonium and N-alkyl pyridinium halides.

[0215] reducing agent

[0216] Lysis buffer and / or reagent composition (for example, dry composition) can include one or more reducing agents. "Reducing agent" can be a compound or a group of compounds. As used herein, "reducing agent (reducing agent)", also referred to as "reducing agent (reductant)", "reducing substance" or "reduction equivalent", can refer to an element or compound that provides electrons to another substance. In particular, a reducing agent is a compound that destroys a disulfide bond by reduction, thereby overcoming those tertiary protein folding and quaternary protein structures (oligomeric subunits) stabilized by disulfide bonds. The example of a suitable reducing agent includes but is not limited to 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythritol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, sulfite, thioglycolic acid and HED. In some embodiments of the method, composition and kit provided herein, lysis buffer and / or reagent composition (for example, dry composition) do not include one or more reducing agents.

[0217] Reagent composition

[0218] The reagent compositions described herein (e.g., dry compositions) can be provided in a "dry form" or in a form that is not suspended in a liquid medium. The "dry form" of the composition can include a dry powder, a freeze-dried composition, a spray-dried or precipitated composition. The "dry form" composition can contain one or more lyoprotectants, such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly (ethylene glycol) or poly (propylene glycol); and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch and carboxymethyl cellulose. As used herein, the terms "lyophilization", "freeze-dried" and "freeze drying" refer to the process of first freezing the material to be dried and then removing the ice or freezing solvent by sublimation in a vacuum environment. "Lyophilate" refers to a freeze-dried substance.

[0219] The reagent composition (for example, dry composition) can be frozen or lyophilized or spray-dried. The reagent composition can be heat dried. The reagent composition can include one or more additives (for example, amino acid, polymer, sugar or sugar alcohol). Sugar or sugar alcohol can include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof. Polymer can include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptide, collagen peptide or any combination thereof. Freeze-dried reagent can include poly-rA, EGTA, EDTA, Tween 80 and / or Tween 20.

[0220] The frozen or lyophilized or spray-dried or heat-dried composition or the aqueous composition used to prepare the frozen or lyophilized or spray-dried composition may contain one or more of the following: (i) non-aqueous solvents such as ethylene glycol, glycerol, dimethyl sulfoxide and dimethylformamide. (ii) surfactants such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic F127 (polyoxyethylene-polyoxypropylene copolymer) is also known as poloxamer, poloxamine and sodium lauryl sulfate. (iii) disaccharides such as trehalose, sucrose, lactose and maltose. (iv) polymers (which may have different MW) such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll and albumin. (v) amino acids such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine and γ-aminobutyric acid.

[0221] The reagent composition (e.g., dry composition) can include one or more protective agents and one or more amplification reagents. One or more protective agents can include cyclodextrin compounds. Cyclodextrin (CD) can be used for complexing with a lysing agent (e.g., SDS). Cyclodextrin (CD) can be a truncated cone-like cyclic oligosaccharide with a hydrophobic inner cavity and a hydrophilic outer surface. The most commonly used natural cyclodextrins include 6, 7, and 8 glucose units, known as α, β, and γ-CD. Natural CD can have solubility. Chemically modified CDs such as hydroxypropyl derivatives increase solubility in aqueous media by up to 50%. It is the trade name of WACKER cyclodextrin derivatives, including various α, β and γ-CD derivatives. β-CD can form a strong inclusion complex with sodium dodecyl sulfate (SDS) (stronger than α-CD and β-CD), and the stoichiometric ratio is mainly 1:1. The binding constant of β-CD with SDS can range from 2100M -1 Up to 2500M -1 .

[0222] Reagent test kit

[0223] 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 may comprise: a lysis buffer, comprising one or more lysing agents capable of lysing a biological entity to release a sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of containing a target nucleic acid sequence, optionally one or more lysing agents comprising a detergent, and wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The kit may comprise: a reagent composition comprising one or more amplification reagents, the amplification reagent comprising one or more components for amplification for amplifying a target nucleic acid sequence under isothermal amplification conditions, wherein one or more components for amplification comprise: (i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing with a sequence of a first chain of a target nucleic acid sequence, and the reverse primer is capable of hybridizing with a sequence of a second chain of a target nucleic acid sequence; and (ii) an enzyme having a hyperthermophilic biopolymerase 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.

[0224] The kit may include: at least one component that provides real-time detection activity for nucleic acid amplification products. The real-time detection activity may be provided by a molecular beacon. The reagent composition (eg, a dried composition) may include a reverse transcriptase and / or a reverse transcription primer.

[0225] The molar ratio of one or more protective agents to one or more amplification reagents can be between about 10: 1 and about 1: 10 (e.g., about 2: 1). In some embodiments, one or more additives include Tween 20, Triton X-100, Tween 80, nonionic detergents (e.g., nonionic surfactants) or any combination thereof. In some embodiments, one or more protective agents include cyclodextrin compounds. In some embodiments, one or more cleavage reagents constitute about 0.001% (w / v) to about 1.0% (w / v) (e.g., about 0.2% (w / v)) of the treated sample. In some embodiments, one or more cleavage agents include detergents. Detergents can include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. In some embodiments, it is advantageous that one or more protective agents can isolate one or more cleavage agents, thereby preventing one or more amplification reagents from being denatured by one or more cleavage agents.

[0226] The kit may include, for example, one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, as described herein. In the case of amplifying one target, a pair of primers (forward and reverse) may be included in the kit. In the case of amplifying multiple target sequences, more than one primer pair may be included in the kit. The kit may include a control polynucleotide, and in the case of amplifying multiple target sequences, more than one control polynucleotide may be included in the kit.

[0227] The enzyme having hyperthermophilic biopolymerase activity may have an amino acid sequence at least about 90% or 95% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof. For example, the enzyme having hyperthermophilic biopolymerase activity may include the amino acid sequence of SEQ ID NO: 7.

[0228] The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may comprise: (1) the sequence of a first primer and its reverse complement, (2) the sequence of a second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length.

[0229] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a viral particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoa. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoa.

[0230] Test kit can also include one or more components in any number of independent vessels, chambers, containers, packets, test tubes, vials, microtiter plates, etc., or components can be combined in such containers in various combinations. For example, the components of test kit can be present in one or more containers. In some embodiments, all components are provided in a container. In some embodiments, enzyme (for example, one or more polymerases and / or one or more reverse transcriptases) can be provided in a container separated from primers. Component can be, for example, freeze-dried, heat-dried, lyophilized or in a stable buffer. In some embodiments, one or more polymerases and / or one or more reverse transcriptases are in a single container in a lyophilized form or a heat-dried form, and primers are freeze-dried, heat-dried, lyophilized or in a buffer in different containers. In some embodiments, polymerase and / or reverse transcriptase and primers are in a single container in a lyophilized form or a heat-dried form.

[0231] The kit may also include, for example, dNTPs used in the reaction, or modified nucleotides, vessels, cuvettes or other containers for the reaction, or water or buffer for rehydrating vials of freeze-dried or heat-dried components. For example, the buffer used may be suitable for both polymerase and primer annealing activity.

[0232] The kit may also include instructions for performing one or more of the methods described herein and / or a description of one or more of the components described herein. The instructions and / or description may be in printed form and may be included in a kit insert. The kit may also include a written description of an Internet location that provides such instructions or descriptions.

[0233] The kit may also contain reagents for detection methods, such as reagents for FRET, lateral flow devices, test strips, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads.

[0234] Example

[0235] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended in any way to limit the scope of the disclosure.

[0236] Example 1

[0237] FluBPB2 assay: non-specific product formation (no 2'OM protection)

[0238] This example shows that non-specific product formation between a reverse primer and a molecular beacon can generate a false positive signal in the absence of an assay target sequence. Figure 5A-5EDepicted are data showing the concentration-dependent trend between the FluB PB2 reverse primer and the occurrence of false-positive signals in no-target conditions (NTC). Figure 5A-5E Data showing non-specific product formation of FluB PB2 (without 2'OM protection) are depicted. FluB PB2 assay primers and probes are shown in Table 4. FluB PB2 reverse primer was titrated to 600 nM ( Figure 5A )、500nM( Figure 5B )、400nM( Figure 5C )、300nM( Figure 5D ) and 200nM( Figure 5E ). As the reverse primer concentration increased, the rate of false positive NTCs increased and the detection of these NTCs occurred earlier.

[0239] Table 4: FluB PB2 assay primers and probes

[0240]

[0241] Example 2

[0242] FluB PB2 assay: prevention of nonspecific product formation via 2'OM protection

[0243] This example shows that preventing the formation of undesired extension products mitigates false positive signals caused by read-through of primers. Figure 6B-6D Depicted are the results of three variants of the FluB PB2 molecular beacon containing a 2'OM modification. Fig. 6A ), it was observed that these molecular beacons had significantly fewer false positive signals in no-target conditions (NTCs). Figure 6A-6D Depicted are data related to the prevention of nonspecific product formation in the FluB PB2 assay by 2'OM-modified beacons. Depicted are data related to the prevention of nonspecific product formation in the FluB PB2 assay by 2'OM-modified beacons. Fig. 6A ; LNA3.13), 2'OM modified form 1 ( Figure 6B ; LNA3.13m1), 2'Om modified form 2 ( Figure 6C ; LNA3.13m2) and 2'OM modified version 3 ( Fig.6D ; LNA3.13m3) results. In some embodiments, the probes provided herein (e.g., molecular beacons) comprise a 5' modification (e.g., 5HEX). In some embodiments, the probes provided herein (e.g., molecular beacons) comprise a 3' modification (e.g., 3IAbRQSp).

[0244] Table 5: FluB PB2 assay probe

[0245]

[0246]

[0247] Example 3

[0248] False Positive Assessment for Neisseria Gonorrhoeae ("NG", "GC") and Chlamydia Trachomatis ("CT") Assays

[0249] This assay demonstrates prevention of false positives in assays for Neisseria gonorrhoeae and Chlamydia trachomatis via the protected probes provided herein.

[0250] First, 20 urine samples (10% in 1.11× GRBS) and 20 ProbeTec TM Swabs (1.67% in 1× GRBS) were evaluated for false positives using unmodified CT and GC ROX beacons. Figure 7A-7D Depicted is the use of 8U 9dN polymerase ( Fig. 7A , Figure 7C ) and 12U 9dN polymerase ( Figure 7B , Fig.7D ) with NTC 10% urine sample ( Figure 7A-7B ) and NTC vaginal swab samples ( Figure 7C-D ) are data related to false positive evaluation in the Neisseria gonorrhoeae and Chlamydia trachomatis assays performed by . Tables 6 and 7 depict the results of testing NTC urine samples and NTC vaginal swab samples, respectively, with different amounts of 9dN polymerase.

[0251] Table 6: False Positive Assessment – ​​10% Urine

[0252] 9dN 8U 12U Neisseria gonorrhoeae assay 1 / 20(5％) 4 / 20(20％) Chlamydia trachomatis assay 8 / 20(40％) 7 / 20(35％)

[0253] Table 7: False Positive Assessment – ​​1.67% Vaginal Matrix

[0254] 9dN 8U 12U Neisseria gonorrhoeae assay 8 / 20(40％) 9 / 20(45％) Chlamydia trachomatis assay 6 / 20(30％) 4 / 20(20％)

[0255] Next, a false positive comparison of the nominal probe and the modified (protected) probe was performed using NTC urine samples (N=20). Figure 8A-8D Plotted with the nominal probe ( Fig. 8A , Figure 8C ) and protected probes ( Figure 8B , Fig.8D ) Chlamydia trachomatis assay using NTC15% urine sample ( Figures 8A-8B ) and Neisseria gonorrhoeae assay ( Figure 8C-Figure 8D) in the assay. In the Chlamydia trachomatis assay, the use of the protected probe resulted in a reduction in false positives from 7 / 20 (35%) to 1 / 20 (5%), with the remaining single false positive attributed to suspected target template contamination. Therefore, this higher false positive frequency was mitigated by O-methyl modification of the beacon. In the Neisseria gonorrhoeae assay, identical performance was observed between the two probes (e.g., no false positives were observed).

[0256] False positive comparisons of the nominal probe and the modified (protected) probe were then performed using vaginal swabs (N=20). Figure 9A-9D Plotted with the nominal probe ( Fig.9A , Fig. 9C ) and protected probes ( Fig. 9B , Fig.9D ) Chlamydia trachomatis assay using vaginal swab samples ( Figure 9A-9B ) and Neisseria gonorrhoeae assay ( Figure 9C-D ). A higher number of false positives were observed with vaginal swab samples, which were mitigated for both assays by O-methyl modification of the beacon. In the Chlamydia trachomatis assay, use of the protected probe resulted in a reduction in false positives from 9 / 20 (45%) to 0 / 20 (0%). In the Neisseria gonorrhoeae assay, use of the protected probe resulted in a reduction in false positives from 1 / 20 (5%) to 0 / 20 (0%).

[0257] Next NTC testing was performed with modified probes using NTC 10% urine samples (N=40).For C. trachomatis, the assay was switched to the HEX beacon (O-methyl modified) and 0% FP was observed for C. trachomatis and 2.5% FP was observed for N. gonorrhoeae (ROX). Figures 10A-10D Depicted is a Chlamydia trachomatis assay performed with NTC urine samples and using protected probes ( Figure 10A-10B ) and Neisseria gonorrhoeae assay ( Figure 10C-10D ) in the data related to false positive assessment.

[0258] Next NTC vaginal swab samples were used for NTC testing with modified probes (1.67% VM (N=20)). 3-in-1 swabs were expressed in 1×GRBS (1.67%) and assayed using CT HEX (2′O-methyl modified beacon) and GC ROX (2′O-methyl modified). Figure 11A-11B Depicts the Chlamydia trachomatis assay performed with NTC vaginal swab samples and using protected probes ( Fig.11A ) and Neisseria gonorrhoeae assay ( Fig. 11B 0% FP was observed for both assays.

[0259] Example 4

[0260] FluA assay: a beacon of RNA modifications

[0261] This example shows the use of RNA base incorporation for protection of molecular beacons. Figure 13A-13C Depicted are data related to RNA base incorporation for protection of molecular beacons in a FluA assay. The assay was performed with NTC primer-only titration ( Figure 13A-13B ) or probes are used to screen targets ( Fig. 13C ). Under the forward and reverse primer conditions, the RNA-modified probe (LP2(rna3)) had the same false positive rate as the nominal probe (LP2), the RNA-modified probe LP2(rna1) detected no false positives, and the RNA-modified probe LP2(rna2) detected 1 / 4 of the false positives (Figure 14A). Under the reverse primer only condition, RNA modification of the probe helped to mitigate false positive detections ( Fig. 13B ). When using a target screening probe ( Fig. 13C ), the RNA-modified probe LP2(rna3) had the same FP rate as the nominal probe LP2 (3 out of 4 NTCs detected). This shows that RNA modifications negatively affect the binding of target products to the probe (the lack of false positives observed with LP2(rna1) and LP2(rna2) may be due to this). This example shows that RNA base modifications of the probe can provide protection against nonspecific product formation.

[0262] In at least some previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0263] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art may convert from the plural to the singular and / or from the singular to the plural where appropriate for the context and / or application. For clarity, various singular / plural arrangements may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0264] Those skilled in the art will understand that, in general, the terms used herein, and especially in the appended claims (e.g., the bodies of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "includes but is not limited to", etc.). Those skilled in the art will further understand that if a specific number of an introduced claim statement is intended, such an intent will be explicitly stated in the claim, and in the absence of such a statement, no such intent is present. For example, as an aid to understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim statements. However, the use of such words should not be interpreted as meaning that introduction of a claim statement by the indefinite article "a" or "an" will limit any particular claim containing such introduced claim statement to embodiments containing only one such statement, even when the same claim includes the introductory words "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim statements. In addition, even if a particular number of introduced claim statements is explicitly stated, one skilled in the art will recognize that such a statement should be interpreted to mean at least the stated number (e.g., merely stating "two statements" without other modifiers means at least two statements or two or more statements). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, generally such syntactic structure is intended so that one skilled in the art will understand the meaning of the convention (e.g., “a system having at least one of A, B, and C” would include but is not limited to systems having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to “at least one of A, B, or C, etc.” is used, generally such syntactic structure is intended so that one skilled in the art will understand the meaning of the convention (e.g., “a system having at least one of A, B, or C” would include but is not limited to systems having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will further appreciate that, in fact, any disjunctive words and / or phrases presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one, either or both terms.

[0265] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0266] As will be understood by those skilled in the art, for any and all purposes, such as in providing written description, all scopes disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges of the scope.Any listed scope can be easily identified as fully describing and enabling the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As non-limiting examples, each scope discussed herein can be easily decomposed into lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", etc. include stated numbers, and refer to the scope that can be subsequently decomposed into sub-ranges as discussed above.Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles.Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0267] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method for detecting a target nucleic acid sequence in a sample, comprising: amplifying the target nucleic acid sequence in the amplification reaction mixture under isothermal amplification conditions to produce a nucleic acid amplification product; and The nucleic acid amplification product is detected using a signal generating oligonucleotide, wherein the signal generating oligonucleotide is capable of hybridizing to the nucleic acid amplification product and comprises one or more polymerase stoppers.

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

3. The method according to any one of claims 1 to 2, wherein: The signal generating oligonucleotide comprises a 5' subdomain and a 3' subdomain, The signal generating oligonucleotide comprises a loop domain located between the 5' subdomain and the 3' subdomain, Intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain can form a paired stem domain, The one or more polymerase terminators are located in the loop domain, and The 5' subdomain, the paired stem domains and / or the 3' subdomain do not comprise the one or more polymerase stoppers.

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

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

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

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

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

9. The method according to any one of claims 1 to 8, wherein: The first region comprises at least a portion of the 5' subdomain and / or the loop domain, The spacer region comprises at least a portion of the loop domain, and The second region comprises at least a portion of the loop domain and / or the 3' subdomain.

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

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

12. The method according to claim 11, wherein: The stable abasic site includes 1',2'-dideoxy; The chemically captured abasic sites include abasic sites that react with alkoxyamines or sodium borohydride; The abasic site comprises an apurinic site, an apyrimidinic site, or both; and / or The abasic site is created by an alkylating agent or an oxidizing agent.

13. The method of any one of claims 1-12, wherein the one or more polymerase terminators comprise: one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromodeoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more isodeoxy Cytidine (Iso-dC), one or more isodeoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(o)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups or any combination thereof.

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

15. The method of any one of claims 1-14, 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. The method of any one of claims 1-15, wherein the signal generating oligonucleotide comprises a label, optionally the label comprises a quenchable label, and further optionally the quenchable label is a fluorophore.

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

18. The method according to any one of claims 1 to 17, in, When the forward primer binds to the signal generating oligonucleotide to form a first undesired duplex, the one or more polymerase terminators are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal generating oligonucleotide, Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal generating oligonucleotide.

19. The method according to any one of claims 1 to 18, in, When the reverse primer binds to the signal generating oligonucleotide to form a second undesired duplex, the one or more polymerase terminators are capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal generating oligonucleotide, Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase stoppers of the signal generating oligonucleotide.

20. The method according to any one of claims 1 to 19, in, When the exogenous nucleic acid binds to the signal generating oligonucleotide to form a third undesired duplex, the one or more polymerase terminators are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal generating oligonucleotide, Optionally, the one or more polymerase stoppers 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, Optionally, the exogenous nucleic acid is selected from the group consisting of: a sample nucleic acid, a primer configured to hybridize to a second target nucleic acid sequence, a primer configured to hybridize to an internal control, or any combination thereof.

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

22. The method of any one of claims 1-21, wherein amplifying the target nucleic acid sequence comprises: A target nucleic acid sequence comprising a first strand and a second strand complementary to each other is amplified under isothermal amplification conditions, wherein the amplification comprises contacting a nucleic acid comprising the target nucleic acid sequence with: i) a forward primer and a reverse primer, wherein the forward primer is capable of hybridizing to a sequence of 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 biopolymerase activity, thereby producing the nucleic acid amplification product.

23. The method of any one of claims 21-22, wherein the nucleic acid is double-stranded DNA.

24. The method of any one of claims 21-23, 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 sample ribonucleic acid, and optionally the amplification comprises generating the nucleic acid by a reverse transcription reaction.

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

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

27. The method according to any one of claims 1 to 26, in, If the forward primer binds to the signal generating oligonucleotide to form a first undesired duplex, the forward primer of the first undesired duplex is extended toward the 5' end of the signal generating oligonucleotide by an enzyme having a hyperthermophilic biopolymerase activity to generate a first undesired extension product, wherein the first undesired extension product is capable of being amplified by an enzyme having hyperthermophilic biopolymerase activity in the presence of the forward primer and the reverse primer to form a first undesired amplification product; and wherein the one or more polymerase terminators are capable of terminating polymerase extension of the forward primer of the first undesired duplex to produce a first stalled extension product, The first stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of the forward primer and the reverse primer to produce the first undesired amplification product.

28. The method of any one of claims 1-27, 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. The method according to any one of claims 1 to 28, in, If the reverse primer binds to the signal generating oligonucleotide to form a second undesired duplex, the reverse primer of the second undesired duplex is extended toward the 5' end of the signal generating oligonucleotide by an enzyme having a hyperthermophilic biopolymerase activity to generate a second undesired extension product, wherein the second undesired extension product is capable of being amplified by an enzyme having hyperthermophilic biopolymerase activity in the presence of the reverse primer to form a second undesired amplification product; and wherein the one or more polymerase terminators are capable of terminating polymerase extension of the reverse primer of the second undesired duplex to produce a second stalled extension product, wherein the second stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of the reverse primer to produce the second undesired amplification product.

30. The method of any one of claims 1-29, 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. The method according to any one of claims 1 to 30, in, If the exogenous nucleic acid binds to the signal generating oligonucleotide to form a third undesired duplex, the exogenous nucleic acid of the third undesired duplex is extended to the 5' end of the signal generating oligonucleotide by an enzyme having a hyperthermophilic biopolymerase activity to generate a third undesired extension product, wherein the third undesired extension product is capable of being amplified by an enzyme having hyperthermophilic biopolymerase activity in the presence of the reverse primer to form a third undesired amplification product; and wherein the one or more polymerase terminators are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex to produce a third stalled extension product, The third stalled extension product cannot be amplified by an enzyme having hyperthermophilic polymerase activity in the presence of the reverse primer to produce the third undesired amplification product.

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

33. The method of any one of claims 1-32, 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 1-33, wherein detecting the nucleic acid amplification product comprises using a real-time detection method.

35. The method according to any one of claims 1 to 34, wherein: The label is capable of generating a signal when the signal generating oligonucleotide hybridizes with the nucleic acid amplification product; and / or When the signal generating oligonucleotide hybridizes with the nucleic acid amplification product, the label generates a signal. Optionally, the signal is fluorescence.

36. The method according to any one of claims 1 to 35, wherein: The detection 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 the signal generating oligonucleotide, optionally, the label is a fluorophore and the signal is fluorescence; Detecting a signal comprises detecting fluorescence emitted by the label; The method comprises determining the presence, absence and / or amount of the target nucleic acid sequence in the sample; Determining the presence, absence and / or amount of the target nucleic acid sequence in the sample comprises determining the presence, absence and / or amount of dsDNA and / or nucleic acid 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 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 method according to any one of claims 1 to 36, wherein: The label is capable of generating a false positive signal when the signal generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product and / or the third undesired amplification product, optionally the signal and the false positive signal are indistinguishable; and / or When the signal generating oligonucleotide hybridizes to the first undesired amplification product, the second undesired amplification product and / or the third undesired amplification product, the label generates a false positive signal, optionally the signal and the false positive signal are indistinguishable.

38. The method according to any one of claims 1 to 37, wherein: The generation of the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product reduces the likelihood of accurately determining the presence, absence, and / or amount of the target nucleic acid sequence in the sample; and / or Detection of such false positive signals reduces the likelihood of accurately determining the presence, absence and / or amount of the target nucleic acid sequence in the sample.

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

40. The method according to any one of claims 1 to 39, wherein: The generation of the first stalled extension product, the second stalled extension product, and / or the third stalled extension product does not generate a false positive signal; and / or The signal generating oligonucleotide hybridizing to the first arrested extension product, the second arrested extension product and / or the third arrested extension product does not generate a false positive signal.

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

42. The method according to any one of claims 1 to 41, wherein: The appearance of detectable levels 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 terminators.

43. The method of any one of claims 1-42, 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 a detectable level within at least about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the start of the amplification step.

44. The method according to any one of claims 1 to 43, wherein: The generation of the false positive signal, the first undesired amplification product, the second undesired amplification product, and / or the third undesired amplification product is reduced by at least about 1.1 fold compared to a comparable method wherein the signal generating oligonucleotide does not comprise the one or more polymerase stoppers.

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

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

2.

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

48. The method of any one of claims 1-47, wherein the one or more amplification reagents comprise: Reverse transcriptase; An enzyme having a hyperthermophilic polymerase activity, optionally wherein the enzyme having a hyperthermophilic polymerase activity has a reverse transcriptase activity Forward primer; Reverse primer; Reverse transcription primers; and / or dNTPs.

49. The method of any one of claims 1-48, wherein the reagent composition is lyophilized, heat dried, and / or comprises one or more additives, wherein the one or more additives include: Tween 20, Triton X-100, and / or Tween 80; Amino acids; A sugar or sugar alcohol, optionally comprising sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof; and / or A polymer, optionally comprising polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptide, collagen peptide or any combination thereof, Optionally, contacting the reagent composition with the treated sample comprises dissolving the reagent composition in the treated sample.

50. The method of any one of claims 1-49, wherein the one or more lysis reagents comprise: From about 0.001% (w / v) to about 1.0% (w / v) of the treated sample, optionally about 0.2% (w / v) of the treated sample; and / or Detergent, optionally the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant and an amphoteric surfactant.

51. The method according to any one of claims 1 to 50, wherein the method: Performed in a single reaction vessel; excluding the use of any enzyme other than the reverse transcriptase and the enzyme having hyperthermophilic biopolymerase activity; excluding the use of any enzyme other than the enzyme having hyperthermophilic biopolymerase activity; does not involve thermal and / or enzymatic denaturation of the nucleic acid during the amplification step; and / or Contacting the nucleic acid with a single-stranded DNA binding protein is not included.

52. The method of any one of claims 1 to 51, wherein: The target nucleic acid sequence comprises a length of no longer than about 20 nucleotides to no longer than about 90 nucleotides, optionally, the target nucleic acid sequence comprises a length of about 30 nucleotides; The forward primer, the reverse primer and / or the reverse transcription primer are about 8 to 16 bases in length; The nucleic acid amplification product is about 20 to 40 bases in length; and / or The spacer sequence comprises a portion of the target nucleic acid sequence, and optionally the spacer sequence is 1 to 10 bases long.

53. The method of any one of claims 1-52, wherein: The isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, further optionally about 55°C to about 75°C, optionally about 56°C to about 67°C; The amplification (a) is performed for a period of about 5 minutes to about 60 minutes, optionally the amplification is performed for a period of about 15 minutes; and / or (b) is performed under isothermal amplification conditions without a helicase, without a single-stranded binding protein, without a cleavage agent, and without a recombinase; The amplification is performed using a method selected from the group consisting of: polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification and transcription-mediated amplification (TMA), optionally the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); The enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof, optionally the enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7, and optionally the enzyme having hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 7, optionally the enzyme having hyperthermophilic polymerase activity has low exonuclease activity or no exonuclease activity; and / or The sample ribonucleic acid is simultaneously contacted with the reverse transcriptase and the enzyme having a hyperthermophilic polymerase activity, optionally the sample ribonucleic acid is simultaneously contacted with the reverse transcriptase, the enzyme having a hyperthermophilic polymerase activity, and the forward primer and the reverse primer, and optionally the sample ribonucleic acid is simultaneously contacted with the reverse transcriptase, the enzyme having a hyperthermophilic polymerase activity, the forward primer, the reverse primer, and the reverse transcription primer.

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

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

56. The method of any one of claims 1-55, wherein the sample is a biological sample or an environmental sample, wherein the environmental sample is or is obtained from: a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a salt water sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof; and / or The biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof.

57. The method of any one of claims 1-56, wherein: 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 for two or more different organisms, 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 amplification does not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA), optionally the amplification does not include LAMP; The amplification comprises one or more of the following: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR and IMDA, optionally the amplification does not comprise LAMP; and / or The method does not include one or more of: (i) dilution of the processed sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) adding a ribonuclease inhibitor to the treated sample; (vii) adding a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal denaturation and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of RNase H to the treated sample or the amplification reaction mixture.

58. A signal generating oligonucleotide capable of hybridizing with a nucleic acid amplification product, the signal generating oligonucleotide comprising: a 5' subdomain, a 3' subdomain, an intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain, and a loop domain between the 5' subdomain and the 3' subdomain, wherein the intramolecular nucleotide base pairing between the 5' subdomain and the 3' subdomain is capable of forming a paired stem domain, wherein the loop domain comprises one or more polymerase terminators, and wherein the 5' subdomain, the paired stem domains and / or the 3' subdomain do not comprise the one or more polymerase stoppers.

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

60. The signal generating oligonucleotide of any one of claims 58-59, wherein the nucleic acid amplification product comprises: (1) The sequence of the forward primer and its reverse complement, (2) the sequence of the reverse primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases long.

61. The signal generating oligonucleotide according to any one of claims 58 to 60, wherein: The signal generating oligonucleotide comprises a first region comprising the sequence of at least a portion 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 a sequence of at least a portion of the spacer sequence, optionally, the first region, the second region and / or the spacer region comprises one or more polymerase stoppers.

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

63. The signal generating oligonucleotide according to any one of claims 58 to 62, 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, Optionally, the nucleic acid amplification product is produced by amplifying the target nucleic acid sequence with the forward primer and the reverse primer.

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

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

66. The signal generating oligonucleotide according to any one of claims 58 to 65, wherein: The first region comprises at least a portion of the 5' subdomain and / or the loop domain, The spacer region comprises at least a portion of the loop domain, and The second region comprises at least a portion of the loop domain and / or the 3' subdomain.

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

68. The signal generating oligonucleotide of any one of claims 58-67, wherein the one or more polymerase stoppers comprise one or more of an abasic site, a stable abasic site, a chemically trapped abasic site, or any combination thereof.

69. The signal generating oligonucleotide of claim 68, wherein: The stable abasic site includes 1',2'-dideoxy; The chemically captured abasic sites include abasic sites that react with alkoxyamines or sodium borohydride; The abasic site comprises an apurinic site, an apyrimidinic site, or both; and / or The abasic site is created by an alkylating agent or an oxidizing agent.

70. The signal generating oligonucleotide of any one of claims 58-69, wherein the one or more polymerase terminators comprise: one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo-deoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more isodeoxy Cytidine (Iso-dC), one or more isodeoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(o)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups or any combination thereof.

71. The signal generating oligonucleotide according to any one of claims 58-70, wherein: The signal generating oligonucleotide comprises one or more phosphorothioate bonds 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, optionally (a) the label is located in the 3' terminal domain and the quencher is located in the 5' terminal domain, and / or (b) the label is located in the 5' terminal domain and the quencher is located in the 3' terminal domain; and / or The label is capable of generating a signal when the signal generating oligonucleotide hybridizes to the nucleic acid amplification product, optionally the signal is fluorescent.

72. The signal generating oligonucleotide according to any one of claims 58 to 71, in, When the forward primer binds to the signal generating oligonucleotide to form a first undesired duplex, the one or more polymerase terminators are capable of terminating polymerase extension of the forward primer of the first undesired duplex toward the 5' end of the signal generating oligonucleotide, Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the forward primer of the first undesired duplex beyond the one or more polymerase stoppers of the signal generating oligonucleotide.

73. The signal generating oligonucleotide according to any one of claims 58 to 72, in, When the reverse primer binds to the signal generating oligonucleotide to form a second undesired duplex, the one or more polymerase terminators are capable of terminating polymerase extension of the reverse primer of the second undesired duplex toward the 5' end of the signal generating oligonucleotide, Optionally, the one or more polymerase stoppers are capable of terminating polymerase extension of the reverse primer of the second undesired duplex beyond the one or more polymerase stoppers of the signal generating oligonucleotide.

74. The signal generating oligonucleotide according to any one of claims 58 to 73, in, When the exogenous nucleic acid binds to the signal generating oligonucleotide to form a third undesired duplex, the one or more polymerase terminators are capable of terminating polymerase extension of the exogenous nucleic acid of the third undesired duplex toward the 5' end of the signal generating oligonucleotide, Optionally, the one or more polymerase stoppers 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.

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

7.

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

77. The kit of any one of claims 75-76, wherein the nucleic acid amplification product is about 20 to 40 bases in length, and wherein the nucleic acid amplification product comprises: (1) the sequence of the forward primer and its reverse complement, (2) the sequence of the reverse primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the forward primer and its reverse complement and (2) the sequence of the reverse primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases long.

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

79. A kit according to any one of claims 75 to 78, wherein 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 virus, herpes virus 6, herpes virus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A virus, influenza B virus, influenza C virus, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV) and hantavirus; wherein 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 species, Legionella pneumophila, Legionella dumov, Mycoplasma fermentans, Ehrlichia species, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, Streptococcus agalactiae, and Listeria monocytogenes; wherein the fungi include one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis and Trichophyton rubrum; and / or The protozoa include one or more of Trypanosoma cruzi, Leishmania species, Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora species, and Eimeria species.

80. The kit of any one of claims 75-79, wherein the reagent composition is lyophilized and / or heat dried and comprises one or more additives, wherein the one or more additives include: Amino acids; A sugar or sugar alcohol, optionally comprising sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol or any combination thereof; and / or A polymer, optionally the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinyl pyrrolidone, hydroxyethyl cellulose, Ficoll, albumin, polypeptides, collagen peptides or any combination thereof.

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

82. The method of claim 81, wherein the at least one pair of primers 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. The method of any one of claims 81-82, wherein: The at least one pair of primers capable of hybridizing to the target nucleic acid sequence of Neisseria gonorrhoeae is SEQ ID NOs: 2 and 3, SEQ ID NOs: 2 and 16, SEQ ID NOs: 17 and 3, or SEQ ID NOs: 17 and 16.

84. The method of any one of claims 81-83, wherein determining the presence or amount of the amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more of the signal generating oligonucleotides, wherein each of the one or more of the 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, optionally 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; and / or 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. A method for detecting Chlamydia trachomatis in a sample, comprising: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises a sequence of any one 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 SEQ ID NOs: 20-23, 25-26, 28-29, and 31; if the sample contains Chlamydia trachomatis, producing an amplicon of the target nucleic acid sequence; and The presence or amount of the amplicon is determined as an indication of the presence of Chlamydia trachomatis in the sample.

86. The method of claim 85, wherein the at least one pair of primers 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. The method of any one of claims 85-86, wherein: The at least one pair of primers capable of hybridizing to the 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.

88. The method of any one of claims 85-87, wherein determining the presence or amount of an amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more of the signal generating oligonucleotides, wherein each of the one or more of the 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% identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 18-19, 24, 27, 30, and 32, optionally 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; and / or 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. A method for detecting influenza B virus in a sample, comprising: contacting the sample with at least one pair of primers, wherein the at least one pair of primers is capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer of the at least one pair of primers comprises any one of the sequences SEQ ID NOs: 36-37 and 41 or a sequence that exhibits at least about 85% identity to any one of the sequences SEQ ID NOs: 36-37 and 41; if the sample comprises influenza B virus, generating an amplicon of the target nucleic acid sequence; and The presence or amount of the amplicon is determined as an indication of the presence of influenza B virus in the sample.

90. The method of claim 89, wherein the at least one pair of primers 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. The method of any one of claims 89-90, wherein: The at least one pair of primers capable of hybridizing to the target nucleic acid sequence of influenza B virus is SEQ ID NOs: 36 and 37, or SEQ ID NOs: 36 and 41.

92. The method of any one of claims 89-91, wherein determining the presence or amount of an amplicon of the target nucleic acid sequence comprises contacting the amplicon with one or more of the signal generating oligonucleotides, wherein each of the one or more of the 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, optionally 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; and / or 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. The method of any one of claims 89-92, comprising contacting the 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.

94. The method of any one of claims 81-93, wherein the sample is a biological sample or an environmental sample, wherein the environmental sample is or is obtained from: a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a waste water sample, a salt water sample, a sample exposed to atmospheric air or other gases, a culture thereof, or any combination thereof; and / or The biological sample is or is obtained from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof.

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

96. The method of any one of claims 81-95, comprising: contacting said sample comprising a biological entity with a lysis buffer to produce a treated sample, wherein said lysis buffer comprises one or more lysis agents capable of lysing the biological entity to release sample nucleic acid contained therein, and wherein said sample nucleic acid is suspected of comprising said target nucleic acid sequence; and A reagent composition comprising the at least one pair of primers is contacted with the treated sample to produce the amplification reaction mixture, wherein the reagent composition comprises one or more amplification reagents.

97. The method of any one of claims 81-96, wherein the one or more amplification reagents comprise: Reverse transcriptase; An enzyme having a hyperthermophilic biopolymerase activity, optionally wherein the enzyme having a hyperthermophilic biopolymerase activity has a reverse transcriptase activity; Reverse transcription primers; and / or dNTPs.

98. The method of any one of claims 81-97, wherein generating an amplicon of the target nucleic acid sequence comprises: The target nucleic acid sequence in the amplification reaction mixture is amplified under amplification conditions, thereby generating amplicons of the target nucleic acid sequence.

99. The method of claim 98, wherein: The amplification is performed using a method selected from the group consisting of: polymerase chain reaction (PCR), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), replicase-mediated amplification, immunoamplification, nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification and transcription-mediated amplification (TMA), optionally the PCR is real-time PCR and / or quantitative real-time PCR (QRT-PCR); The amplification does not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nickase amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), branch amplification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA), optionally the amplification does not include LAMP; The amplification comprises one or more of the following: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR and IMDA, optionally the amplification does not comprise LAMP; and / or The method does not include one or more of: (i) dilution of the processed sample; (ii) dilution of the amplification reaction mixture; (iii) thermal denaturation of the treated sample; (iv) sonication of the treated sample; (v) sonication of the amplification reaction mixture; (vi) adding a ribonuclease inhibitor to the treated sample; (vii) adding a ribonuclease inhibitor to the amplification reaction mixture; (viii) purification of the sample; (ix) purification of the sample nucleic acid; (x) purification of the nucleic acid amplification product; (xi) removal of one or more cleavage agents from the treated sample or the amplification reaction mixture; (xii) thermal denaturation and / or enzymatic denaturation of the sample nucleic acid before and / or during amplification; and (xiii) addition of RNase H to the treated sample or the amplification reaction mixture.

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

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

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

103. The composition of claim 102, wherein: The at least one pair of primers capable of hybridizing to the target nucleic acid sequence of Neisseria gonorrhoeae includes 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. The composition according to any one of claims 102-103, further comprising one or more of the signal generating oligonucleotides, wherein each of the one or more of the signal generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14, or a sequence exhibiting at least about 85% identity with a sequence selected from the group consisting of SEQ ID NO: 1 and 12-14.

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

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

107. A composition for detecting Chlamydia trachomatis in a sample, comprising: At least one pair of primers capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein each primer in the at least one pair of primers comprises any one of the sequences 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 SEQ ID NOs: 20-23, 25-26, 28-29 and 31.

108. The composition of claim 107, wherein: The at least one pair of primers capable of hybridizing to the 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.

109. The composition according to any one of claims 107-108, further comprising one or more of the signal generating oligonucleotides, wherein each of the one or more of the signal generating oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NO: 15, 18-19, 24, 27, 30 and 32, or a sequence exhibiting at least about 85% identity with a sequence selected from the group consisting of SEQ ID NO: 15, 18-19, 24, 27, 30 and 32.

110. The composition of any one of claims 107-109, 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. 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. A composition for detecting influenza B virus in a sample, comprising: At least one pair of primers capable of hybridizing to a target nucleic acid sequence of influenza B virus, wherein each primer in the at least one pair of primers comprises any one of the sequences of SEQ ID NOs: 36-37 and 41 or a sequence exhibiting at least about 85% identity to any one of the sequences of SEQ ID NOs: 36-37 and 41.

113. The composition of claim 112, wherein: The at least one pair of primers 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. The composition of any one of claims 112-113, further comprising a primer that exhibits at least about 85% identity to SEQ ID NO:

38.

115. The composition according to any one of claims 112-114, further comprising one or more of the signal generating oligonucleotides, wherein each of the one or more of the 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 with a sequence selected from the group consisting of SEQ ID NOs: 39-40 and 42-45.

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

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

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

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

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

121. The method or composition of any one of claims 118-120, wherein the one or more polymerase terminators comprise one or more of an abasic site, a stable abasic site, a chemically trapped abasic site, or any combination thereof.

122. The method or composition of any one of claims 118-121, wherein: The stable abasic site includes 1',2'-dideoxy; The chemically captured abasic sites include abasic sites that react with alkoxyamines or sodium borohydride; The abasic site comprises an apurinic site, an apyrimidinic site, or both; and / or The abasic site is created by an alkylating agent or an oxidizing agent.

123. The method or composition of any one of claims 118-122, wherein: The one or more polymerase terminators include: one or more nitroindole, one or more inosine, one or more acridine, one or more 2-aminopurine, one or more 2-6-diaminopurine, one or more 5-bromo-deoxyuridine, one or more inverted thymidine (inverted dT), one or more inverted dideoxythymidine (ddT), one or more dideoxycytidine (ddC), one or more 5-methylcytidine, one or more 5-hydroxymethylcytidine, one or more 2'-O-methyl RNA bases, one or more unmethylated RNA bases, one or more 5-bromo-deoxyuridine ... one or more isodeoxycytidine (Iso-dC), one or more isodeoxyguanosine (Iso-dG), one or more C3 (OC3H6OPO3) groups, one or more photocleavable (PC) [OC3H6-C(o)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups, or any combination thereof; The signal generating oligonucleotide comprises one or more phosphorothioate bonds 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 The signal generating oligonucleotide comprises a label, optionally the label comprises a quenchable label, and further optionally 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, wherein 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, 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, optionally wherein: 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 consists of 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: 1-3, 12-14 and 16-17.

125. A signal generating oligonucleotide or primer of up to about 100 nucleotides in length capable of hybridizing to a target nucleic acid sequence of Chlamydia trachomatis, wherein the signal generating oligonucleotide or primer comprises 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, optionally wherein: The signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 15 and 18-32, or consists of 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, wherein the signal generating oligonucleotide or primer comprises 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, optionally wherein: The signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 36-45, or consists of 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: 36-45.

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, wherein the signal generating oligonucleotide or primer comprises 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, optionally wherein: The signal generating oligonucleotide or primer consists of a sequence selected from the group consisting of SEQ ID NOs: 33-35, or consists of 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: 33-35.

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

129. The composition of claim 128, comprising: A lysis buffer comprising one or more lysis agents capable of lysing a biological entity to release sample nucleic acid contained therein, wherein the sample nucleic acid is suspected of comprising a target nucleic acid sequence, optionally wherein the one or more lysis agents comprise a detergent, and wherein the detergent comprises one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant and an amphoteric surfactant; and / or The reagent composition comprises one or more amplification reagents, wherein the amplification reagents comprise one or more components for amplification for amplifying the target nucleic acid sequence under isothermal amplification conditions.

130. The composition of any one of claims 128-129, wherein the one or more components for amplification comprise: An enzyme with hyperthermophilic polymerase activity capable of producing a nucleic acid amplification product, optionally the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 7 or a functional fragment thereof, optionally the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7, and optionally, the enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 7.

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