Methods for isolating genomic DNA for amplification of short nucleic acid targets - Patents.com

Abstract

Disclosed herein are methods, compositions, and kits for use in detecting target nucleic acid sequences in a sample. Provided herein are acidic compositions for separating dsDNA. In some embodiments, the acidic composition comprises a monovalent salt and / or a divalent salt, one or more surfactants, and an acidic agent. In some embodiments, the pH of the acidic composition is less than 4. Also provided herein are reagent compositions comprising a buffer and one or more amplification agents.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 307,085, filed February 5, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Sequence Listing Reference This application is filed with an electronic Sequence Listing. The Sequence Listing is provided as file number 68EB-317333-WO, created on February 2, 2023, and is 8.0 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. The present disclosure relates generally to methods and compositions for amplifying (eg, isothermal amplification) nucleic acids. [Background technology]

[0002] Nucleic acid-based diagnostics can be useful for the rapid detection of infections, diseases, and / or genetic mutations. For example, identifying bacterial or viral nucleic acids in a sample can be useful for diagnosing certain types of infectious diseases. Other examples include identifying single nucleotide polymorphisms for disease control or forensics, and identifying genetic mutations indicative of genetically modified foods. Nucleic acid-based diagnostic assays often require the amplification of specific portions of nucleic acids in a sample. A common technique for nucleic acid amplification is polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermal cycling) to proceed through the following steps: denaturation (e.g., separating the strands of a double-stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short strands of complementary DNA sequence), and extension of the primers along their complementary targets by a polymerase. Such thermal cycling can be a time-consuming process that generally requires specialized machinery. Therefore, there is a need for faster nucleic acid amplification methods that can be performed without thermal cycling. Such methods can be useful, for example, for on-site testing and point-of-care diagnostics. Most isothermal amplification methods require either a separate heat denaturation step or the presence of helicases, single-stranded DNA binding proteins (SSBs), or nicking enzymes for genomic DNA strand separation. These requirements typically add either extra steps or significant expenditures on equipment and / or consumables, making such methods less desirable than purely chemical approaches. Numerous chemical methods, including alkaline treatment, formamide, glycerol, and DMSO, have previously been identified to separate DNA strands for use in downstream applications. However, denaturants such as urea and formamide must be removed from the solution prior to any amplification, which can be a time-consuming and costly step. While the pH of alkaline solutions can be easily adjusted without chemical purification, their application is limited by the fact that magnesium ions are not readily soluble in aqueous solutions that are sufficiently alkaline to support DNA strand separation. There is a need for compositions and methods of nucleic acid detection in which chemical denaturation results in simple and rapid denaturation of DNA (without the need for sample separation, purification, or extraction steps) and is compatible with downstream amplification and detection steps. Summary of the Invention

[0003] Disclosed herein is a method for amplifying a target nucleic acid sequence in a sample. In some embodiments, the method includes: (a) contacting a sample containing a biological entity with an acidic composition to produce an acidic mixture, wherein the acidic composition is capable of dissolving the biological entity to release sample nucleic acids contained therein, the sample nucleic acid comprising double-stranded DNA (dsDNA) suspected of comprising a target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length, the acidic composition comprising (i) a monovalent salt and / or a divalent salt, (ii) one or more surfactants, (iii) one or more chelating agents, and (iv) an acidic agent, wherein the acidic composition has a pH of less than 4, thereby denaturing the dsDNA to produce single-stranded DNA (ssDNA); (b) contacting a reagent composition (e.g., a dry composition, a wet composition) comprising a buffer with the acidic mixture to produce a neutral mixture, wherein the neutral mixture comprises ssDNA and the reagent composition comprises one or more amplification reagents; and (c) amplifying the target nucleic acid sequence in the neutral mixture, thereby producing nucleic acid amplification products. In some embodiments, the dsDNA suspected of containing the target nucleic acid sequence is 100 base pairs or less in length. In some embodiments, one or more of the sample nucleic acids is 100 base pairs or less in length.

[0004] The step of amplifying the target nucleic acid sequence may include producing a detectable level of nucleic acid amplification product within about 20 minutes, about 15 minutes, about 10 minutes, or about 5 minutes. In some embodiments, the acidic composition has a pH of about 1 to about 6.9, e.g., about 1 to about 3.9, or about 2. In some embodiments, the neutral mixture has a pH of about 7 to about 9, e.g., about 8.8. The method may include detecting the target nucleic acid sequence in the sample, wherein detecting the target nucleic acid sequence in the sample includes (d) detecting the nucleic acid amplification product, wherein the detecting is performed less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the acidic mixture.

[0005] The step of contacting the reagent composition (e.g., a dry composition, a wet composition) with the acidic mixture may include dissolving the reagent composition in the acidic mixture. The reagent composition can be a wet composition or a dry composition. In some embodiments, the one or more amplification reagents include one or more of an enzyme having hyperthermophilic polymerase activity, a first primer, a second primer, and dNTPs. In some embodiments, the reagent composition is a wet composition (e.g., an aqueous composition suspended in a liquid medium). In some embodiments, the reagent composition is lyophilized and / or heat-dried (e.g., a dry composition) and includes one or more additives, wherein the one or more additives include an amino acid; a sugar or a sugar alcohol. In some embodiments, the sugar or sugar alcohol includes sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the one or more additives include a polymer. In some embodiments, the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof.

[0006] In some embodiments, the dsDNA suspected of containing the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other. In some embodiments, the step of amplifying the target nucleic acid sequence comprises amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying comprises contacting the ssDNA with: i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence, and the second primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product, wherein the nucleic acid amplification product comprises (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length. An enzyme with hyperthermophilic polymerase activity can have an amino acid sequence that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof. In some embodiments, an enzyme with hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, an enzyme with hyperthermophilic polymerase activity has reduced or no exonuclease activity.

[0007] In some embodiments, the first primer and / or the second primer is about 8 to 16 bases in length. In some embodiments, the first primer and / or the second primer comprises one or more of DNA bases, modified DNA bases, or combinations thereof. In some embodiments, the nucleic acid amplification product is about 20 to 40 bases in length. In some embodiments, the spacer sequence comprises a portion of the target nucleic acid sequence. In some embodiments, the spacer sequence is 1 to 10 bases in length. The dsDNA may comprise genomic DNA (gDNA), plasmid DNA, or both. The sample can be, for example, a biological sample or an environmental sample. In some embodiments, the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a fresh water sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof. In some embodiments, the biological sample is or is derived from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof.

[0008] In some embodiments, the acidic agent comprises an organic acid, an inorganic acid, or both. In some embodiments, the acidic agent is selected from hydrochloric acid, glycine hydrochloride, acetic acid, citric acid, and phosphoric acid. In some embodiments, the acidic agent is present in the acidic composition at a concentration of less than about 20 mM. For example, the acidic agent may be present in the acidic composition at a concentration in the range of about 1 mM to about 100 mM, e.g., about 10 mM. The monovalent salt and / or divalent salt can include a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or any combination thereof. In some embodiments, the monovalent salt is selected from ammonium sulfate, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, potassium fluoride, potassium chloride, potassium bromide, and potassium iodide. In some embodiments, the divalent salt is selected from the group consisting of magnesium sulfate, calcium chloride, magnesium chloride, copper(II) chloride, zinc chloride, calcium oxide, magnesium oxide, barium oxide, sodium sulfate, calcium sulfate, copper(II) sulfate, potassium carbonate, and sodium carbonate. In some embodiments, the monovalent salt and / or divalent salt is present in the acidic composition at a concentration in the range of about 1 mM to about 14 mM, e.g., about 5 mM or about 4 mM.

[0009] The one or more surfactants can include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. In some embodiments, the one or more surfactants are present in the acidic composition at a concentration ranging from about 0.01% to about 2% by weight of the acidic composition (% wt / vol). In some embodiments, the one or more surfactants are present in the acidic composition at a concentration ranging from about 0.1% by weight of the acidic composition (% wt / vol). In some embodiments, the acidic composition further comprises a chelating agent. In some embodiments, the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), and Tris. In some embodiments, the chelating agent is present in the acidic composition at a concentration ranging from about 0.1 mM to about 14 mM. In some embodiments, contacting the sample with the acidic composition is carried out at a temperature ranging from about 18° C. to about 99° C., e.g., at a temperature of about 78° C. In some embodiments, contacting the sample with the acidic composition is carried out at a temperature ranging from about 18° C. to about 25° C. In some embodiments, contacting the sample with the acidic composition is carried out for a period of about 5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some embodiments, the buffer comprises MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium buffer, or any combination thereof. In some embodiments, the neutral mixture has a pH in the range of about 7 to about 9, for example, a pH of about 8.8.

[0010] In some embodiments, the target nucleic acid sequence comprises a length of about 20 nucleotides or less, about 30 nucleotides or less, about 40 nucleotides or less, about 50 nucleotides or less, about 60 nucleotides or less, or about 90 nucleotides or less. In some embodiments, the target nucleic acid sequence comprises a length of about 30 nucleotides. In some embodiments, the amplifying step is performed under isothermal amplification conditions. In some embodiments, the isothermal amplification conditions include a constant temperature of about 30°C to about 72°C, e.g., a constant temperature of about 67°C. In some embodiments, the amplifying step is performed for a period of about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60 minutes, e.g., a period of about 15 minutes.

[0011] The amplifying step may be performed under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions. In some embodiments, step (d) further comprises determining the amount of dsDNA comprising the target nucleic acid sequence in the sample. In some embodiments, the detecting nucleic acid amplification product comprises using a real-time detection method. In some embodiments, the detecting nucleic acid amplification product comprises contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product, wherein the signal-generating oligonucleotide comprises a fluorophore, a quencher, or both. In some embodiments, the detecting nucleic acid amplification product comprises detecting a fluorescent signal. In some embodiments, the fluorescent signal is from a molecular beacon. In some embodiments, the method is performed in a single reaction vessel. In some embodiments, the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences. In some embodiments, the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. In some embodiments, the two or more target nucleic acid sequences are specific to two or more different organisms.

[0012] In some embodiments, the amplifying step does not include the use of any enzyme other than an enzyme having hyperthermophilic polymerase activity. In some embodiments, the amplifying step includes one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplifying step does not include one or more of the following: Archaeal polymerase amplification (APA), LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplifying step does not include LAMP.

[0013] In some embodiments, the method does not include one or more of the following: (i) diluting the acidic mixture; (ii) diluting the neutral mixture; (iii) heat denaturing the acidic mixture; (iv) sonicating the acidic mixture; (v) sonicating the neutral mixture; (vi) adding a ribonuclease inhibitor to the acidic mixture; (vii) adding a ribonuclease inhibitor to the neutral mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more detergents from the acidic or neutral mixture. (xii) thermally and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; (xiii) adding RNase H to the acidic or neutral mixture; (xiv) contacting the sample, acidic or neutral mixture with a helicase, single-stranded binding protein, nicking enzyme, restriction enzyme or recombinase; (xv) contacting the sample, acidic or neutral mixture with glycerol, formamide or urea; (xvi) purifying ssDNA from the acidic or neutral mixture; (xvii) a thermal denaturation step; and (xviii) enriching, purifying and / or isolating dsDNA.

[0014] In some embodiments, the acidic composition and / or the reagent composition includes a reducing agent, a chelating agent, or both. In some embodiments, the acidic composition does not include a reducing agent, a chelating agent, or both. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), Tris, or any combination thereof. In some embodiments, the reducing agent is 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythritol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, a salt of sulfite, thioglycolic acid, hydroxyethyl disulfide (HED), or any combination thereof. In some embodiments, the acidic composition is free of both anionic and cationic surfactants. In some embodiments, the acidic composition further comprises a tween surfactant, including but not limited to Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85. In some embodiments, the tween surfactant comprises about 0.01% (w / v) of the acidic composition.

[0015] The present disclosure includes an acidic composition for dissolving biological entities and denaturing the dsDNA contained therein.In some embodiments, the acidic composition comprises a monovalent salt and / or a divalent salt; one or more surfactants; and an acidic agent, wherein the acidic agent is present in the acidic composition at a concentration of less than 100mM, and the acidic composition has a pH of less than 4.The monovalent salt can be present in the acidic composition at a concentration of less than 30mM.The divalent salt can be present in the acidic composition at a concentration of less than 15mM. In some embodiments, the acidic composition does not contain a reducing agent, a chelating agent, or both. In some embodiments, the chelating agent is EDTA, EGTA, NTA, Tris, or any combination thereof. In some embodiments, the reducing agent is selected from 2-mercaptoethanol, DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, sulfite, thioglycolic acid, HED, or any combination thereof. In some embodiments, the acidic composition does not contain either an anionic surfactant or a cationic surfactant. In some embodiments, the acidic composition does not contain glycerol, formamide, or urea.

[0016] In some embodiments, the acidic composition has a pH in the range of about 1 to about 3.9, e.g., a pH of about 2. In some embodiments, the acidic agent comprises an organic acid and / or an inorganic acid. In some embodiments, the acidic agent is selected from hydrochloric acid, glycine hydrochloride, acetic acid, citric acid, sulfuric acid, and phosphoric acid. In some embodiments, the acidic agent is present in the acidic composition at a concentration in the range of about 1 mM to about 100 mM, e.g., about 10 mM. In some embodiments, the monovalent salt and / or divalent salt comprises a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or any combination thereof. In some embodiments, the monovalent salt is selected from ammonium sulfate, ammonium chloride, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, potassium fluoride, potassium chloride, potassium bromide, and potassium iodide. In some embodiments, the divalent salt is selected from magnesium sulfate, calcium chloride, magnesium chloride, copper(II) chloride, zinc chloride, calcium oxide, magnesium oxide, barium oxide, sodium sulfate, calcium sulfate, copper(II) sulfate, potassium carbonate, and sodium carbonate. In some embodiments, the monovalent salt and / or divalent salt is present in the acidic composition at a concentration in the range of about 1 mM to about 14 mM, e.g., about 5 mM or about 4 mM. In some embodiments, the one or more surfactants include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. In some embodiments, the one or more surfactants are present in the acidic composition at a concentration in the range of about 0.01% to about 2% by weight relative to the volume of the acidic composition (% wt / vol), e.g., about 0.1% by weight relative to the volume of the acidic composition (% wt / vol). In some embodiments, the acidic composition further comprises a tween surfactant. In some embodiments, the tween surfactant is selected from Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85. In some embodiments, the tween surfactant comprises about 0.01% to about 1% (wt / vol) of the acidic composition.In some embodiments, the acidic composition further comprises a chelating agent selected from ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), Tris, and any combination thereof. In some embodiments, the chelating agent is present in the acidic composition at a concentration in the range of about 0.1 mM to about 14 mM.

[0017] The present disclosure includes a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit includes: (a) an acidic composition provided herein, the acidic composition being capable of dissolving biological entities to release sample nucleic acids contained therein, the sample nucleic acid comprising dsDNA suspected of containing the target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length; and (b) a reagent composition comprising a buffer and one or more amplification reagents for amplifying the target nucleic acid sequence under isothermal amplification conditions, the one or more amplification reagents comprising: (i) a first primer and a second primer, the first primer being capable of hybridizing to a first strand sequence of the target nucleic acid sequence, and the second primer being capable of hybridizing to a second strand sequence of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity capable of producing a nucleic acid amplification product. In some embodiments, the dsDNA suspected of containing the target nucleic acid sequence is 100 base pairs or less in length. In some embodiments, one or more of the sample nucleic acids is 100 base pairs or less in length.

[0018] In some embodiments, the buffer is selected from MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium buffer, and any combination thereof. The kit may include at least one component that provides real-time detection activity of the nucleic acid amplification product. In some embodiments, the real-time detection activity is provided by a molecular beacon. In some embodiments, the enzyme having hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% identical or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. In some embodiments, the enzyme having hyperthermophilic polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid amplification product is about 20-40 bases in length. In some embodiments, the nucleic acid amplification product comprises (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length.

[0019] In some embodiments, the first primer and / or the second primer are approximately 8-16 bases in length. In some embodiments, the first primer and / or the second primer comprise one or more DNA bases, modified DNA bases, or combinations thereof. In some embodiments, the reagent composition is lyophilized and / or heat-dried (e.g., a dry composition) and comprises one or more additives. The one or more additives may comprise an amino acid; a sugar, or a sugar alcohol. The sugar or sugar alcohol may comprise sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the one or more additives comprise a polymer. The polymer may comprise polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof.

[0020] In some embodiments, the mixture of the acidic composition and the reagent composition has a pH of about 7 to about 9, for example, a pH of about 8.8. In some embodiments, the mixture of the sample, the acidic composition, and the reagent composition has a pH of about 7 to about 9, for example, a pH of about 8.8. In some embodiments, the buffer comprises Tris. In some embodiments, the mixture of the sample, the acidic composition, and the reagent composition comprises Tris at a concentration in the range of about 30 mM Tris to about 50 mM Tris. In some embodiments, the kit comprises a sterile container containing the acidic composition and the reagent composition.

[0021] In the methods, compositions, kits, and systems disclosed herein, the biological entity can include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the biological entity includes a virus, a bacterium, a fungus, a protozoa, a portion thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoa. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoa. Non-limiting examples of viruses include hepatitis B virus, herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, rotavirus, human adenovirus, and genital human papillomavirus (HPV).Non-limiting examples of bacteria include Salmonella enterica, Streptococcus pyogenes, Clostridium difficile, Streptococcus agalactiae, Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, and the like. pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, or Listeria monocytogenes.In some embodiments, the fungus is Coccidioides posadasii, Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, or Trichophyton rubrum. In some embodiments, the protozoan is Trichomonas vaginalis, Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp. [Brief explanation of the drawings]

[0022] [Figure 1A-1B] Figure 1B shows non-limiting exemplary data on the effect of a low pH solution (ARBT solution; Figure 1B) on the dissociation of gDNA for archaeal polymerase amplification (APA) compared to a non-acidic control (TE solution; Figure 1A). The legend indicates the number of target sequence copies in the sample. NTC, no target control. LOD, limit of detection. [Figure 2]

[0023] Figure 1 shows non-limiting exemplary data regarding the effect of pre-incubation temperature on gDNA dissociation. The legend indicates the number of target sequence copies in the sample and the pre-incubation temperature. NTC, no target control. LOD, limit of detection. [Figure 3A-3B]

[0023] Figure 3A shows non-limiting exemplary data regarding the effect of elution buffer pH on N. gonorrhoeae (Ng) gDNA detection (in wet reactions) with an acidic elution buffer (Figure 3B) compared to a non-acidic control elution buffer (Figure 3A). The legend indicates the number of target sequence copies in the sample. NTC, no target control. LOD, limit of detection. [Figure 4A-4B] Figure 4 shows non-limiting exemplary data regarding the effect of elution buffer pH (Figure 4B) on N. gonorrhoeae (Ng) gDNA detection (in reactions using dried lyophilized pellets) with an acidic elution buffer (Figure 4B) compared to a non-acidic control elution buffer (Figure 4A). The legend indicates the number of target sequence copies in the sample. NTC, no target control. LOD, limit of detection. [Figure 5A-5B] Figure 5A shows non-limiting, exemplary data regarding the effect of temperature and pH on N. gonorrhoeae (Ng) gDNA stability, with incubations performed at room temperature (RT; Figure 5A) or 78°C (Figure 5B) prior to mixing with amplification components. The legend indicates incubation under acidic (ARBT) or non-acidic (TE) conditions with the indicated incubation times and temperatures. [Figures 6A-6B] Figure 6 shows non-limiting, exemplary data regarding the effect of temperature and pH on N. gonorrhoeae (Ng) gDNA stability in a 10% urine matrix, with incubations performed at room temperature (RT; Figure 6B) or 78°C (Figure 6A) prior to mixing with amplification components. The legend indicates incubation under acidic (ARBT) or non-acidic (TE) conditions with the indicated incubation times and temperatures. NTC, no target control. [Figure 7]

[0023] Figure 1 shows non-limiting exemplary data regarding the effect of temperature and pH on N. gonorrhoeae (Ng) gDNA stability (in a dry reaction format). Legend indicates incubation time and temperature. NTC, no target control. [Figure 8]FIG. 1 shows non-limiting exemplary melting curves of lambda DNA as a function of pH (adapted from Biophys J. 2001 Feb; 80(2): 874-881. Effect of pH on the overstretching transition of double-stranded DNA: evidence of force-induced DNA melting). DETAILED DESCRIPTION OF THE INVENTION

[0023] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments set forth in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form part of this disclosure. All patents, published patent applications, other publications, and GenBank sequences and other databases referenced herein are incorporated by reference in their entirety with respect to the relevant art.

[0024] Disclosed herein are methods, compositions, and kits for lysing biological entities and separating double-stranded polynucleotides contained therein for the amplification and detection of one or more target nucleotide sequences. Disclosed herein is a method for amplifying a target nucleic acid sequence in a sample. In some embodiments, the method includes: (a) contacting a sample containing a biological entity with an acidic composition to produce an acidic mixture, the acidic composition being capable of dissolving the biological entity and releasing sample nucleic acids contained therein, the sample nucleic acid including dsDNA suspected of containing a target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length, the acidic composition comprising (i) a monovalent salt and / or a divalent salt, (ii) one or more surfactants, and (iii) an acidic agent, the acidic composition having a pH of less than 4, thereby denaturing the dsDNA to produce single-stranded DNA (ssDNA); (b) contacting a reagent composition (e.g., a dry composition, a wet composition) including a buffer with the acidic mixture to produce a neutral mixture, the neutral mixture including the ssDNA, and the reagent composition comprising one or more amplification reagents; and (c) amplifying the target nucleic acid sequence in the neutral mixture, thereby producing a nucleic acid amplification product.

[0025] The present disclosure includes an acidic composition for dissolving biological entities and denaturing the dsDNA contained therein.In some embodiments, the acidic composition comprises a monovalent salt and / or a divalent salt; one or more surfactants; and an acidic agent, wherein the acidic agent is present in the acidic composition at a concentration of less than 100mM, and the acidic composition has a pH of less than 4.The monovalent salt can be present in the acidic composition at a concentration of less than 30mM.The divalent salt can be present in the acidic composition at a concentration of less than 15mM. The present disclosure includes a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit includes: (a) an acidic composition provided herein, the acidic composition being capable of dissolving biological entities to release sample nucleic acids contained therein, the sample nucleic acid comprising dsDNA suspected of comprising a target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length; and (b) a reagent composition (e.g., a dry composition, a wet composition) comprising a buffer and one or more amplification reagents for amplifying the target nucleic acid sequence under isothermal amplification conditions, the one or more amplification reagents comprising: (i) a first primer and a second primer, the first primer being capable of hybridizing to a first strand sequence of the target nucleic acid sequence, and the second primer being capable of hybridizing to a second strand sequence of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity capable of producing a nucleic acid amplification product. In some embodiments, the dsDNA suspected of containing the target nucleic acid sequence is 100 base pairs or less in length.

[0026] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below. As used herein, the term "acid" shall be given its ordinary meaning, and in some embodiments, an "acid" may be either an Arrhenius acid, a Bronsted-Lowry acid, or a Lewis acid. Arrhenius acids are acids that react with hydronium ions (HO) in solution. + A soluble base is a substance or fluid that increases the concentration of hydroxide ions (OH). A Bronsted-Lowry acid is a substance that can act as a proton donor. A Lewis acid is an electron pair acceptor. As used herein, the term "base" refers to either a substance that can accept a hydrogen cation (proton) or, more generally, a substance that can donate a pair of valence electrons. A soluble base quantitatively converts hydroxide ions (OH - A base is called an alkali if it contains and releases protons (hydrogen ions). The Brønsted-Lowry theory defines a base as a proton (hydrogen ion) acceptor, while the more common Lewis theory defines a base as an electron pair donor, allowing for the inclusion of Lewis acids other than protons.

[0027] As used herein, the term "buffer" shall be given its ordinary meaning and shall refer to an aqueous solution or composition that resists a change in pH when an acid or base is added to the solution or composition. This resistance to pH change is due to the buffering properties of such a solution. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have an unlimited capacity to maintain the pH of a solution or composition. Rather, buffers are typically capable of maintaining the pH within a certain range, e.g., between pH 7 and pH 9. Typically, buffers can maintain the pH within one logarithm above or below its pKa. Buffers and buffer solutions are typically made from buffer salts or from nonionic buffer components such as Tris and HEPES. Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations thereof.

[0028] Provided herein are methods and compositions for amplifying nucleic acids. Conventional nucleic acid amplification methods typically require a thermal cycling process, nucleic acid denaturation, proteins (e.g., enzymes) (e.g., helicases, recombinases) that promote strand unwinding, strand separation, and / or strand exchange, and / or endonuclease agents (e.g., restriction enzymes, nicking enzymes), and often require a reaction time of at least 20 to 30 minutes. The nucleic acid amplification method provided herein can be performed without thermal cycling, without thermal and / or enzymatic denaturation of the sample nucleic acid, without the addition of proteins (e.g., enzymes) that promote strand unwinding, strand separation, and / or strand exchange, without the use of endonuclease agents, and within a reaction time of approximately 10 to 15 minutes.

[0029] Chemical methods for separating genomic strands of DNA for amplification of short nucleic acid targets. Provided herein are methods and compositions for direct pathogen lysis in clinical samples, enabling isothermal amplification and real-time detection of nucleic acids using archaeal polymerases. Rapid point-of-care (POC) diagnostics that do not require sample purification can be developed. Viral particles, bacterial cells, or other pathogens still need to be lysed so that their DNA and / or RNA can be released from the cells and made available for amplification reactions. Traditional chemical lysis methods (e.g., using strong bases, ionic detergents, and chaotropic agents) are incompatible with enzymatic function because they also inactivate DNA polymerases or other enzymes. Therefore, an effective chemical lysis method that is compatible with enzymatic function and does not require any nucleic acid purification steps to remove the lysis reagents would be of significant value.

[0030] Provided herein are, for example, rapid and simple methods for separating complementary strands of dsDNA (e.g., genomic DNA (gDNA)) for subsequent targeting of short sequence stretches in amplification reactions. In some embodiments, strand separation is achieved by the use of an acidic agent (e.g., an inorganic acid) such as HCl, which can be performed simultaneously with sample lysis. Without being bound by any particular theory, upon release of dsDNA by lysis, protonation and spontaneous loss of hydrogen bonds between complementary bases allows dissociation of the DNA strands in the lysis solution (e.g., an acidic composition). In some embodiments, the denatured DNA sample is mixed with reaction components (e.g., a reagent composition) for subsequent amplification at a constant temperature without any separation or purification steps. In some embodiments, the amplification reaction components include an amplification buffer (e.g., a reagent composition) that has sufficient capacity to neutralize acid and raise the pH to the pH required for rapid amplification. In some embodiments, the amplification reaction contains a high concentration of primers complementary to the target gDNA to ensure that the formation of gDNA-primer complexes occurs more rapidly and frequently than the spontaneous reformation of the original double-stranded gDNA. In some embodiments, amplification can be initiated by primers extending on the gDNA target by incorporation of nucleotides by one or more DNA polymerases.

[0031] Disclosed herein is the surprising finding that exposing genomic DNA to a low pH solution as an initial step in archaeal polymerase amplification does not adversely affect amplification, as commonly believed. Instead, as shown in the Examples, this approach can increase the speed of isothermal amplification while improving the sensitivity of genomic DNA detection by up to 100-fold. The methods, compositions, and kits provided herein can advantageously provide point-of-care molecular diagnostics for pathogens (e.g., DNA pathogens) in clinical samples. After pathogen lysis, genomic DNA strands are separated, in some embodiments, without the need for a separate thermal or enzymatic step. In some embodiments, a sample preparation method using acid dissociation for archaeal polymerase amplification (APA) is provided. Without being bound by any particular theory, in some embodiments, acidic conditions function to denature or dissociate genomic DNA material to facilitate isothermal APA amplification. In some embodiments, the presence of an acidic agent in the acidic compositions disclosed herein (e.g., resuspension buffer, elution buffer, lysis buffer) can also improve biological entity lysis efficiency (e.g., cell lysis efficiency). The disclosed acid dissociation approach can be used to denature DNA in a variety of nucleic acid detection methods, such as isothermal amplification methods including LAMP, NEAR, RCA, MDA, and RPA, to improve priming in genomic DNA for improved amplification efficiency. The methods, compositions, and kits provided herein are not limited to isothermal amplification methods and can be applied to applications requiring single-stranded DNA or DNA dissociation, such as forensic analysis.

[0032] Currently available methods do not use acidic conditions in the sample preparation process for DNA dissociation or pathogen lysis for direct DNA amplification. Most isothermal amplification methods require a prior heat denaturation step, chemical denaturation, or enzymes to unwind genomic DNA, which are not well suited to point-of-care settings. Therefore, there is a need for a simple and rapid denaturation method, which the compositions and methods provided herein address. Although dsDNA is known to spontaneously dissociate in acidic solutions, its use is widely considered incompatible with subsequent DNA amplification due to the detrimental effects resulting from hydrolytic depurination. Depurination, one of the discoveries for which Thomas Lindahl received the Nobel Prize in Chemistry in 2015, is a naturally occurring process under physiological conditions. It is a chemical reaction of purine deoxyribonucleosides in which the β-N-glycosidic bond between the purine base guanine or adenine and the deoxyribose moiety is hydrolytically cleaved. Cleavage of the base-sugar bond results in the loss of genetic information in DNA, the formation of abasic sites, and alterations to the DNA structure. DNA damage caused by depurination has been shown to be problematic for the amplification of PCR products because a single DNA lesion in the template is sufficient to stop PCR enzymes (e.g., DNA polymerase).

[0033] The methods, compositions, and kits disclosed herein address the aforementioned needs and overcome the challenges presented by acid-catalyzed DNA damage by pairing the use of acidic separation with the amplification of short DNA sequences (e.g., approximately 30 nucleotides in length). In some embodiments, an acidic agent is included in the elution / lysis solution that dissociates genomic DNA after lysis of a biological entity (e.g., lysis of a pathogen cell wall). The inclusion of a high-capacity buffer provided herein in the amplification step can ensure that the acid is neutralized and optimal pH conditions are maintained. In some embodiments, the acidic composition (e.g., resuspension buffer, elution solution, lysis solution) also contains low concentrations of monovalent salts and / or divalent magnesium, which, without being bound by any particular theory, have been reported to inhibit depurination, possibly by neutralizing the negative charges on the phosphate groups on the DNA backbone. Unlike alkaline treatment, where magnesium is insoluble at high pH, ​​magnesium is readily solubilized at acidic pH.

[0034] Disclosed herein is a method for amplifying a target nucleic acid sequence in a sample. In some embodiments, the method includes: (a) contacting a sample containing a biological entity with an acidic composition to produce an acidic mixture, the acidic composition being capable of dissolving the biological entity to release sample nucleic acids contained therein, the sample nucleic acid including dsDNA suspected of containing a target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length, the acidic composition comprising (i) a monovalent salt and / or a divalent salt, (ii) one or more surfactants, and (iii) an acidic agent, the acidic composition having a pH of less than 4, thereby denaturing the dsDNA to produce single-stranded DNA (ssDNA); (b) contacting a reagent composition (e.g., a dry composition, a wet composition) including a buffer with the acidic mixture to produce a neutral mixture, the neutral mixture including the ssDNA, and the reagent composition comprising one or more amplification reagents; and (c) amplifying the target nucleic acid sequence in the neutral mixture, thereby producing a nucleic acid amplification product. In some embodiments, the dsDNA suspected of containing the target nucleic acid sequence is 100 base pairs or less in length. In some embodiments, one or more of the sample nucleic acids is 100 base pairs or less in length. In some embodiments, the dsDNA suspected of containing the target nucleic acid sequence is longer than 100 base pairs in length. In some embodiments, one or more of the sample nucleic acids is longer than 100 base pairs in length. The dsDNA suspected of containing the sample nucleic acid and / or target nucleic acid sequence can be approximately 100nt, 500nt, 1kb, 5kb, 10kb, 50kb, 100kb, 200kb, 300kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1Mb, 2Mb, 3Mb, 4Mb, 5Mb, 6Mb, 7Mb, 8Mb, 9Mb, 10Mb, 20Mb, 30Mb, 40Mb, 50Mb, 60Mb, 70Mb, 80Mb, 90Mb, or 100Mb in length, or a number or range between any two of these values. The dsDNA suspected of containing the sample nucleic acid and / or target nucleic acid sequence may be completely denatured after contact with the acidic composition.In some embodiments, a portion of the sample nucleic acid molecule and / or dsDNA molecule suspected of containing the target nucleic acid sequence is denatured (e.g., locally denatured) after contact with the acidic composition. In some embodiments, a portion of the sample nucleic acid molecule and / or dsDNA molecule suspected of containing the target nucleic acid sequence is denatured (e.g., locally denatured) in the acidic mixture, and a portion of the molecule remains in double-stranded form.

[0035] The step of amplifying the target nucleic acid sequence may include producing a detectable level of nucleic acid amplification product within about 20 minutes, 15 minutes, or about 10 minutes (e.g., within about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 minute, or a number or range between any two of these values). In some embodiments, steps (b) and (c) are performed simultaneously (e.g., amplification begins once contact of the reagent composition and the acidic mixture occurs). In some embodiments, the reagent composition comprises two or more dry compositions (comprising the same or different components) or two or more wet compositions (comprising the same or different components). In some embodiments, the acidic composition comprises two or more acidic agents (comprising the same or different components).

[0036] The acidic composition may have a pH of about 1 to about 6.9 (e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, The acidic composition may have a pH of about 1 to about 3.9, e.g., 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or a number or range between any two of these values. In some embodiments, the acidic composition has a pH of about 1 to about 3.9, e.g., a pH of about 2, or about 2, about 2.1, about 2.2, about 2.3, about 2.4, and about 2.5. In some embodiments, the acidic composition has a pH of about 2.2. In some embodiments, the neutral mixture has a pH of about 7 to about 9 (e.g., about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or a number or range between any two of these values). In some embodiments, the neutral mixture has a pH of about 8.8.

[0037] The method may include detecting a target nucleic acid sequence in the sample, wherein detecting the target nucleic acid sequence in the sample includes (d) detecting a nucleic acid amplification product, wherein the detecting is performed in less than about 20 minutes, less than about 15 minutes, or less than about 10 minutes (e.g., about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 minute, or a number or range between any two of these values) from the time the reagent composition contacts the acidic mixture. Contacting the reagent composition (e.g., dry composition, wet composition) with the acidic mixture can include dissolving the reagent composition in the acidic mixture. The one or more amplification reagents can include one or more of an enzyme having hyperthermophilic polymerase activity, a first primer, a second primer, and dNTPs. In some embodiments, the reagent composition (e.g., dry composition) is freeze-dried and / or heat-dried and includes one or more additives, where the one or more additives include an amino acid; a sugar or a sugar alcohol. In some embodiments, the sugar or sugar alcohol includes sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the one or more additives include a polymer. In some embodiments, the polymer includes polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof.

[0038] The dsDNA suspected of containing the target nucleic acid sequence can include a first strand and a second strand that are complementary to each other. In some embodiments, the step of amplifying the target nucleic acid sequence comprises amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying comprises contacting the ssDNA with: i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence, and the second primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product, wherein the nucleic acid amplification product comprises (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length.

[0039] An enzyme with hyperthermophilic polymerase activity can have an amino acid sequence that is at least about 90% or 95% identical to the sequence of SEQ ID NO: 1 or a functional fragment thereof, for example, an enzyme with hyperthermophilic polymerase activity can be a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, an enzyme with hyperthermophilic polymerase activity has reduced or no exonuclease activity. The first primer and / or the second primer may be approximately 8 to 16 bases in length. The first primer and / or the second primer may contain one or more of DNA bases, modified DNA bases, or combinations thereof. The nucleic acid amplification product may be approximately 20 to 40 bases in length (e.g., approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases in length). The spacer sequence may comprise a portion of the target nucleic acid sequence. The spacer sequence may be 1 to 10 bases in length. The dsDNA may include genomic DNA (gDNA), plasmid DNA, or both.

[0040] The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the biological entity includes a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoan. The virus may be hepatitis B virus, herpes simplex, herpes virus 6, herpes virus 7, Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, rotavirus, human adenovirus, or genital human papillomavirus (HPV). The bacteria can be Salmonella enterica, Streptococcus pyogenes, Clostridium difficile, Streptococcus agalactiae, Mycobacterium tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma species, Legionella pneumophila, Legionella dumophila, Mycoplasma fermentans, Ehrlichia species, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae, S. agalactiae, or Listeria monocytogenes. The fungus can be Coccidioides posadaci, Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, or Trichophyton rubrum. The protozoan can be Trichomonas vaginalis, Trypanosoma cruzi, Leishmania species, Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora species, or Eimeria species.

[0041] The sample can be a biological sample or an environmental sample. An environmental sample can be or be derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof. A biological sample can be or be derived from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of a skin or mucosal surface, a culture thereof, or any combination thereof.

[0042] The step of contacting the sample with the acidic composition may be carried out at temperatures of about 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 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, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C The step of contacting the sample with the acidic composition may be carried out at a temperature of about 78°C. The step of contacting the sample with the acidic composition may be carried out at a temperature within the range of about 18°C ​​to about 25°C (e.g., room temperature). The step of contacting the sample with the acidic composition can be carried out for a period of about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or about 60 minutes, or a number or range between any two of these values.

[0043] The buffering agent can include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium buffer, or any combination thereof. In some embodiments, the neutral mixture has a pH within the range of about 7 to about 9 (e.g., about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, about 9.0, or a number or range between any two of these values). In some embodiments, the neutral mixture has a pH of about 8.8. The buffer may be at about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM M, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35m M, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50m M, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65 mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, or a number or range of concentrations between any two of these values. Buffers present in the reagent compositions (e.g., dry compositions, wet compositions) and neutral mixtures provided herein can vary and include, for example, citrate buffer, maleate, phosphate, glycine, glycylglycine, carbonate, ethanolamine, ADA, imidazole, hydrazine, HEPBS, TABS,Borate, N-(2-acetamido)-aminoethanesulfonic acid (ACES), salt of acetic acid (acetate), N-(2-acetamido)-iminodiacetic acid (ADA), 2-aminoethanesulfonic acid, taurine (AES), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (Ammediol or AMPD), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonate Acid (AMPSO), N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), Sodium bicarbonate, N,N'-bis(2-hydroxyethyl)-glycine (Bicine), [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (bis-tris), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (bis-tris-propane), Boric acid, Dimethylarsenic acid acid) (cacodylate), 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), sodium carbonate, cyclohexylaminoethanesulfonic acid (CHES), citric acid salt (citrate), 3-[N-bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), formate salt (formate salt of formic acid), glycine, glycylglycine, N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid (HEPPS, EPPS), N-(2-hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), malic acid salt (malate), maleate salt of maleic acid), 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), salts of phosphoric acid (phosphates),Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, salts of succinic acid (succinate), 3-{[tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid (TAPSO), triethanolamine (TEA), 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), N-[tris(hydroxymethyl)-methyl]-glycine (tricine), and tris(hydroxymethyl)-aminomethane (tris), or any combination thereof.

[0044] The methods, compositions, and kits disclosed herein can pair the use of acidic separation with amplification of short target nucleic acid sequences (e.g., about 30 nucleotides in length). The target nucleic acid sequence can be about 20 nucleotides or less, about 30 nucleotides or less, about 40 nucleotides or less, about 50 nucleotides or less, about 60 nucleotides or less, or about 100 nucleotides or less (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, The target nucleic acid sequence can have a length of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 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 nucleotides. The target nucleic acid sequence can have a length of about 30 nucleotides. The amplifying step may be performed under isothermal amplification conditions. Isothermal amplification conditions may include a constant temperature of about 30°C to about 72°C. Isothermal amplification conditions may include a constant temperature of about 67°C. The amplifying step may be performed for a period of about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or about 60 minutes, or any number or range between any two of these values. The amplifying step may be performed for a period of about 15 minutes. The amplifying step may be performed under helicase-free, single-stranded binding protein-free, cleavage agent-free, and recombinase-free isothermal amplification conditions.

[0045] In some embodiments, step (d) further comprises determining the amount of dsDNA containing the target nucleic acid sequence in the sample. Detecting the nucleic acid amplification product may comprise using a real-time detection method. Detecting the nucleic acid amplification product may comprise contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product, wherein the signal-generating oligonucleotide comprises a fluorophore, a quencher, or both. Detecting the nucleic acid amplification product may comprise detecting a fluorescent signal. The fluorescent signal may be from a molecular beacon. The method may be performed in a single reaction vessel. The amplifying step can include multiplex amplification of two or more target nucleic acid sequences. The detecting step can include multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. In some embodiments, the two or more target nucleic acid sequences are specific to two or more different organisms. In some embodiments, the two or more different organisms include Chlamydia trachomatis and Neisseria gonorrhea.

[0046] In some embodiments of the methods described herein, the amplifying step / amplification does not include the use of any enzyme other than an enzyme having hyperthermophilic polymerase activity. The amplifying step can include one or more of the following: Archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplifying step does not include one or more of the following: Archaeal polymerase amplification (APA), LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA.

[0047] In some embodiments, the method does not include one or more of the following: (i) diluting the acidic mixture (e.g., the processed sample); (ii) diluting the neutral mixture (e.g., the amplification reaction mixture); (iii) heat denaturing the acidic mixture; (iv) sonicating the acidic mixture; (v) sonicating the neutral mixture; (vi) adding an RNase inhibitor to the acidic mixture; (vii) adding an RNase inhibitor to the neutral mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more nucleic acid amplification products from the acidic mixture or the neutral mixture. (xii) thermally and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; (xiii) adding RNase H to the acidic or neutral mixture; (xiv) contacting the sample, acidic or neutral mixture with a helicase, single-stranded binding protein, nicking enzyme, restriction enzyme or recombinase; (xv) contacting the sample, acidic or neutral mixture with glycerol, formamide or urea; (xvi) purifying ssDNA from the acidic or neutral mixture; (xvii) a heat denaturation step; and (xviii) enriching, purifying and / or isolating dsDNA.

[0048] Compositions, kits, and methods for nucleic acid detection, in which lysis agents used to lyse biological entities (e.g., virus particles, bacteria) are prevented from inactivating amplification reagents (e.g., polymerases), and deleterious ribonuclease activity is inhibited at some or all stages, are described in PCT Patent Application Publication No. WO2022198086, the entire contents of which are incorporated herein by reference. In some embodiments, the acidic compositions (e.g., lysis buffers) provided herein contain one or more reducing agents (e.g., DTT) described therein, and / or the reagent compositions provided herein contain one or more protecting agents (e.g., cyclodextrin compounds) described therein. In some embodiments, the methods and compositions of the present disclosure enable isothermal amplification and real-time detection of nucleic acids for direct pathogen lysis in clinical samples, without the need for sample separation or purification for point-of-care molecular diagnostics. In some embodiments, acidic compositions (e.g., lysis buffers) containing strong ionic detergents that can be used to lyse pathogens in clinical samples are provided. In some embodiments, the acidic composition (e.g., lysis buffer) can include a chelating agent. The amplification reagents can include a protectant for the lysis reagent, can be dried (e.g., lyophilized, heat-dried), and can be used in a point-of-care setting to amplify the released nucleic acids.

[0049] The methods and compositions provided herein can be applied to other amplification methods that do not require purification or separation, such as PCR, RT-PCR, or other isothermal amplification methods. The methods and compositions provided herein can also be applied to genome sequencing or any nucleic acid (DNA or RNA) amplification or detection method that requires a sample preparation step. The methods and compositions provided herein can also find use in genotyping, diagnostics, and forensics. The methods and compositions of the present disclosure are not limited to isothermal amplification methods, but rather can be applied to other amplification / detection methods, such as RT-PCR, WGS sequencing, and RNA purification / extraction without separation.

[0050] Disclosed herein are methods for detecting a target nucleic acid sequence in a sample. In some embodiments, the method includes: (a) contacting a sample containing a biological entity with a lysis buffer (e.g., an acidic composition) provided herein to generate an acidic mixture (e.g., a processed sample), where the lysis buffer is capable of lysing the biological entity to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing the target nucleic acid sequence. The method may also include: (b) contacting a reagent composition (e.g., a dry composition, a wet composition) with the acidic mixture to generate a neutral mixture (e.g., an amplification reaction mixture), where the reagent composition includes one or more amplification reagents. The method may also include (c) amplifying the target nucleic acid sequence in the neutral mixture, thereby generating a nucleic acid amplification product. The method may include (d) detecting the nucleic acid amplification product, wherein the detecting is performed in less than or less than about 20 minutes (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or a number or range between any two of these values) from the time the reagent composition is contacted with the acidic mixture. In some embodiments, steps (b) and (c) are performed simultaneously (e.g., amplification begins once contact between the reagent composition and the acidic mixture occurs). In some embodiments, the reagent composition comprises two or more dry compositions (comprising the same or different components) or two or more wet compositions (comprising the same or different components). In some embodiments, the lysis buffer comprises two or more lysis buffers (comprising the same or different components).

[0051] The sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid. The sample ribonucleic acid may include cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or any combination thereof. The one or more amplification reagents may include a reverse transcriptase and / or an enzyme having hyperthermophilic polymerase activity. In some embodiments, the enzyme having hyperthermophilic polymerase activity has reverse transcriptase activity. Contacting a reagent composition (e.g., a dry composition, a wet composition) with an acidic mixture (e.g., a sample to be treated) may include dissolving the reagent composition in the acidic mixture. The reagent composition may include one or more of a reverse transcriptase, an enzyme having hyperthermophilic polymerase activity, a first primer, a second primer, and a reverse transcription primer. Amplification may be performed under isothermal amplification conditions. Detecting the nucleic acid amplification product may include using a real-time detection method. The sample nucleic acid may include a nucleic acid comprising a target nucleic acid sequence. The target nucleic acid sequence may comprise a first strand and a second strand that are complementary to each other.

[0052] Amplifying a target nucleic acid sequence can include amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other under isothermal amplification conditions, wherein the amplification includes contacting a nucleic acid comprising the target nucleic acid sequence with: i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the second primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product, wherein the nucleic acid amplification product comprises (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length. In some embodiments, the amplification does not involve using an enzyme other than an enzyme having hyperthermophilic polymerase activity, and the amplification step does not involve denaturing the nucleic acid. In some embodiments, the method does not involve contacting the nucleic acid with a single-stranded DNA binding protein before or during step (c). In some embodiments, the method does not involve thermal or enzymatic denaturation of the sample nucleic acid.

[0053] The nucleic acid may be dsDNA. The nucleic acid may be a product of a reverse transcription reaction. The nucleic acid may be a product of a reverse transcription reaction generated from a sample ribonucleic acid. Step (c) may include generating the nucleic acid by a reverse transcription reaction. The sample nucleic acid may include a sample ribonucleic acid. The method may include contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. Amplifying the target nucleic acid sequence includes (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to generate dsDNA, the dsDNA comprising the target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; and (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplification involves hybridizing the dsDNA to (i) a first primer and a second primer, the first primer being capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the second primer being capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence. and (ii) an enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product, the nucleic acid amplification product comprising (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length.

[0054] In some embodiments, the method does not include the use of enzymes other than the reverse transcriptase and the enzyme having hyperthermophilic polymerase activity. Step (d) may further include determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. The enzyme with hyperthermophilic polymerase activity may have an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. The enzyme with hyperthermophilic polymerase activity may be a polymerase that comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme with hyperthermophilic polymerase activity has low or no exonuclease activity. Amplification of the target nucleic acid sequence can be carried out at a constant temperature of about 55°C to about 75°C, for example, about 65°C. The first primer, the second primer, or both may be about 8 to 16 bases in length. The first primer, the second primer, or both may contain one or more of DNA bases, modified DNA bases, or combinations thereof. The nucleic acid amplification product may be about 20 to 40 bases in length. The spacer sequence may contain a portion of the target nucleic acid sequence. The spacer sequence may be 1 to 10 bases in length.

[0055] In some embodiments, the method includes contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product. The signal-generating oligonucleotide may include a fluorophore, a quencher, or both. Detecting the nucleic acid amplification product may include detecting a fluorescent signal. The fluorescent signal may be from a molecular beacon. The method may be performed in a single reaction vessel. The sample ribonucleic acid may be simultaneously contacted with a reverse transcriptase and an enzyme having hyperthermophilic polymerase activity. The sample ribonucleic acid may be simultaneously contacted with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, and a first and second primer. In some embodiments, the sample ribonucleic acid is simultaneously contacted with the reverse transcriptase, the enzyme having hyperthermophilic polymerase activity, a first primer, a second primer, and a reverse transcription primer. Reverse transcription of the sample ribonucleic acid may occur by adding a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. In some embodiments, the oligo(dT) primer is 12-18 nucleotides in length and binds to the endogenous poly(A) tail at the 3' end of the mRNA. The random hexanucleotide primer can bind to various complementary sites on the sample ribonucleic acid. The target-specific oligonucleotide primer typically selectively primes the sample ribonucleic acid of interest. In some embodiments, the first primer and / or the second primer is a reverse transcription primer.

[0056] The amplification step may comprise multiplex amplification of two or more target nucleic acid sequences. The detection step may comprise multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences. The two or more target nucleic acid sequences may be specific to two or more different organisms. The lysis buffer (e.g., acidic composition) provided herein may be used upstream of various amplification reactions, such as, for example, isothermal amplification reactions. In some embodiments, the method does not include one or more of, or none of, the following: (i) diluting the acidic mixture (e.g., the processed sample); (ii) diluting the neutral mixture (e.g., the amplification reaction mixture); (iii) heat denaturing the acidic mixture; (iv) sonicating the acidic mixture; (v) sonicating the neutral mixture; (vi) adding an RNase inhibitor to the acidic mixture; (vii) adding an RNase inhibitor to the neutral mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysing agents (e.g., one or more detergents) from the acidic or neutral mixture; (xii) heat and / or enzymatic denaturation of the sample nucleic acid before or during amplification; and (xiii) adding RNase H to the acidic or neutral mixture. In some embodiments, the sample is held at the amplification temperature (e.g., 67°C). In some embodiments, the sample (e.g., a sample containing RNA) is held at a temperature between room temperature and the reaction temperature for 1-2 minutes prior to the amplification reaction to facilitate the reverse transcription reaction.

[0057] In some embodiments, step (a), step (b), step (c) and / or step (d) are carried out for a period of about 20 minutes, 15 minutes, 10 minutes, 5 minutes, 2.5 minutes or about 1 minute. In some embodiments, step (a), step (b), step (c) and / or step (d) comprises sonication, osmotic shock, chemical treatment, heating, or any combination thereof. The term "isothermal amplification reaction" shall be given its ordinary meaning and shall include reactions in which the temperature does not change significantly during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10°C during the main enzymatic reaction step in which amplification occurs, for example, by no more than 5°C, and no more than 2°C. Depending on the method of isothermal amplification of nucleic acids, different enzymes can be used for amplification. Exemplary isothermal amplification compositions and methods are described in WO2017176404, the entire contents of which are incorporated herein by reference.

[0058] Disclosed herein are methods for amplifying nucleic acids. In some embodiments, the methods include contacting a sample nucleic acid under isothermal amplification conditions with: a) at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid; and b) at least one component providing hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the methods include contacting the sample nucleic acid under isothermal amplification conditions with: a) non-enzyme components comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid; and b) enzymatic components consisting of a hyperthermophile polymerase or a polymerase comprising an amino acid sequence at least about 90% identical or at least 95% identical to a hyperthermophile polymerase, thereby generating a nucleic acid amplification product. In some embodiments, the method comprises contacting a sample nucleic acid under isothermal amplification conditions with a) non-enzymatic components comprising at least one oligonucleotide, wherein the at least one oligonucleotide comprises a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) enzymatic activity consisting of i) a hyperthermophile polymerase activity and optionally ii) a reverse transcriptase activity, thereby producing a nucleic acid amplification product.

[0059] Disclosed herein are methods for processing nucleic acids. In some embodiments, the methods include amplifying nucleic acids, wherein the amplifying step comprises contacting the sample nucleic acid under isothermal amplification conditions with a) at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) at least one component providing hyperthermophile polymerase activity, thereby generating nucleic acid amplification products. In some embodiments, the methods for processing nucleic acids include amplifying nucleic acids, wherein the amplifying step comprises contacting the sample nucleic acid under isothermal amplification conditions with a) non-enzymatic components comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) enzymatic components consisting of a hyperthermophile polymerase or a polymerase comprising an amino acid sequence at least about 90% identical or at least 95% identical to a hyperthermophile polymerase, thereby generating nucleic acid amplification products. In some embodiments, methods for processing nucleic acids include amplifying the nucleic acids, the amplifying step comprising contacting the sample nucleic acid under isothermal amplification conditions with a) non-enzymatic components including at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) enzymatic activity consisting of i) a hyperthermophile polymerase activity and optionally ii) a reverse transcriptase activity, thereby producing nucleic acid amplification products. In some embodiments, the enzymatic activity consists of i) a hyperthermophile polymerase activity and ii) a reverse transcriptase activity.

[0060] Disclosed herein is a method for determining the presence, absence, or amount of a target sequence in a sample nucleic acid. In some embodiments, the method includes: a) amplifying a target sequence in the sample nucleic acid, the target sequence comprising a first strand and a second strand, the first strand and the second strand being complementary to each other, the amplification comprising: amplifying the sample nucleic acid under helicase-free isothermal amplification conditions with: i) a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprising or consisting of a first polynucleotide that is sequentially complementary to a sequence of the first strand, and the second oligonucleotide comprising or consisting of a second polynucleotide that is sequentially complementary to a sequence of the second strand; and ii) at least one component providing a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, the nucleic acid amplification product comprising: 1) the first oligonucleotide; and b) contacting the sample nucleic acid with at least one component comprising or consisting of: 1) a first nucleotide sequence that is contiguous with or substantially identical to a first polynucleotide of the second oligonucleotide, 2) a second nucleotide sequence that is contiguous with or substantially identical to a second polynucleotide of the second oligonucleotide, and 3) a spacer sequence comprising 1 to 10 bases flanked by the first nucleotide sequence and the second nucleotide sequence; and b) detecting nucleic acid amplification products, wherein the detection of the nucleic acid amplification products includes the use of a real-time detection method and is carried out within 10 minutes or less from the time of contacting the sample nucleic acid with (a)(i) and (a)(ii), thereby determining the presence, absence, or amount of the target sequence in the sample nucleic acid.

[0061] The present disclosure includes a kit for determining the presence, absence, or amount of a target sequence in a sample nucleic acid. In some embodiments, the kit includes: a) components for amplifying a target sequence in the sample nucleic acid under helicase-free isothermal amplification conditions, the components including: i) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises or consists of a first polynucleotide that is sequentially complementary to a sequence in a first strand of the target sequence, and the second oligonucleotide comprises or consists of a second polynucleotide that is sequentially complementary to a sequence in a second strand of the target sequence, and the first strand and the second strand of the target sequence are complementary to each other; and ii) components including at least one component providing hyperthermophile polymerase activity; and b) at least one component providing real-time detection activity of nucleic acid amplification products.

[0062] The enzymatic activity can comprise or consist of i) hyperthermophilic polymerase activity and ii) reverse transcriptase activity. In some embodiments, the method does not include enzymatic and / or thermal denaturation of the sample nucleic acid before or during amplification. In some embodiments, the sample nucleic acid is not contacted with an endonuclease before or during amplification. In some embodiments, the sample nucleic acid is not contacted with an unwinding agent before or during amplification. In some embodiments, the sample nucleic acid is not contacted with a helicase before or during amplification. In some embodiments, the sample nucleic acid is not contacted with a recombinase before or during amplification. In some embodiments, the sample nucleic acid is not contacted with a single-stranded DNA binding protein before or during amplification. In some embodiments, the sample nucleic acid is unmodified before amplification. In some embodiments, the unmodified sample nucleic acid is derived from disrupted cells. In some embodiments, the sample nucleic acid comprises DNA. In some embodiments, the sample nucleic acid comprises genomic DNA. In some embodiments, the sample nucleic acid comprises RNA. In some embodiments, the sample nucleic acid comprises viral RNA. In some embodiments, the sample nucleic acid comprises bacterial RNA. The sample nucleic acid can comprise single-stranded nucleic acid, double-stranded nucleic acid, or both. For example, a double-stranded nucleic acid can comprise a first strand and a second strand. In some embodiments, the at least one oligonucleotide comprises or consists of a first oligonucleotide and a second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide each comprise 8 to 16 bases. In some embodiments, the first oligonucleotide comprises or consists of a first polynucleotide that is complementary to a target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide that is complementary to a target sequence in the second strand of the sample nucleic acid. In some embodiments, the sample nucleic acid is obtained from a subject before amplification. In some embodiments, unpurified sample nucleic acid is amplified. In some embodiments, purified sample nucleic acid is amplified. In some embodiments, the method further comprises purifying the sample nucleic acid before amplification.

[0063] In some embodiments, the hyperthermophile polymerase activity is provided by a hyperthermophile polymerase or a functional fragment thereof. In some embodiments, the hyperthermophile polymerase activity is provided by a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase or a functional fragment thereof. In some embodiments, the hyperthermophile polymerase activity is provided by an archaeal hyperthermophile polymerase or a functional fragment thereof. In some embodiments, the hyperthermophile polymerase activity is provided by a polymerase comprising the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof, or a polymerase comprising an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. In some embodiments, the hyperthermophile polymerase activity is provided by a polymerase with reduced or no exonuclease activity. In some embodiments, amplification is performed at a constant temperature of about 55°C to about 75°C, e.g., about 55°C to about 65°C, or about 65°C, or about 60°C.

[0064] In some embodiments, the nucleic acid amplification product is detectable in 20, 15, 14, 13, 12, 11, or 10 minutes or less. In some embodiments, the nucleic acid amplification product comprises or consists of a polynucleotide that is contiguously complementary to or substantially identical to a target sequence in the sample nucleic acid. The nucleic acid amplification product may be about 20-40 bases in length. In some embodiments, the nucleic acid amplification product comprises or consists of: i) a first nucleotide sequence that is contiguously complementary to or substantially identical to a first polynucleotide of the first oligonucleotide; ii) a second nucleotide sequence that is contiguously complementary to or substantially identical to a second polynucleotide of the second oligonucleotide; and iii) a spacer sequence flanked by the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the spacer sequence comprises 1-10 bases, e.g., 1-5 bases. In some embodiments, the spacer sequence is neither complementary nor identical to the first polynucleotide of the first oligonucleotide, nor complementary nor identical to the second polynucleotide of the second oligonucleotide. In some embodiments, the spacer sequence is contiguous with or substantially identical to a portion of the target sequence in the sample nucleic acid.

[0065] In some embodiments, the method further comprises detecting the nucleic acid amplification product. In some embodiments, detecting the nucleic acid amplification product is performed within 10 minutes or less from the time the sample nucleic acid is contacted with the components providing hyperthermophile polymerase activity and the at least one oligonucleotide. In some embodiments, detecting the nucleic acid amplification product comprises using a real-time detection method. In some embodiments, detecting the nucleic acid amplification product comprises detecting a fluorescent signal. In some embodiments, the fluorescent signal is from a molecular beacon. In some embodiments, the method further comprises contacting the nucleic acid amplification product with i) a polynucleotide complementary to a sequence in the amplification product and ii) a signal-generating oligonucleotide comprising a fluorophore and a quencher. In some embodiments, one or more of the at least one oligonucleotide comprises a polynucleotide that is not complementary to a sequence in the sample nucleic acid that hybridizes to the signal-generating oligonucleotide, and the method further comprises contacting the amplification product with a signal-generating oligonucleotide comprising a fluorophore and a quencher. In some embodiments, the method is performed in a single reaction volume. In some embodiments, the method is performed in a single reaction vessel. In some embodiments, the method comprises multiplex amplification. In some embodiments, the enzymatic activity comprises i) hyperthermophile polymerase activity and ii) reverse transcriptase activity. In some embodiments, the first oligonucleotide comprises or consists of a first polynucleotide that is complementary to a target sequence in a first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide that is complementary to a target sequence in a second strand of the sample nucleic acid. In some embodiments, the first oligonucleotide comprises or consists of a first polynucleotide that is sequentially complementary to a target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide that is sequentially complementary to a target sequence in the second strand of the sample nucleic acid.

[0066] In some embodiments, the amplifying step comprises contacting the sample nucleic acid under helicase-free and recombinase-free isothermal amplification conditions. In some embodiments, the at least one component providing hyperthermophile polymerase activity comprises or consists of a hyperthermophile polymerase or a functional fragment thereof, or a polymerase comprising or consisting of an amino acid sequence at least about 90% identical to a hyperthermophile polymerase or a functional fragment thereof. In some embodiments, part (a)(ii) further comprises at least one component providing reverse transcriptase activity. In some embodiments, the at least one component providing hyperthermophile polymerase activity further provides reverse transcriptase activity. In some embodiments, the sample nucleic acid is amplified under helicase-free and recombinase-free isothermal amplification conditions. In some embodiments, the real-time detection activity is provided by a molecular beacon. In some embodiments, the kit further comprises instructions for performing the methods provided herein for determining the presence, absence, or amount of a target sequence in a sample nucleic acid. In some embodiments, the methods and compositions described herein include a storage-stable lysis buffer. In some embodiments, the lysis buffer is resistant to the formation of precipitates over a period of time under storage conditions (e.g., a storage-stable lysis buffer). Compositions, kits, and methods in which the lysis buffer resists precipitation are described in U.S. Patent Application No. 63 / 307,092, filed February 5, 2022, entitled "NON-OPAQUE LYTIC BUFFER COMPOSITION FORMULATIONS," the entire contents of which are incorporated herein by reference.

[0067] Nucleic Acids, Subjects, Samples, and Nucleic Acid Processing Provided herein are methods and compositions for amplifying nucleic acids. The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein. This term refers to nucleic acids of any composition, including DNA (e.g., complementary DNA (cDNA) and genomic DNA (gDNA)), RNA (e.g., message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and / or DNA or RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single- or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be found in plasmids, phages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or in vivo. It may be or be derived from other nucleic acids that can be replicated or replicated in vitro or in a host cell, cell, cell nucleus, mitochondria, or cell cytoplasm. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.The term can also include nucleotide analogs, single-stranded polynucleotides ("sense" or "antisense," "plus" or "minus" strand, "forward" or "reverse" reading frame, "forward" or "reverse" strand), and RNA or DNA equivalents, derivatives, variants, and analogs synthesized from double-stranded polynucleotides. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain and generally includes regions preceding and following the coding region (leader and trailer) involved in transcription / translation of the gene product and regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons). Nucleotides or bases generally refer to the purine and pyrimidine molecular units of nucleic acids (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)). In RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid can be expressed as the number of bases.

[0068] In some embodiments of the methods provided herein, one or more nucleic acid targets are amplified. The target nucleic acid may be referred to as a target sequence, a target polynucleotide, and / or a target polynucleotide sequence, and may include double-stranded and single-stranded nucleic acid molecules. The target nucleic acid may be, for example, DNA or RNA. If the target nucleic acid is an RNA molecule, the molecule may be, for example, double-stranded, single-stranded, or the RNA molecule may include a single-stranded target sequence. If the target nucleic acid is double-stranded, the target nucleic acid generally comprises a first strand and a second strand. The first strand and the second strand may be referred to as the forward strand and the reverse strand, and are generally complementary to each other. If the target nucleic acid is single-stranded, the complementary strand may be generated, for example, by polymerization and / or reverse transcription, to make the target nucleic acid double-stranded and have a first / forward strand and a second / reverse strand.

[0069] A target sequence can refer to either the sense or antisense strand of a nucleic acid sequence, a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence can also be a subsequence within a larger polynucleotide. For example, a target sequence can be a short sequence (e.g., 20-50 bases) within a nucleic acid fragment, chromosome, or plasmid targeted for amplification. In some embodiments, a target sequence can refer to a sequence within a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence can refer to the entire sequence targeted for amplification, or to a subsequence within the target nucleic acid to which the oligonucleotide binds. An amplification product can be a larger molecule containing the target sequence as well as at least one other sequence or other nucleotide. An amplification product can be approximately the same length as the target sequence, e.g., exactly the same length as the target sequence. An amplification product can comprise or consist of the target sequence. The target sequence length and / or guanosine cytosine (GC) concentration (percentage) may depend, in part, on the temperature at which the amplification reaction is performed, which may in turn depend, in part, on the stability of the polymerase used in the reaction. Trial assays may be performed to determine the appropriate target sequence length and GC concentration for a set of reaction conditions. For example, if the polymerase is stable up to 60°C-65°C, the target sequence may be, for example, 19-50 nucleotides in length, or, for example, about 40-50, 20-45, 20-40, or 20-30 nucleotides in length. The GC concentration under these conditions may be, for example, less than 60%, less than 55%, less than 50%, or less than 45%.

[0070] Target nucleic acids may include, for example, genomic nucleic acids, plasmid nucleic acids, mitochondrial nucleic acids, cellular nucleic acids, extracellular nucleic acids, bacterial nucleic acids, and viral nucleic acids. In some embodiments, target nucleic acids may include genomic DNA, chromosomal DNA, plasmid DNA, mitochondrial DNA, genes, any type of cellular RNA, messenger RNA, bacterial RNA, viral RNA, or synthetic oligonucleotides. Genomic nucleic acids may include any nucleic acid derived from any genome, for example, animal genomes, plant genomes, insect genomes, viral genomes, and bacterial genomes (e.g., genomes present in spores). In some embodiments, genomic target nucleic acids are present within a specific genomic locus or multiple genomic loci. A genomic locus may include any or a combination of open reading frame DNA, non-transcribed DNA, intronic sequences, exonic sequences, promoter sequences, enhancer sequences, flanking sequences, or any sequences considered to be associated with a given genomic locus.

[0071] The target sequence may comprise one or more types of repetitive elements (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellites, minisatellites, etc.). In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as a repetitive element (e.g., multiple repeats, inverted repeats, palindromic sequences, tandem repeats, microsatellite repeats, minisatellite repeats, etc.). For example, the target sequence may occur multiple times as a repetitive element, and one, some, or all occurrences of the target sequence within the repetitive element can be amplified (e.g., using a single pair of primers) using the methods described herein. In some embodiments, the target sequence is present within the sample nucleic acid (e.g., within a nucleic acid fragment, within a chromosome, within a genome, within a plasmid) as duplicates and / or paralogs. The target nucleic acid may include a microRNA. MicroRNAs, miRNAs, or small temporal RNAs (stRNAs) are short (e.g., about 21-23 nucleotides in length), single-stranded RNA sequences involved in gene regulation. MicroRNAs can interfere with the translation of messenger RNAs and are partially complementary to them. The target nucleic acid may also include microRNA precursors, such as primary transcripts (pri-miRNAs) and pre-miRNA stem-loop RNAs that are further processed into miRNAs. The target nucleic acid may also include small interfering RNAs (siRNAs), which are short (e.g., about 20-25 nucleotides in length), at least partially double-stranded RNA molecules involved in RNA interference (e.g., viral replication or downregulation of gene expression).

[0072] Nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, for example, isolated from a sample obtained from a subject. The subject can be any living or non-living organism, including, but not limited to, humans, non-human animals, plants, bacteria, fungi, viruses, or protists. Any human or non-human animal can be selected, including, but not limited to, mammals, reptiles, birds, amphibians, fish, ungulates, ruminants, bovines (e.g., cows), equines (e.g., horses), caprines and ovines (e.g., sheep, goats), suidae (e.g., pigs), camelids (e.g., camels, llamas, alpacas), monkeys, apes (e.g., gorillas, chimpanzees), ursidae (e.g., bears), poultry, dogs, cats, mice, rats, fish, dolphins, whales, and sharks. The subject can be male or female, and the subject can be of any age (eg, embryo, fetus, infant, child, adult).

[0073] A sample or test sample may be any specimen isolated or obtained from a subject or a portion thereof. Non-limiting examples of specimens include fluids or tissues derived from a subject, including, but not limited to, blood or blood products (such as serum or plasma), umbilical cord blood, bone marrow, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), serum, plasma, urine, aspirate, biopsy sample, intestinal puncture sample, cells (e.g., blood cells) or portions thereof (e.g., mitochondria, nuclei, or extracts), female reproductive tract washings, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage, semen, lymph, bile, tears, sweat, breast milk, mammary fluid, hard tissue (e.g., liver, spleen, kidney, lung, or ovary), or the like, or combinations thereof. The term blood, as conventionally defined, includes whole blood, blood products, or any fraction of blood, such as serum, plasma, or buffy coat. Plasma refers to the fraction of whole blood obtained by centrifugation of blood that has been treated with an anticoagulant. Serum refers to the aqueous portion of the fluid that remains after a blood sample has clotted. Fluid or tissue samples are often collected according to standard protocols commonly followed by hospitals or clinics. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 milliliters) is often collected and can be stored according to standard procedures before or after preparation.

[0074] The sample or test sample can include nucleic acid from spores, viruses, cells, prokaryotes, or eukaryotes, or any sample containing free nucleic acid. For example, the methods described herein can be used to detect nucleic acid outside of spores (e.g., without the need for lysis). The sample can be isolated from any material suspected of containing the target sequence, such as from a subject as described above. In some embodiments, the target sequence is present in air, plants, soil, or other material suspected of containing biological organisms. Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more sources by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from biological samples can be used, including art-known DNA preparation methods and various commercially available reagents or kits, such as Qiagen's QIAamp Circulating Nucleic Acid Kit, QiaAmp DNA Mini Kit, or QiaAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), GenomicPrep™ Blood DNA Isolation Kit (Promega, Madison, Wisconsin), and GFX™ Genomic Blood DNA Purification Kit (Amersham, Piscataway, New Jersey), or combinations thereof. U.S. Patent No. 7,888,006 provides a DNA purification method, but does not disclose the compositions (e.g., lysis buffer, protectant) and methods provided herein.

[0075] In some embodiments, a cell lysis procedure is performed. Cell lysis may be performed before initiating the amplification reaction described herein (e.g., to release DNA and / or RNA from cells for amplification). Cell lysis procedures and reagents are known in the art and can be performed by chemical methods (e.g., detergents, hypotonic solutions, enzymatic procedures, etc., or a combination thereof), physical methods (e.g., French press and sonication, etc.), or electrolytic lysis. For example, chemical methods generally involve disrupting cells using a lysing agent, extracting nucleic acids from the cells, followed by treatment with a chaotropic salt. In some embodiments, cell lysis involves the use of a detergent (e.g., ionic, nonionic, anionic, zwitterionic). In some embodiments, cell lysis involves the use of an ionic detergent (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), deoxycholate, cholate, sarkosyl). Physical methods, such as freeze / thaw trituration and the use of a cell press, may also be useful. High-salt lysis procedures can also be used. For example, alkaline lysis procedures can be used. The latter procedure traditionally incorporates the use of a phenol-chloroform solution, although an alternative phenol-chloroform-free procedure involving three solutions can also be used. In the latter procedure, for example, one solution may contain 15 mM Tris (pH 8.0), 10 mM EDTA, and 100 μg / ml RNase A, a second solution may contain 0.2 N NaOH and 1% SDS, and a third solution may contain 3 M KOAc, pH 5.5. In some embodiments, a cell lysis buffer is used in conjunction with the methods and components described herein.

[0076] Nucleic acids for performing the methods described herein can be provided without processing a sample containing the nucleic acid. For example, nucleic acids for performing the amplification methods described herein can be provided without prior nucleic acid purification. In some embodiments, target sequences are amplified directly from a sample (e.g., without any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids for performing the methods described herein are provided after processing a sample containing the nucleic acid. For example, nucleic acids can be extracted, isolated, purified, or partially purified from a sample. The term "isolated" generally refers to nucleic acids that have been removed from their original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed) and thus have been altered from their original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid" can refer to nucleic acids removed from a subject (e.g., a human subject). Isolated nucleic acids can be provided that have fewer non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. A composition containing isolated nucleic acids may contain no more than about 50%-99% of non-nucleic acid components. A composition comprising an isolated nucleic acid may be free of more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of non-nucleic acid components. The term "purified" generally refers to a nucleic acid that contains less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of non-nucleic acid components present before the nucleic acid is subjected to a purification procedure. A composition comprising a purified nucleic acid may be free of more than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% of other non-nucleic acid components.

[0077] Nucleic acids can be provided for performing the methods described herein without modifying the nucleic acid, for example, by denaturing, digesting, nicking, unwinding, incorporating and / or ligating heterologous sequences, adding epigenetic modifications, adding labels (e.g., 32P, 33 P, 125 I, or 35 Examples of such labels include radioactive labels such as S; enzyme labels such as alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, antigens, haptens, and fluorescent dyes. Thus, in some embodiments, unmodified nucleic acids are amplified.

[0078] The methods disclosed herein for detecting target nucleic acid sequences (single-stranded or dsDNA and / or RNA) in a sample can detect target nucleic acid sequences (e.g., DNA or RNA) with high sensitivity. In some embodiments, the methods can be used to detect target DNA / RNA present in a sample containing multiple RNAs / DNAs (including a target RNA / DNA and multiple non-target RNAs / DNAs), where the target RNA / DNA is 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 It is present in one or more copies per non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meaning and shall refer to DNA, or RNA, or a combination of DNA and RNA.

[0079] The detection threshold of a method for detecting target RNA / DNA in a sample can be, for example, 10 nM or lower. The term "detection threshold" shall be given its ordinary meaning and shall describe the minimum amount of target RNA / DNA that must be present in a sample for detection to occur. As an illustrative example, if the detection threshold is 10 nM, a signal can be detected when the target RNA / DNA is present in the sample at a concentration of 10 nM or higher. In some embodiments, the methods of the present disclosure provide a method for detecting target RNA / DNA at a concentration of 5 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower, 0.05 nM or lower, 0.01 nM or lower, 0.005 nM or lower, 0.001 nM or lower, 0.0005 nM or lower, 0.0001 nM or lower, 0.00005 nM or lower, 0.00001 nM or lower, or 0.00001 nM. or lower, 10 pM or lower, 1 pM or lower, 500 fM or lower, 250 fM or lower, 100 fM or lower, 50 fM or lower, 500 aM (attomolar) or lower, 250 aM or lower, 100 aM or lower, 50 aM or lower, 10 aM or lower, or 1 aM or lower. A composition or method disclosed herein can have attomolar (aM) detection sensitivity, femtomolar (fM) detection sensitivity, picomolar (pM) detection sensitivity, or nanomolar (nM) detection sensitivity.

[0080] A sample may contain sample nucleic acids (e.g., multiple sample nucleic acids). The term "multiple" is used herein to mean two or more. Thus, in some embodiments, a sample contains two or more (e.g., three or more, five or more, ten or more, twenty or more, fifty or more, one hundred or more, five hundred or more, one thousand or more, or five thousand or more) sample nucleic acids (e.g., DNA / RNA). The disclosed methods can be used as highly sensitive methods for detecting target nucleic acids present in a sample (e.g., in a complex mixture of nucleic acids such as DNA / RNA). In some embodiments, a sample contains 5, 10, 20, 25, 50, 100, 500, 10, 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 seeds or 10 7 The sample may contain DNA / RNA from 50 or more species, each differing in sequence from the others. In some embodiments, the sample contains DNA / RNA from cells (e.g., eukaryotic, mammalian, or human cells) or cell lysates (e.g., eukaryotic cell lysates, mammalian cell lysates, human cell lysates, prokaryotic cell lysates, plant cell lysates, etc.).

[0081] As used herein, the term "sample" shall be given its ordinary meaning and shall include any sample containing RNA and / or DNA (e.g., for determining whether target DNA and / or target RNA is present in a population of RNA and / or DNA). A sample may be derived from any source; for example, a sample may be a synthetic combination of purified DNA and / or RNA. A sample may be a cell lysate, a DNA / RNA-enriched cell lysate, or DNA / RNA isolated and / or purified from a cell lysate. A sample may be derived from a patient (e.g., for diagnostic purposes). A sample may be derived from permeabilized cells, crosslinked cells, tissue sections, or combinations thereof. A sample may be derived from tissue prepared by crosslinking followed by delipidation and adjusting to a uniform refractive index. A sample may contain a target nucleic acid (e.g., target DNA / RNA) and multiple species of non-target DNA / RNA. In some embodiments, the target DNA / RNA may be 10, 20, 25, 50, 100, 500, 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , or 10 7 Present in the sample at 1 copy per non-target DNA / RNA.

[0082] Patient-related samples include blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny, and samples that have been manipulated in some way after procurement (e.g., treatment with reagents); washed; or enriched for certain cell populations (e.g., cancer cells) or specific types of molecules (e.g., RNA). Samples may include biological samples, including, but not limited to, clinical samples such as blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, and bone marrow. Biological samples may also include biological fluids derived therefrom (e.g., cancerous cells, infected cells, etc.), such as samples containing RNA obtained from such cells (e.g., RNA-containing cell lysates or other cell extracts).

[0083] The source of the sample may be a diseased (or suspected diseased) cell, fluid, tissue, or organ, or may be a normal (non-diseased) cell, fluid, tissue, or organ. In some embodiments, the source of the sample is a cell, tissue, or organ infected (or suspected to be infected) with a pathogen. For example, the source of the sample may be an individual, which may or may not be infected, and the sample may be any biological sample collected from the individual (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage fluid, sputum, stool sample, cerebrospinal fluid, fine needle aspirate, swab sample (e.g., buccal swab, cervical swab, nasal swab), interstitial fluid, synovial fluid, nasal secretion, tears, buffy coat, mucosal sample, epithelial cell sample (e.g., epithelial cell scraping), etc.). The sample may be a cell-free liquid sample or a liquid sample containing cells. Pathogens may be viruses, fungi, helminths, protozoans, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites, etc. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny headworms, and tapeworms (Cestodes). Protozoan infections include infections by Giardia spp., Trichomonas spp., African trypanosomiasis, amebic dysentery, babesiosis, balantidiosis, Chagas disease, coccidiosis, malaria, and toxoplasmosis. Examples of pathogens, such as parasitic / protozoan pathogens, include, but are not limited to, Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi, and Toxoplasma gondii. Fungal pathogens include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza virus, dengue fever, West Nile virus, herpes virus, yellow fever virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, and papillomavirus. Pathogenic viruses include papovaviruses (e.g., HPV, polyomavirus); hepadnaviruses; herpes viruses (e.g., HSV (e.g., HSV I, HSV II), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, and pityriasis rosea). Rosea, Kaposi's sarcoma-associated herpesvirus); adenoviruses (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); poxviruses (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; variola virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); parvoviruses (e.g., adeno-associated virus (AAV), parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; and Phycodnaviridae. Non-limiting examples of pathogens include Mycobacterium tuberculosis and Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, and Haemophilus influenzae type B.B), Treponema pallidum, Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, human serum bulbo-like virus, respiratory syncytial virus, measles virus, adenovirus, human T-cell leukemia virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, reovirus, poliovirus, Simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlowiilaidlawii, M. salivarium, and M. pneumoniae.

[0084] amplification Provided herein are methods for amplifying nucleic acids. In some embodiments, nucleic acids are amplified using a suitable amplification process. Nucleic acid amplification involves enzymatic synthesis of nucleic acid amplicons (copies) that contain sequences complementary to the nucleotide sequence being amplified. Amplification can be performed in a single vessel, a single chamber, and / or a single volume (i.e., continuous volume). In some embodiments, amplification and detection (e.g., detection methods described herein) are performed in a single vessel, a single chamber, and / or a single volume (i.e., continuous volume).

[0085] The terms "amplify," "amplification," "amplification reaction," or "amplifying" refer to any in vitro process for multiplying copies of a target nucleic acid. Amplification can also refer to an "exponential" increase in the target nucleic acid. "Amplifying" can also refer to a linear increase in the number of target nucleic acids, but is distinct from a single, single primer extension step. In some embodiments, a limited amplification reaction, also known as preamplification, can be performed. Preamplification is a method in which a limited amount of amplification occurs because a small number of cycles, e.g., 10 cycles, are performed. Preamplification allows for some amplification but stops amplification before the exponential phase, typically producing approximately 500 copies of the desired nucleotide sequence. Preamplification can be used to limit inaccuracies associated with reactant depletion in a particular amplification reaction and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a single primer extension step can be performed prior to linear or exponential amplification.

[0086] A general description of the amplification process is provided herein. For example, when a primer (e.g., an oligonucleotide described herein) and a target nucleic acid are contacted, complementary sequences anneal or hybridize to each other. The primer can anneal to the target nucleic acid at or near (e.g., adjacent, abutting, etc.) the sequence of interest. A primer annealed to a target may be referred to as a primer-target hybrid, a hybridized primer-target, or a primer-target duplex. The terms "near" or "adjacent" when referring to a nucleotide sequence of interest refer to the distance (e.g., number of bases) or region between the end of the primer and one or more nucleotides (e.g., a nucleotide sequence) of the target. Generally, adjacent refers to a range of about 1 nucleotide to about 50 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides) from the nucleotide or nucleotide sequence of interest. In some embodiments, a set of primers (e.g., a pair of primers, a forward primer and a reverse primer, a first oligonucleotide and a second oligonucleotide) anneals within about 1-20 nucleotides of a nucleotide or nucleotide sequence of interest to generate an amplification product. In some embodiments, the primers anneal within the nucleotide or nucleotide sequence of interest. After annealing, each primer is extended along the target (i.e., template strand) by a polymerase to generate a complementary strand. For example, several cycles of primer annealing and extension can be performed until a detectable amount of amplification product is generated. In some embodiments, when the target nucleic acid is RNA, a DNA copy (cDNA) of the target RNA is synthesized by reverse transcription before or during the amplification step.

[0087] Components of an amplification reaction (e.g., one or more amplification reagents) can include, for example, one or more primers (e.g., individual primers, primer pairs, primer sets, oligonucleotides, and multiple primer sets for multiplex amplification), a nucleic acid target (e.g., a target nucleic acid derived from a sample), one or more polymerases, nucleotides (e.g., dNTPs), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, a monovalent ion, and a divalent ion). The amplification reaction may further include one or more of a reverse transcriptase, a reverse transcription primer, and one or more detection agents. Nucleic acid amplification can be performed in the presence of natural nucleotides, such as dideoxyribonucleoside triphosphates (dNTPs) and / or derivatized nucleotides. Natural nucleotides generally refer to adenylate, guanylate, cytidylate, thymidylate, or uridylate. Derivatized nucleotides generally are nucleotides other than natural nucleotides. Ribonucleoside triphosphates are referred to as NTPs or rNTPs, where N can be A, G, C, or U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be referred to herein as A, G, C, T, or U, without specific reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs can be used, such as analogs containing a detectable label (e.g., a fluorescent label or a colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, such as 32 P, 33 P, 125 I, or 35 The amplification can be performed in the presence of a radioactive label such as S; an enzyme label such as alkaline phosphatase; a fluorescent label such as fluorescein isothiocyanate (FITC); or other labels such as biotin, avidin, digoxigenin, an antigen, a hapten, or a fluorescent dye. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., TriLink's CleanAmp™ dNTPs).

[0088] The one or more amplification reagents may include non-enzymatic and enzymatic components. Non-enzymatic components may include, for example, primers, nucleotides, buffers, salts, reducing agents, detergents, and ions. In some embodiments, the non-enzymatic components do not include proteins (e.g., nucleic acid-binding proteins), enzymes, or proteins with enzymatic activity, such as polymerases, reverse transcriptases, helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. In some embodiments, the enzymatic components consist of a polymerase or a polymerase and a reverse transcriptase. Thus, such enzymatic components would exclude other proteins (e.g., nucleic acid-binding proteins and / or proteins with enzymatic activity), such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases.

[0089] In some embodiments, amplification conditions include enzymatic activity (e.g., enzymatic activity provided by a polymerase, or enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include enzymatic activity provided by enzymes other than the polymerase and / or reverse transcriptase, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, and recombinases. The polymerase activity and reverse transcriptase activity may be provided by separate enzymes or separate enzyme types (e.g., a polymerase and a reverse transcriptase), or may be provided by a single enzyme or enzyme type (e.g., a polymerase). Nucleic acid amplification may include non-thermal cycling PCR. In some embodiments, nucleic acid amplification includes an isothermal amplification process, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process performed at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature is maintained essentially constant during the amplification reaction. Isothermal amplification conditions generally do not include a thermal cycling (i.e., cycling between upper and lower temperature limits) component to the amplification process. When amplifying under isothermal conditions, the reaction can be maintained at an essentially constant temperature, meaning that the temperature does not need to be maintained at exactly one temperature. For example, isothermal amplification processes may experience small temperature fluctuations (e.g., ±1-5°C) due to environmental or equipment-based variables. Often, the entire reaction volume is maintained at an essentially constant temperature, and isothermal reactions, as used herein, generally do not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0090] The isothermal amplification reaction herein can be carried out at an essentially constant temperature. In some embodiments, the isothermal amplification reaction herein is carried out at a temperature of about 55°C to about 75°C, for example, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C, or a temperature about these values, or a value or range between any two of these values. In some embodiments, a temperature element (e.g., a heat source) is maintained at an essentially constant temperature, for example, about 75°C or less, about 70°C or less, about 65°C or less, or about 60°C or less.

[0091] The amplification process herein can be carried out for a certain period of time, for example, until a detectable nucleic acid amplification product is produced. The nucleic acid amplification product can be detected by any suitable detection process and / or detection process described herein. The amplification process can be carried out within about 20 minutes or less, or within about 10 minutes or less. For example, the amplification process can be carried out within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or within any value or range between any two of these values. In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation during the amplification step (and / or other steps). In some embodiments, nucleic acid targets can be amplified without exposure to agents or conditions that promote unwinding during the amplification step (and / or other steps). Agents or conditions that denature nucleic acids and / or promote strand separation and / or promote unwinding can include, for example, thermal conditions (e.g., high temperature), pH conditions (e.g., high or low pH), chemical agents, and proteins (e.g., enzymatic agents).

[0092] In some embodiments, the methods disclosed herein do not involve heat denaturation (e.g., heating a solution containing nucleic acids to an elevated temperature, such as 75°C, 80°C, 90°C, or 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of nucleic acids. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions provided herein do not include a helicase, topoisomerase, ligase, exonuclease, endonuclease, restriction enzyme, nicking enzyme, recombinase, RNA replicase, and / or a nucleic acid binding protein (e.g., a single-stranded binding protein). In some embodiments, the compositions and methods provided herein do not include intercalating agents, alkylating agents, and / or chemicals such as formamide, glycerol, urea, dimethyl sulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods of the present disclosure do not include contacting nucleic acids with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding). In some embodiments, the amplification step (e.g., step (c)) does not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophilic polymerase) and / or low pH conditions (e.g., contact with acid).

[0093] Nucleic acid targets can be amplified without exposure to agents or conditions that promote strand separation and / or unwinding, such as helicases, topoisomerases, ligases, exonucleases, endonucleases, restriction enzymes, nicking enzymes, recombinases, RNA replicases, nucleic acid binding proteins (e.g., single-strand binding proteins), or any combination thereof. For example, nucleic acid targets can be amplified without exposure to helicases, including, but not limited to, DNA helicases and RNA helicases. Amplification conditions that do not include the use of helicases are helicase-free amplification conditions.

[0094] Nucleic acid targets can be amplified without exposure to recombinases, including, but not limited to, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX. In some embodiments, nucleic acid targets are amplified without exposure to recombinase accessory proteins, such as recombinase loading factors (e.g., T4 uvsY). Nucleic acid targets can be amplified without exposure to nucleic acid binding proteins (e.g., single-strand binding protein or single-stranded DNA binding protein (SSB)), such as T4 gp32. In some embodiments, nucleic acid targets are amplified without exposure to topoisomerases. Nucleic acid targets can be amplified with or without exposure to agents or conditions that destabilize nucleic acids. As used herein, the term "destabilization" shall be given its ordinary meaning and shall refer to the disruption of the overall organization and geometric orientation (e.g., double helix structure) of a nucleic acid molecule by one or more of tilt, rotation, twist, slip, and flip effects (e.g., as described in Lenglet et al., (2010) Journal of Nucleic Acids Volume 2010, Article ID 290935, page 17). Destabilization generally does not refer to the melting or separation (e.g., denaturation) of nucleic acid strands. Nucleic acid destabilization can be achieved, for example, by exposure to agents such as intercalating or alkylating agents, and / or chemicals such as formamide, urea, DMSO, or betaine. In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, the nucleic acid target is amplified without exposure to ligase and / or RNA replicase.

[0095] In some embodiments, nucleic acid targets can be amplified without cleavage or digestion. For example, nucleic acid targets can be amplified without prior exposure to one or more cleavage agents, resulting in an intact nucleic acid being amplified. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents during amplification. In some embodiments, nucleic acid targets are amplified without exposure to one or more cleavage agents after amplification. Amplification conditions that do not include the use of a cleavage agent may be referred to herein as cleavage agent-free amplification conditions. The term "cleavage agent" generally refers to an agent, possibly a chemical or enzyme, that can cleave nucleic acids at one or more specific or non-specific sites. Specific cleavage agents often specifically cleave specific sites according to a specific nucleotide sequence. Cleavage agents can include endonucleases (e.g., restriction enzymes and nicking enzymes); exonucleases (DNAses, RNAses (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases); and chemical cleavage agents.

[0096] Nucleic acid targets can be amplified without the use of restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without prior exposure to restriction enzymes and / or nicking enzymes. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes during amplification. In some embodiments, nucleic acids are amplified without exposure to restriction enzymes and / or nicking enzymes after amplification. Nucleic acid targets can be amplified without exonuclease treatment. Exonucleases include, for example, DNAse, RNAse (e.g., RNAse H), 5'-3' exonucleases (e.g., exonuclease II), 3'-5' exonucleases (e.g., exonuclease I), and poly(A)-specific 3'-5' exonucleases. In some embodiments, nucleic acids are amplified without exonuclease treatment before, during, and / or after amplification. Amplification conditions that do not include the use of exonucleases are exonuclease-free amplification conditions. In some embodiments, the nucleic acid is amplified without DNAse and / or RNAse treatment.

[0097] Amplified nucleic acids may be referred to herein as nucleic acid amplification products or amplicons. In some embodiments, amplification products include naturally occurring nucleotides, non-naturally occurring nucleotides, nucleotide analogs, and the like, as well as combinations of the foregoing. Amplification products typically have a nucleotide sequence that is identical or substantially identical to the sequence of a sample nucleic acid (e.g., a target sequence) or its complement. A "substantially identical" nucleotide sequence in an amplification product will generally have a high degree of sequence identity (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity) to the nucleotide sequence being amplified or its complement, with variations being the result of poor polymerase fidelity or other variables.

[0098] In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is contiguously complementary or substantially identical to a target sequence in the sample nucleic acid. Contiguously complementary generally refers to, for example, a nucleotide sequence in a first strand in which each base pairs in order (e.g., reading from 5' to 3') with a correspondingly ordered base in a second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered contiguously complementary. In other words, contiguously complementary generally refers to every contiguous base in the nucleotide sequence of the first strand being complementary to the corresponding contiguous base in the nucleotide sequence of the second strand. For example, a first strand having the sequence 5'-ATGCATGCATGC-3' (SEQ ID NO: 3) would be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 4), and every contiguous base in the first strand is complementary to every corresponding contiguous base in the second strand. However, a first strand having the sequence 5'-ATGCATAAAAAAGCATGC-3' (SEQ ID NO: 5) would not be considered contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 4) because the sequence of six adenines (six As) in the middle of the first strand would not pair with bases in the second strand. Contiguous complementary sequences are optionally about 5 to about 25 contiguous bases in length, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range between any two of these values. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is contiguous complementary to or substantially identical to a target sequence in the sample nucleic acid. Thus, in some embodiments, the nucleic acid amplification product does not include any additional sequence (e.g., at the 5' and / or 3' end or within the product) that is not contiguous with or substantially identical to the target sequence, such as additional sequences incorporated into the amplification product by tail primers or ligation, and / or additional sequences that provide cleavage agent recognition sites (e.g., nicking enzyme recognition sites). Generally, unless the target sequence includes tandem repeats, the amplification product will not include products in the form of tandem repeats.

[0099] The nucleic acid amplification product can comprise sequences complementary to or substantially identical to one or more primers used in the amplification reaction, hi some embodiments, the nucleic acid amplification product comprises a first nucleotide sequence that is contiguous to or identical to a first primer sequence and a second nucleotide sequence that is contiguous to or identical to a second primer sequence. Nucleic acid amplification products can include spacer sequences. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is contiguous with or substantially identical to a portion of a target sequence in a sample nucleic acid and is flanked by sequences in the amplification product that are complementary to or substantially identical to one or more primers used in the amplification reaction. The spacer sequence flanked by sequences in the amplification product is generally located between a first sequence (complementary to or substantially identical to the first primer) and a second sequence (complementary to or substantially identical to the second primer). Thus, an amplification product typically includes a first sequence, followed by a spacer sequence, followed by a second sequence. The spacer sequence is generally neither complementary nor substantially identical to the sequence of the primers. The spacer sequence may be or include approximately 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists of or consists essentially of a first nucleotide sequence that is contiguously complementary to or identical to the first primer sequence, a second nucleotide sequence that is contiguously complementary to or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence that is not contiguously complementary to or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product), and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other mechanism incorporated into the amplification product. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguously complementary to or identical to the first and second primer sequences (e.g., at the 5' and / or 3' ends or within the product), and is not part of a spacer sequence, e.g., a tail or loop primer, ligation, or other mechanism incorporated into the amplification product.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or another extra base (e.g., at the 5' and / or 3' end or within the product) introduced into the product due to, for example, errors or promiscuity in the amplification process.

[0100] Nucleic acid amplification products may be up to 50 bases in length, including 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number or range of bases between any two of these values. In some embodiments, nucleic acid amplification products of a given target sequence have the same or substantially the same length (e.g., within 1-10 bases). Thus, nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band on an electrophoresis gel) and generally do not produce multiple signals representing multiple lengths (e.g., a ladder or smear on an electrophoresis gel). In multiplex reactions, nucleic acid amplification products of different target sequences may have different lengths.

[0101] The methods and components described herein can be used for multiplex amplification, which generally refers to the amplification of more than one nucleic acid of interest (e.g., the amplification of more than one target sequence). For example, multiplex amplification can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample. Multiplex amplification can also refer to the simultaneous or sequential amplification of one or more sequences present in multiple samples. For example, multiplex amplification can be used to amplify at least two amplifiable target sequences (e.g., the amplification reaction includes appropriate primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is configured to detect at least two target sequences, but only one of the target sequences is present in the sample being tested, so that both sequences are amplifiable, but only one sequence is amplified. In some embodiments, when two target sequences are present, the amplification reaction results in the amplification of both target sequences. A multiplex amplification reaction including appropriate primers and enzymes can result in the amplification of one, some, or all of the target sequences. In some embodiments, amplification reactions are set up to detect two sequences using a single pair of primers, one sequence being a target sequence and one sequence being a control sequence (e.g., a synthetic sequence that is amplifiable with the same primers as the target sequence but has spacer bases or a sequence that differs from the target). In some embodiments, amplification reactions are set up to detect multiple sets of sequences using corresponding primer pairs, each set including a target sequence and a control sequence.

[0102] Primer Nucleic acid amplification is generally performed in the presence of one or more primers. A primer is generally characterized as an oligonucleotide comprising a nucleotide sequence capable of hybridizing or annealing to a target nucleic acid at or near (e.g., adjacent to) a specific region of interest (i.e., target sequence). A primer can, for example, enable specific determination of the nucleotide sequence of a target nucleic acid or detection of the target nucleic acid or a characteristic thereof (e.g., the presence or absence of a sequence). A primer can be naturally occurring or synthetic. The term specific or specific generally refers to the binding or hybridization of one molecule, such as a primer for a target polynucleotide, with another molecule. That is, the term specific or specific refers to the recognition, contact, and formation of a stable complex between two molecules, compared to substantially less recognition, contact, or complex formation between either of those two molecules and other molecules. The term annealing or hybridization generally refers to the formation of a stable complex between two molecules. The terms primer, oligo, or oligonucleotide can be used interchangeably herein when referring to a primer.

[0103] Primers can be designed and synthesized using any suitable process and may be of any length suitable for hybridizing to a target sequence and carrying out the amplification processes described herein. Primers are often designed according to the sequence of the target nucleic acid. In some embodiments, primers may be about 5 to about 30 bases in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases in length. Primers may be composed of naturally occurring and / or non-naturally occurring nucleotides (e.g., modified nucleotides, labeled nucleotides), or mixtures thereof. Modifications and modified bases include, for example, phosphorylation (e.g., 3' phosphorylation, 5' phosphorylation); attachment chemistry or linker modification (e.g., Acrydite™, adenylation, azide (NHS ester), digoxigenin (NHS ester), cholesteryl-TEG, I-Linker™, amino modifiers (e.g., amino modifier C6, amino modifier C12, amino modifier C6dT, Uni-Link™ amino modifier), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG), thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); fluorophores (e.g., Freedom™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO™ dyes, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy750 (NHS ester)); Iowa Black® dark quencher modifications (e.g., Iowa Black® FQ, Iowa Black® RQ); dark quencher modifications (e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl);spacers (C3 spacer, PC spacer, hexanediol, spacer 9, spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA) ), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases);and click chemistry modifications. In some embodiments, modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, phosphorothioate linkages, 3' phosphate groups, and spacer bases (such as C3 spacers or other spacer bases). For example, a primer may contain one or more O-methyl RNA bases (e.g., 2'-O-methyl RNA bases). 2'-O-methyl RNA is a post-transcriptional modification of RNA commonly found in tRNA and other small RNA molecules. Primers containing 2'-O-methyl RNA bases can be directly synthesized. This modification can, for example, increase the Tm of an RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in a primer to, for example, increase stability and binding affinity with a target sequence. In some embodiments, a primer may contain one or more phosphorothioate linkages (e.g., phosphorothioate bond modifications). Phosphorothioate (PS) bonds replace non-bridging oxygen atoms in the phosphate backbone of a primer with sulfur atoms. This modification typically renders the internucleotide linkage resistant to nuclease degradation. Phosphorothioate bonds can be introduced at the 5' or 3' end of a primer, within the last 3-5 nucleotides, for example, to inhibit exonucleolytic degradation. In some embodiments, phosphorothioate bonds throughout the primer can help reduce endonucleolytic attack. Primers may, for example, contain a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases and, in certain cases, can be used to block extension by DNA polymerase. In some embodiments, primers contain one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated internally or at the 5' end of a primer. Multiple C3 spacers can be added to either end of the primer to introduce long hydrophilic spacer arms for attachment of, for example, fluorophores or other pendant groups;

[0104] A primer may comprise DNA bases, RNA bases, or both, and one or more of the DNA and RNA bases may be modified or unmodified. For example, a primer may be a mixture of DNA bases and RNA bases. A primer may comprise DNA bases (e.g., modified DNA bases and / or unmodified DNA bases). In some embodiments, a primer comprises unmodified DNA bases. In some embodiments, a primer comprises modified DNA bases. A primer may comprise RNA bases (e.g., modified RNA bases and / or unmodified RNA bases). In some embodiments, a primer comprises unmodified RNA bases. In some embodiments, a primer comprises modified RNA bases. In some embodiments, a primer does not comprise RNA bases. In some embodiments, a primer does not comprise DNA bases. In some embodiments, a primer does not comprise a cleavage agent recognition site (e.g., does not comprise a nicking enzyme recognition site). In some embodiments, a primer does not comprise a tail (e.g., does not comprise a tail comprising a nicking enzyme recognition site).

[0105] In some embodiments, all or a portion of the primer sequence may be complementary or substantially complementary to the target nucleic acid. Substantially complementary, in the context of sequences, generally refers to nucleotide sequences that will hybridize to each other. The stringency of hybridization conditions can be varied to allow for varying amounts of sequence mismatch. The target sequence and primer sequence may be at least 75% complementary to each other, including, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to each other. A primer that is substantially complementary to a target nucleic acid sequence is typically also substantially identical to the complement of the target nucleic acid sequence (i.e., the sequence of the antisense strand of the target nucleic acid). The primer and the antisense strand of the target nucleic acid may be at least 75% identical in sequence, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to each other.

[0106] In some embodiments, the primers comprise a pair of primers. A pair of primers may comprise a forward primer and a reverse primer (e.g., primers that bind to the sense and antisense strands of a target nucleic acid). In some embodiments, the primers consist of a pair of primers (i.e., a forward primer and a reverse primer). Thus, in some embodiments, amplification of a target sequence is performed using a pair of primers, and no additional primers or oligonucleotides are included in the amplification of the target sequence (e.g., the amplification reaction components do not include additional primer pairs for a given target sequence, nested primers, bumper primers, oligonucleotides other than primers, probes, etc.). In some embodiments, the primers consist of a pair of primers. In some embodiments, the amplification reaction may include additional primer pairs for amplifying different target sequences, such as in multiplex amplification. In some embodiments, the primers consist of a pair of primers, but in some embodiments, the amplification reaction may include additional primers, oligonucleotides, or probes for a detection process that is not considered part of the amplification. In some embodiments, primers are used in sets. An amplification primer set may comprise a pair of forward and reverse primers for a given target sequence. In the case of multiplex amplification, the primers that amplify a first target sequence are considered to be a primer set, and the primers that amplify a second target sequence are considered to be a different primer set.

[0107] The amplification reaction components may 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 the sample nucleic acid. In some embodiments, the first primer (first oligonucleotide) comprises a first polynucleotide that is contiguously complementary to the target sequence in the first strand of the sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide that is contiguously complementary to the target sequence in the second strand of the sample nucleic acid. Contiguously complementary, with respect to primer-target, generally refers to a nucleotide sequence of a primer in which each base pairs in sequence with a corresponding, ordered base in the target sequence, with no gaps, additional sequence, or unpaired bases within the sequence considered contiguously complementary. In some embodiments, the primer does not include any additional sequence (e.g., at the 5' and / or 3' end or within the primer) that is not contiguous with the target sequence, such as additional sequence present in a tail primer or loop primer, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site). In some embodiments, the amplification reaction components do not include primers that include additional sequence (i.e., sequence other than sequence that is contiguous with the target sequence), such as a tail primer, a loop primer, a step-loop structure, a primer that can form a hairpin structure, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site).

[0108] In some embodiments, a 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 modifier that alters the binding properties of the primer. In some embodiments, a primer may include a detectable molecule or entity (e.g., a fluorophore, a radioisotope, a colorimetric reagent, a particle, an enzyme, etc.).

[0109] polymerase In some embodiments, amplification reaction components (e.g., one or more amplification reagents) include one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides extending the 3' hydroxyl end of a primer molecule, such as an amplification primer described herein, into a nucleic acid target sequence (e.g., to which the primer anneals). Non-limiting examples of polymerases include thermophilic or hyperthermophilic polymerases that can exhibit activity at high reaction temperatures (e.g., greater than 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C). Hyperthermophilic polymerases are sometimes referred to as hyperthermophilic polymerases. Polymerases may or may not have strand displacement capabilities. In some embodiments, the polymerase can incorporate from about 1 to about 50 nucleotides in a single synthesis, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range of nucleotides between any two of these values ​​in a single synthesis.

[0110] The amplification reaction components may include one or more DNA polymerases selected from the following: 9°N DNA polymerase; 9°Nm™ DNA polymerase; Therminator™ DNA polymerase; Therminator™ II DNA polymerase; Therminator™ III DNA polymerase; Therminator™ γ DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' exo-); T4 DNA polymerase; T7 DNA polymerase; Deep VentR™ (exo-) DNA polymerase; Deep VentR™ DNA polymerase; DyNAzyme™ EXT DNA; DyNAzyme™ II Hot Start DNA polymerase; Phusion™ High Fidelity DNA polymerase; VentR™ DNA polymerase; VentR™ (exo-) DNA polymerase; RepliPHI™ Phi29 DNA polymerase; rBst DNA polymerase, large fragment (IsoTherm™ DNA polymerase); MasterAmp™ AmpliTherm™ DNA polymerase; Tag DNA polymerase; Tth DNA polymerase; Tfl DNA polymerase; Tgo DNA polymerase; SP6 DNA polymerase; Tbr DNA polymerase; DNA polymerase beta; and ThermoPhi DNA polymerase.

[0111] In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases (e.g., hyperthermophilic DNA polymerases that are thermostable at high temperatures). The half-life of the hyperthermophilic DNA polymerase may be about 5-10 hours at 95°C and about 1-3 hours at 100°C. For example, the amplification reaction components may include one or more hyperthermophilic DNA polymerases from Archaea (e.g., a hyperthermophilic DNA polymerase from Thermococcus or a hyperthermophilic DNA polymerase from Thermococcaceaen archaean). In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from the genus Pyrococcus, Methanococcaceae, Methanococcus, or Thermus. In some embodiments, the amplification reaction components include one or more hyperthermophilic DNA polymerases from Thermus thermophiles.

[0112] In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase or a functional fragment thereof. A functional fragment generally retains one or more functions of a full-length polymerase, such as the ability to polymerize DNA (e.g., in an amplification reaction). In some cases, the functional fragment performs a function (e.g., polymerizing DNA in an amplification reaction) at a level that is at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the functional level of the full-length polymerase. The level of polymerase activity can be assessed using a detectable nucleic acid amplification method, such as the detectable nucleic acid amplification methods described herein. In some embodiments, an amplification reaction component comprises a hyperthermophile DNA polymerase comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, an amplification reaction component (e.g., one or more amplification reagents) comprises a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase or a functional fragment thereof, hi some embodiments, an amplification reaction component comprises a polymerase comprising an amino acid sequence at least about 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment thereof.

[0113] The polymerase may have reverse transcriptase activity. In such embodiments, the amplification reaction can, for example, amplify an RNA target in a single step without the use of a separate reverse transcriptase. Non-limiting examples of polymerases with reverse transcriptase activity include Bst (large fragment), 9°N DNA polymerase, 9°Nm™ DNA polymerase, Therminator™, and Therminator™ II. In some embodiments, the amplification reaction components include one or more separate reverse transcriptases. In some embodiments, more than one polymerase is included in the amplification reaction. For example, the amplification reaction can include a polymerase with reverse transcriptase activity and a second polymerase without reverse transcriptase activity. In some embodiments, one or more polymerases with exonuclease activity are used during amplification. In some embodiments, one or more polymerases with no or low exonuclease activity are used during amplification. In some embodiments, the polymerase with no or low exonuclease activity comprises one or more modifications (e.g., amino acid substitutions) that reduce or eliminate the exonuclease activity of the polymerase. For example, a modified polymerase with low exonuclease activity may have 10% or less exonuclease activity compared to an unmodified polymerase, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the exonuclease activity compared to an unmodified polymerase. In some embodiments, the polymerase has no or low 5'-3' exonuclease activity and / or no or low 3'-5' exonuclease activity. In some embodiments, the polymerase has no or low single-strand-dependent exonuclease activity and / or no or low double-strand-dependent exonuclease activity. Non-limiting examples of modifications that can reduce or eliminate the exonuclease activity of a polymerase include one or more amino acid substitutions at or corresponding to positions 141 and / or 143 and / or 458 of SEQ ID NO:1 (e.g., D141A, E143A, E143D, and A485L).

[0114] Detection and Quantification The methods described herein can include detecting and / or quantifying nucleic acid amplification products. The amplification products can be detected and / or quantified, for example, by any suitable detection and / or quantification method described herein. Non-limiting examples of detection and / or quantification methods include molecular beacons (e.g., real-time, end-point), lateral flow, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), surface capture, 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN), intercalating / binding dyes, absorbance (e.g., colorimetric, turbidity), electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), mass spectrometry, nucleic acid sequencing, digital amplification, primer extension (e.g., iPLEX™), Affymetrix molecular inversion probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, and molecular spectrometry. Allele-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, reverse dot blot, GeneChip microarray, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, AlphaScreen, SNPstream, gene bit analysis (GBA), multiplex mini-sequencing, SNaPshot, GOOD assay, microarray miniseq, array primer extension (APEX), microarray primer extension, Tag array, coded microspheres, template-directed integration (TDI) incorporation), colorimetric oligonucleotide ligation assay (OLA), sequence-coded OLA, microarray ligation, ligase chain reaction, padlock probes, invader assay, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, cloning and sequencing, use of hybridization probes and quantitative real-time polymerase chain reaction (QRT-PCR), nanopore sequencing, chips, and combinations thereof.In some embodiments, detection of nucleic acid amplification products involves the use of real-time detection methods (i.e., products are detected and / or continuously monitored during the amplification process). In some embodiments, detection of nucleic acid amplification products involves the use of end-point detection methods (i.e., products are detected after the amplification process is completed or stopped). Nucleic acid detection methods can also employ the use of labeled nucleotides that are incorporated directly into the target sequence or into a probe that contains a complementary sequence to the target. Such labels may be radioactive and / or fluorescent in nature and can be resolved in any of the manners discussed herein. In some embodiments, quantification of nucleic acid amplification products can be achieved using one or more detection methods described below. In some embodiments, detection methods can be used in conjunction with measuring signal intensity and / or generating (or referencing) a standard curve and / or look-up table for quantification of nucleic acid amplification products.

[0115] Detection of nucleic acid amplification products may involve the use of molecular beacon technology. The term molecular beacon generally refers to a detectable molecule whose detectable property is detectable under certain conditions, thereby enabling the molecule to function as a specific, useful signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and the time or speed at which a molecule passes through an aperture of a known size. A molecular beacon for detecting nucleic acid molecules may be, for example, a hairpin-shaped oligonucleotide containing a fluorophore at one end and a quenching dye at the opposite end. The loop of the hairpin may contain a probe sequence complementary to the target sequence, and the stem is formed by annealing complementary arm sequences located on either side of the probe sequence. The fluorophore and quenching molecule may be covalently linked to the opposite ends of each arm. Under conditions that prevent the oligonucleotide from hybridizing to its complementary target, or when the molecular beacon is free in solution, the fluorescent molecule and quenching molecule are in close proximity to each other, preventing FRET. When a molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, converting the loop structure into a stable, more rigid conformation, causing the fluorophore and quencher molecules to separate, resulting in fluorescence. Because the probe is specific, fluorescence generally occurs only with the synthesis of the intended amplification product. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is identical to or complementary to a sequence in the target nucleic acid. In some cases, the molecular beacon probe sequence hybridizes to a sequence in the amplification product that is neither identical to nor complementary to a sequence in the target nucleic acid (e.g., hybridizes to a tail amplification primer or a sequence added to the amplification product by ligation). Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with different colored fluorophores and different target sequences, allowing for simultaneous detection of several products in the same reaction (e.g., in a multiplex reaction).In the case of quantitative amplification processes, molecular beacons can specifically bind to the amplified target after each cycle of amplification, and unhybridized molecular beacons are dark, so there is no need to isolate the probe-target hybrid to quantitatively determine the amount of amplification product. The signal obtained is proportional to the amount of amplification product. Detection using molecular beacons can be performed in real time or as an end-point detection method.

[0116] Detection of nucleic acid amplification products may involve the use of lateral flow. Lateral flow typically involves the use of lateral flow devices, including, but not limited to, dipstick assays and thin-layer chromatography plates with various appropriate coatings. Various binding reagents for the sample, binding partners or conjugates containing binding partners for the sample, and a signal generation system are immobilized in the flow path. Detection can be achieved, for example, by enzyme detection, nanoparticle detection, colorimetric detection, and fluorescent detection. Nucleic acids can be captured in lateral flow devices by, for example, antibody-dependent and / or antibody-independent methods. Antibody-dependent capture involves an antibody capture line and a labeled probe of complementary sequence to the target. Antibody-independent capture utilizes a non-covalent interaction between two binding partners, such as the high affinity and irreversible linkage between a biotinylated probe and a streptavidin line. The capture probe can be directly immobilized on the lateral flow membrane. Both antibody-dependent and antibody-independent methods can be used to detect amplification products generated, for example, in multiplex reactions.

[0117] Detection of nucleic acid amplification products may involve the use of FRET, an energy transfer mechanism between two chromophores: a donor molecule and an acceptor molecule. Briefly, a donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emission of the donor molecule as it returns to its ground state can transfer the excitation energy to the acceptor molecule via long-range dipole-dipole interactions. The emission intensity of the acceptor molecule can be monitored and is a function of the distance between the donor and acceptor, the overlap between the donor emission spectrum and the acceptor absorption spectrum, and the orientation of the donor emission dipole moment and the acceptor absorption dipole moment. FRET can be useful, for example, for quantifying the molecular dynamics of DNA-DNA interactions, as described for molecular beacons. To monitor the production of a specific product, a probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe-target hybridization changes the distance or orientation between the donor and acceptor, and a FRET change is observed.

[0118] Detection of nucleic acid amplification products may involve the use of fluorescence polarization (FP). FP techniques are based on the principle that when a fluorescently labeled compound is excited with linearly polarized light, it will emit fluorescence with a degree of polarization inversely proportional to its rotation rate. Thus, when a molecule, such as a fluorescently labeled tracer-nucleic acid conjugate, is excited with linearly polarized light, the fluorophore's rotation is constrained between light absorption and emission, resulting in a highly polarized emission. When a free tracer compound (i.e., not bound to a nucleic acid) is excited with linearly polarized light, its rotation is much faster than that of the corresponding tracer-nucleic acid conjugate, resulting in a more random molecular orientation and depolarized emitted light. Thus, fluorescence polarization provides a quantitative means for measuring the amount of tracer-nucleic acid conjugate produced in an amplification reaction.

[0119] Detection of nucleic acid amplification products may involve the use of surface capture, achieved, for example, by immobilizing specific oligonucleotides on a surface to create a biosensor with both high sensitivity and selectivity. Examples of surfaces that can be used include gold and carbon, and surface capture methods can employ a variety of covalent or non-covalent coupling methods to attach probes to the surface. Detection of nucleic acid amplification products may involve the use of 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN). For example, TAQMAN probes are hydrolysis probes that can increase the specificity of quantitative amplification methods (e.g., quantitative PCR). The principle of TAQMAN probes relies on 1) the 5'-3' exonuclease activity of Taq polymerase, which cleaves a dual-labeled probe upon hybridization to a complementary target sequence, and 2) fluorophore-based detection. The resulting fluorescent signal allows for quantitative measurement of amplification product accumulation during the exponential phase of amplification, and TAQMAN probes can significantly increase the specificity of detection.

[0120] Detection of nucleic acid amplification products may involve the use of intercalating and / or binding dyes, e.g., dyes that specifically stain nucleic acids (e.g., intercalating dyes exhibit enhanced fluorescence upon binding to DNA or RNA). Dyes may include DNA or RNA intercalating fluorophores, including, but not limited to, SYTO® 82, acridine orange, ethidium bromide, Hoechst dyes, PicoGreen®, propidium iodide, SYBR® I (asymmetric cyanine dye), SYBR® II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Detection of nucleic acid amplification products may involve the use of absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection and / or quantification of nucleic acids can be achieved by directly converting absorbance (e.g., UV absorbance measurement at 260 nm) to concentration. Direct measurements of nucleic acids can be converted to concentrations using the Beer-Lambert law, which relates absorbance to concentration using the path length and extinction coefficient of the measurement. Detection of nucleic acid amplification products can involve the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis) and / or mass spectrometry. Mass spectrometry is an analytical technique that can be used to determine the structure and quantity of nucleic acids and can be used to provide rapid analysis of complex mixtures. After amplification, the sample is ionized, and the resulting ions can be separated according to their mass-to-charge ratio in an electric and / or magnetic field, and the mass-to-charge ratio of the ions is measured by a detector. Mass spectrometry methods include, for example, MALDI, MALDI-TOF, and electrospray. These methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can achieve high throughput due to fast signal acquisition from solid surfaces and automated analysis.

[0121] Detection of nucleic acid amplification products may involve the use of nucleic acid sequencing. The entire or partial sequence of the amplification product may be determined, and the determined nucleotide sequence may be referred to as a read. For example, linear amplification products may be directly analyzed without further amplification (e.g., by using single-molecule sequencing). In some embodiments, linear amplification products are subjected to further amplification and then analyzed (e.g., using sequencing by ligation or pyrosequencing). Non-limiting examples of sequencing methods include single-end sequencing, paired-end sequencing, reversible terminator-based sequencing, sequencing by ligation, pyrosequencing, sequencing by synthesis, single-molecule sequencing, multiplex sequencing, solid-phase single-nucleotide sequencing, and nanopore sequencing. Detection of nucleic acid amplification products may involve the use of digital amplification (e.g., digital PCR). Systems for digital amplification and analysis of nucleic acids are available (e.g., Fluidigm® Corporation).

[0122] acidic composition Acidic compositions for dissociating dsDNA disclosed herein (e.g., lysis buffers, elution buffers, resuspension buffers) can be made with low concentrations of acidic agents, such as 10-20 mM HCl. Other inorganic or organic acids known in the art can also be used. The amplification reaction buffer used for pH neutralization can be 30-50 mM Tris pH 8.8 buffer. In some embodiments, the acidic composition can contain an acidic buffer, such as glycine / -HCl, to increase acidic buffering capacity and compensate for the pH variability found in clinical samples (e.g., urine or nasal swabs). Without being bound by any particular theory, in some embodiments, at low pH, a high concentration of positively charged hydrogen ions disrupts the secondary structure of nucleic acids by disrupting non-covalent hydrogen bonds and hydrophobic interactions, destabilizing the double helix structure and causing strand separation.

[0123] The present disclosure includes an acidic composition for dissolving biological entities and denaturing the dsDNA contained therein.In some embodiments, the acidic composition comprises a monovalent salt and / or a divalent salt; one or more surfactants; and an acidic agent, wherein the acidic agent is present in the acidic composition at a concentration of less than 100mM, and the acidic composition has a pH of less than 4.The monovalent salt can be present in the acidic composition at a concentration of less than 30mM.The divalent salt can be present in the acidic composition at a concentration of less than 15mM. In some embodiments, the acidic composition has a pH in the range of about 1 to about 3.9 (e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or a number or range between any two of these values). In some embodiments, the acidic composition has a pH of about 2. In some embodiments, the acidic composition has a pH of about 2, about 2.1, about 2.2, about 2.3, about 2.4, and about 2.5. pH, as used herein, may be determined by any known method of calculating or measuring the pH of an aqueous solution. In some embodiments, the acidic composition does not comprise glycerol, formamide, or urea.

[0124] In some embodiments, the percentage of an acidic composition component disclosed herein is provided as % weight / weight, % moles (m) / volume, % volume / volume, % moles / weight, % weight / volume, or variants thereof. In some embodiments, the percentage (% weight / weight, % moles / volume, % volume / volume, % moles / weight, % weight / volume, or variants thereof) of an acidic composition component disclosed herein (e.g., one or more surfactants) in an acidic composition is about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.0001%, 0.001%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0. 08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83% 3%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.0%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.38%, 1.39%, 1.40%, 1.41%, 1.42%, 1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, 1.51%, 1.52%, 1.53%, 1.54%, 1.55%, 1.56%, 1.57%, 1.58%, 1.59%, 1.60%, 1.61%, 1.62%, 1.63%, 1.64%, 1.65%, 1.66%, 1.67%, 1.6821%、1.22%、1.23%、1.24%、1.25%、1.26%、1.27%、1.28%、1.29%、1.30%、1.31%、1.32%、1.33%、1.34%、1.35%、1.36%、1.37%、1.38%、1.39%、1.40%、1.41%、1.42%、1.43%、1.44%、1.45%、1.46%、1.47%、1.48%、1.49%、1.50%、1.51%、1.52%、1.53%、1.54%、1.55%、1.56%、1.57%、1.58%、1.59%、1.60%、1.61%、1.62%、1.63%、1.64%、1.65%、1.66%、1.67%、1.68%、1.69%、1.70%、1.71%、1.72%、1.73%、1.74%、1.75%、1.76%、1.77%、1.78%、1.79%、1.80%、1.81%、1.82%、1.83%、1.84%、1.85%、1.86%、1.87%、1.88%、1.89%、1.90%、1.91%、1.92%、1.93%、1.94%、1.95%、1.96%、1.97%、1.98%、1.99%, 2%, 3%, 4%, 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or a number or range between any two of these values. In some embodiments, the percentages of the components of the acidic compositions disclosed herein are described with respect to their final concentrations when the acidic composition is contacted with a sample containing a biological entity. Moreover, in some embodiments, although the acidic composition components disclosed herein are described in terms of the working concentration of the acidic composition (e.g., 1×), the present disclosure also contemplates concentrated versions of the disclosed acidic compositions (e.g., 2× acidic compositions).

[0125] Acidic drugs The compositions (e.g., acidic compositions) disclosed herein can include one or more acidic agents. The acidic agents can include organic acids, inorganic acids, or both. The acidic agents can be hydrochloric acid, glycine hydrochloride, acetic acid, citric acid, phosphoric acid, or a combination thereof.

[0126] The acidic agent may vary depending on the embodiment. The inorganic acid may be hydrochloric acid (HCl). The inorganic acid may include one or more of hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, phosphoric acid, phosphinic acid, phosphonic acid, sulfonic acid, sulfuric acid, sulfurous acid, disulfuric acid, carbonic acid, and boric acid. The organic acid may be acetic acid, CHCOOH, CHCOOH, CHCOOH, (COOH), CH( ... 10 The acid may include one or more of (COOH)2, fumaric acid, maleic acid, malonic acid, lactic acid, citric acid, tartaric acid, oxalic acid, ascorbic acid, benzoic acid, salicylic acid, phthalic acid, pyruvic acid, L-aspartic acid, D-aspartic acid, carbonic acid, formic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, glucosamine sulfate, L-threonic acid, camphoric acid, gluconic acid, L-glutamic acid, D-glutamic acid, trifluoroacetic acid, or ranelic acid.

[0127] In some embodiments, the acidic agent is an acidic buffer (e.g., an acidic buffer solution). The acidic buffer solution can be a solution having a pH of less than 7. The acidic buffer solution can be made from a weak acid and one of its salts (e.g., the sodium salt) or can be obtained from a commercial source. One example is a mixture of ethanoic acid and sodium ethanoate in solution. In this case, the solution would have a pH of 4.76 if it contained equimolar concentrations of both the acid and the salt. Thus, as used herein, the term "acidic buffer" shall be given its ordinary meaning and shall also refer to a compound that, when added to an aqueous solution, reduces the pH and makes the resulting solution resistant to changes in pH when the solution is mixed with a solution of lower or higher pH. The acidic buffer can have a pKa below about 7. The acidic buffer can have a pKa below about 7, below about 6, below about 5, below about 4, and below about 3. Acidic buffers having a pKa of all individual values ​​and ranges below about 7 are included in the present disclosure. Examples of suitable acidic buffers for the acidic compositions described herein include, but are not limited to, phosphate, citrate, isocitrate, acetate, succinate, ascorbic acid, formic acid, lactic acid, sulfuric acid, hydrochloric acid, nitric acid, benzoic acid, boric acid, butyric acid, capric acid, caprilic acid, carbonic acid, carboxylic acid, oxalic acid, pyruvic acid, phthalic acid, adipic acid, citramalic acid, fumaric acid, glycolic acid, tartaric acid, isotartaric acid, lauric acid, maleic acid, isomalic acid, malonic acid, orotic acid, propionic acid, methylpropionic acid, polyacrylic acid, succinic acid, salicylic acid, 5-sulfosalicylic acid, valeric acid, isovaleric acid, uric acid, and combinations thereof, such as the combination of hydrochloric acid and glycine having a pH of about 2.2, as well as other suitable acids and bases known in the art. Other suitable acidic buffers are mixtures of an acid and one or more salts or buffering agents. For example, suitable acidic buffers for use herein can be prepared using glycine in combination with an appropriate concentration of hydrochloric acid. The acidic buffer can include glycine-HCl. The acidic buffer can include 10.0 mM glycine, 8.8 mM HCl, or both.The pH of the acidic buffer solution can be from about 1.0 to about 6.0 (eg, 2.2).

[0128] The concentration of the acidic agent present in the acidic composition can vary. The acidic agent can be present in an amount of about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 3 5mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 5 0mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, The acidic agent may be present in the acidic composition at a concentration of less than 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, or a number or range between any two of these values. The acidic agent may be present in the acidic composition at a concentration within the range of about 1 mM to about 100 mM, e.g., about 10 mM or about 8.8 mM.

[0129] salt Without being bound by any particular theory, the acidic composition (e.g., lysis buffer) may, in some embodiments, contain monovalent and / or divalent salts (e.g., magnesium), which may inhibit depurination. In some embodiments, the acidic composition contains low concentrations of monovalent and / or divalent salts. Without being bound by any particular theory, this may be due to neutralization of the negative charges on the phosphate groups on the DNA backbone. The monovalent salt and / or divalent salt of the acidic compositions (e.g., lysis buffer) provided herein can include sodium salts, potassium salts, calcium salts, magnesium salts, or any combination thereof. The monovalent salt can be selected from the group consisting of ammonium sulfate, ammonium chloride, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, potassium fluoride, potassium chloride, potassium bromide, and potassium iodide. The divalent salt can be selected from the group consisting of magnesium sulfate, calcium chloride, magnesium chloride, copper(II) chloride, zinc chloride, calcium oxide, magnesium oxide, barium oxide, sodium sulfate, calcium sulfate, copper(II) sulfate, potassium carbonate, and sodium carbonate. In some embodiments, the monovalent salt is ammonium sulfate. In some embodiments, the divalent salt is magnesium sulfate.

[0130] In some embodiments, the monovalent salt and / or divalent salt is present in the acidic composition at a concentration of less than 15 mM, e.g., about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 29.9 mM, or a number or range between any two of these values. The monovalent salt and / or divalent salt can be present in the acidic composition at a concentration in the range of about 1 mM to about 14 mM, for example, at a concentration of about 5 mM or about 4 mM.

[0131] surfactants The acid compositions (e.g., lysis buffers) provided herein can include one or more lysis agents (e.g., surfactants, detergents), such as cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. Anionic surfactants contain NH4 as a counterion. + , K. + , Na + , or Li+ The cationic surfactant may contain I as a counter ion. - , Br - , or Cl - may also include: The anionic surfactant may be selected from the group consisting of potassium laurate, triethanolamine stearate, ammonium lauryl sulfate, lithium dodecyl sulfate, sodium lauryl sulfate, sodium alkyl sulfate (C8-16), SDS, alkyl polyoxyethylene sulfate, sodium alginate, dioctyl sodium sulfosuccinate, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid and its salts, glyceryl esters, sodium carboxymethylcellulose, bile acids and acids, alkyl sulfonates, aryl sulfonates, alkyl phosphates, alkyl sulfonates, stearic acid and its salts, calcium stearate, phosphates, sodium carboxymethylcellulose, dioctyl sulfosuccinate, dialkyl esters of sodium sulfosuccinic acid, phospholipids, and calcium carboxymethylcellulose.

[0132] The cationic surfactant may be, for example, the following: quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitic acid, lauryldimethylbenzylammonium chloride, acylcarnitine hydrochloride, alkylpyridinium halides, cetylpyridinium chloride, cationic lipids, polymethylmethacrylate trimethylammonium bromide, sulfonium compounds, polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate, hexadecyltrimethylammonium bromide, phosphonium compounds, quaternary ammonium compounds, benzyl-di(2-chloroethyl)ethylammonium bromide, coconuttrimethylammonium chloride, coconuttrimethylammonium bromide, coconutmethyldihydroxyethylammonium chloride, coconutmethyldihydroxyethylammonium bromide, decyltriethylammonium chloride, decyldimethylhydroxyethylammonium chloride, decyldimethylhydroxyethylammonium chloride bromide, C12-15-dimethylhydroxyethylammonium ammonium chloride, C12-15-dimethylhydroxyethylammonium chloride bromide, coconut dimethylhydroxyethylammonium chloride, coconut dimethylhydroxyethylammonium bromide, myristyltrimethylammonium methyl sulfate, lauryldimethylbenzylammonium chloride, lauryldimethylbenzylammonium bromide, lauryldimethyl(ethenoxy)4 ammonium chloride, lauryldimethyl(ethenoxy)4 ammonium bromide, N-alkyl(C12-18)dimethylbenzylammonium chloride, N-alkyl(C14-18)dimethylbenzylammonium chloride, N-tetradecyldimethylbenzylammonium chloride monohydrate, dimethyldidecylammonium chloride, N-alkyl(C12-14)dimethyl 1-naphthylmethylammonium chloride, trimethylammonium halide alkyl-trimethylammonium salt, dialkyl-dimethylammonium salt, lauryltrimethylammonium chloride, ethoxylated alkyamido alkyldialkylammonium salt, ethoxylated trialkylammonium salt,Dialkylbenzenedialkylammonium chloride, N-didecyldimethylammonium chloride, N-tetradecyldimethylbenzylammonium chloride monohydrate, N-alkyl(C12-14)dimethyl 1-naphthylmethylammonium chloride, dodecyldimethylbenzylammonium chloride, dialkylbenzenealkylammonium chloride, lauryltrimethylammonium chloride, alkylbenzylmethylammonium chloride, alkylbenzyldimethylammonium bromide, C8-16 trimethylammonium bromide, C8-16 trimethylammonium chloride, C15 trimethylammonium bromide, C17 trimethylammonium bromide, dodecylbenzyl Cetyltriethylammonium chloride, polydiallyldimethylammonium chloride, dimethylammonium chloride, alkyldimethylammonium halides, tricetylmethylammonium chloride, decyltrimethylammonium bromide, dodecyltriethylammonium bromide, tetradecyltrimethylammonium bromide, methyltrioctylammonium chloride, POLYQUAT 10, tetrabutylammonium bromide, benzyltrimethylammonium bromide, choline esters, benzalkonium chloride, stearalkonium chloride, cetylpyridinium bromide, cetylpyridinium chloride, halide salts of quaternized polyoxyethylalkylamines, MIRAPOL Alkaquat, alkylpyridinium salts, amines, amine salts, imidoazolinium salts, protonated quaternary acrylamides, methylated quaternary polymers, cationic guar gum, benzalkonium chloride, dodecyltrimethylammonium bromide, triethanolamine, or poloxamine.

[0133] The nonionic surfactants may be, for example, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, sorbitan esters, glyceryl esters, glycerol monostearate, polyethylene glycol, polypropylene glycol, polypropylene glycol esters, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene polyoxypropylene copolymers, poloxamers, poloxamines, methylcellulose, hydroxycellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl ... propylmethylcellulose phthalate, amorphous cellulose, polysaccharides, starch, starch derivatives, hydroxyethyl starch, polyvinyl alcohol, triethanolamine stearate, amine oxides, dextran, glycerol, gum acacia, cholesterol, tragacanth, polyvinylpyrrolidone, alkyl sulfates, alkyl sulfonates, fatty acid soaps, hydroxy fatty acids, hydroperoxy fatty acids, polyhydroxy fatty acids, salts of epoxy fatty acids, salts of mono- and polycarboxylic acids, prostanoic acids and prostaglandins, leukotrienes and lipoxins, alkyl phosphates, alkyl phosphonates, sodium dialkyl sulfosuccinates, n-alkyl ethoxylated sulfates, bile salts cholate and deoxycholate, perfluorocarboxylic acids, fluoroacliphatic phosphonates, or fluoroaliphatic sulfates.

[0134] The lysis agents provided herein may be capable of acting as denaturing agents. As used herein, "denaturing agent" or "denaturant" shall be given its ordinary meaning and include any compound or substance that will cause reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined by both the properties and concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturants may be 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 include SDS or polyoxyethylene ethers (e.g., Tween or Triton detergents), strong detergents such as sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including, but not limited to, sulfobetaine (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS), and 3-(3-chloramidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). Organic water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (especially C2-C4 alkanediols such as ethylene glycol) can be used as denaturants. The phospholipids may be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[0135] The one or more surfactants can include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. In some embodiments, the acidic composition is free of both anionic surfactants and cationic surfactants. The one or more surfactants can be present in the acidic composition at a concentration ranging from about 0.01% to about 2% by weight to volume (% wt / vol) of the acidic composition. The one or more surfactants can be present in the acidic composition at a concentration of about 0.1% by weight to volume (% wt / vol) of the acidic composition. In some embodiments, the acidic composition further includes a tween surfactant, such as a tween surfactant selected from Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85. The tween surfactant can comprise about 0.01% (wt / vol) of the acidic composition. Anionic surfactants useful herein include the water-soluble salts of alkyl sulfates and alkyl ether sulfates having 10 to 18 carbon atoms in the alkyl radical, and the water-soluble salts of sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant.

[0136] Suitable cationic surfactants can be broadly defined as derivatives of aliphatic quaternary ammonium compounds having one long alkyl chain containing about 8 to 18 carbon atoms, such as lauryltrimethylammonium chloride, cetylpyridinium chloride, benzalkonium chloride, CTAB, diisobutylphenoxyethyl-dimethylbenzylammonium chloride, coconut alkyltrimethyl-ammonium nitrite, cetylpyridinium fluoride, etc. Certain cationic surfactants can also act as disinfectants in the compositions disclosed herein. Suitable nonionic surfactants that can be used in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds produced by the condensation of an alkylene oxide group (hydrophilic in nature) with an organic hydrophobic compound, which may 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 aliphatic alcohols or alkylphenols; products derived from the condensation of ethylene oxide with the reaction product of propylene oxide and ethylenediamine; long-chain tertiary amine oxides; long-chain tertiary phosphine oxides; long-chain dialkyl sulfoxides; and mixtures of such substances. These substances are useful for stabilizing foam without contributing to excessive viscosity increase in consumer product compositions.

[0137] Zwitterionic surfactants can be broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, where the aliphatic radical may be straight or branched chain, and one of the aliphatic substituents contains about 8 to 18 carbon atoms and one contains an anionic water-solubilizing group, such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of anionic short-chain surfactants include alkyl sulfates, alkyl sulfonates, alkylbenzene sulfonates, saturated or unsaturated fatty acids, and their salts. The polar head group-containing moiety in cationic surfactants can include, for example, quaternary ammonium, pyridinium, sulfonium, and / or phosphonium groups. For example, the polar head group can include trimethylammonium. Exemplary cationic short-chain surfactants include alkyltrimethylammonium halides, alkyltrimethylammonium tosylates, and N-alkylpyridinium halides.

[0138] Alkyl sulfates include sodium octyl sulfate, SDeS, SDS, and sodium tetradecyl sulfate. Alkyl sulfonates include sodium octyl sulfonate, sodium decyl sulfonate, and sodium dodecyl sulfonate. Alkyl benzene sulfonates include sodium octyl benzene sulfonate, sodium decyl benzene sulfonate, and sodium dodecyl benzene sulfonate. Fatty acid salts include sodium octanoate, sodium decanoate, sodium dodecanoate, and the sodium salt of oleic acid. Examples of alkyltrimethylammonium halides include octyltrimethylammonium bromide, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, myristyltrimethylammonium bromide, and CTAB. Examples of alkyltrimethylammonium tosylates include octyltrimethylammonium tosylate, decyltrimethylammonium tosylate, dodecyltrimethylammonium tosylate, myristyltrimethylammonium tosylate, and cetyltrimethylammonium tosylate. For example, examples of N-alkylpyridinium halides include decylpyridinium chloride, dodecylpyridinium chloride, cetylpyridinium chloride, decylpyridinium bromide, dodecylpyridinium bromide, cetylpyridinium bromide, decylpyridinium iodide, dodecylpyridinium iodide, and cetylpyridinium iodide.

[0139] Cationic surfactants include dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, cetyltrimethylammonium bromide, cetyldimethylethylammonium bromide, (C1-C30 alkyl)-trimethylammonium bromide, (C1-C30 alkyl)amines, (C1-C30 alkyl)imidazolines, ethoxylated amines, quaternary compounds, quaternary esters, (C1-C30 alkyl)amine oxides, lauramine oxide, dicetyldimonium chloride, cetrimonium chloride, primary polyethoxylated fatty amines, The polyethoxylated fatty amine salts may include amine salts, secondary polyethoxylated fatty amine salts, tertiary polyethoxylated fatty amine salts, quaternary ammonium salts, tetra(C1-C30 alkyl)ammonium halides, (C1-C30 alkyl)amido-(C1-C30 alkyl)ammonium halides, tri(C1-C30 alkyl)benzylammonium halides, tri(C1-C30 alkyl)hydroxy-(C1-C30 alkyl)ammonium halides, (C1-C30 alkyl)pyridinium chloride, (C1-C30 alkyl)pyridinium bromide, amine oxides, or combinations thereof.

[0140] Anionic surfactants can include the following: SDS, (C6-C30 alkyl)benzenesulfonates, C6-C30 alpha olefin sulfonates, paraffin sulfonates, (C6-C30 alkyl)ester sulfonates, (C6-C30 alkyl)sulfates, (C6-C30 alkylalkoxy)sulfates, (C6-C30 alkyl)sulfonates, (C6-C30 alkylalkoxy)carboxylates, (C6-C30 alkyl alkoxylated)sulfates, mono(C1-C30 alkyl)(ether)phosphates, di(C6-C30 alkyl)(ether)phosphates, (C6-C30 alkyl)salts Cosinate, sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, ethoxylated 4-nonylphenyl ether glycolate, (C1-C30 alkyl) isethionate, taurate, ammonium lauryl sulfate, ammonium laureth sulfate, triethylamine lauryl sulfate, triethylamine laureth sulfate, triethanolamine lauryl sulfate, triethanolamine laureth sulfate, monoethanolamine lauryl sulfate, monoethanolamine laureth sulfate, diethanolamine lauryl sulfate, diethanolamine laureth sulfate, lauric monoglyceride sodium sulfatesulfate), sodium lauryl sulfate, sodium laureth sulfate, potassium lauryl sulfate, potassium laureth sulfate, sodium lauryl phosphate, sodium tridecyl phosphate, sodium behenyl phosphate, sodium laureth-2 phosphate, sodium ceteth-3 phosphate, sodium trideceth-4 phosphate, sodium dilauryl phosphate, sodium ditridecyl phosphate, sodium ditrideceth-6 phosphate, sodium lauroyl sarcosinate, lauroyl sarcosine, cocoyl sarcosine, ammonium cocoyl sulfate, sodium cocoyl sulfate, sodium trideceth sulfate, sodium tridecyl sulfate, ammonium trideceth sulfate, ammonium tridecyl sulfate, sodium cocoyl isethionate, disodium laureth sulfosuccinate, sodium oleoyl methyl taurate, sodium laureth carboxylate, sodium trideceth carboxylate, sodium lauryl sulfate, potassium cocoyl sulfate, potassium lauryl sulfate, monoethanolamine cocoyl sulfate, sodium tridecyl benzenesulfonate, sodium dodecyl benzenesulfonate, or combinations thereof.

[0141] Nonionic surfactants may include, for example, C6-C18 alkyl alcohols, (C6-C18 alkyl)phenols, (C6-C18 alkyl)ethoxylates, (C6-C18 alkyl)phenol (C1-C3 alkoxylates), block oxy(C1-C3 alkylene) condensates of C6-C18 alkylphenols, oxy(C1-C3 alkylene) condensates of alkanols, oxyethylene / oxypropylene block copolymers, amine oxides, phosphine oxides, alkylamine oxides having 8 to 50 carbon atoms, mono- or di(C8-C30) alkyl alkanolamides, (C6-C30 alkyl)polysaccharides, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyisobutylene ... Oxyethylene sorbitol esters, polyoxyethylene nonylphenyl ether, polyoxyethylene acid, polyoxyethylene alcohol, coco monoethanolamide, coco diethanolamide, coco diglycoside, (C8-C30 alkyl) polyglycosides, cocamidopropyl betaine, lauramine oxide, polyoxyethylene (20) sorbitan monolaurate, ethoxylated linear C8-C30 alcohols, cetearyl alcohol, lanolin alcohol, stearic acid, glyceryl stearate, polyethylene glycol 100 stearate, 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol, polyoxyethylene (10) cetyl ether, eicosaethylene glycol octadecyl ether, and HO(CH2CHO). 20 (CH2CH(CH3)O) 70 (CH2CH2O) 20 H.

[0142] The one or more surfactants can comprise about 0.001% (wt / vol) to about 2.0% (wt / vol) of the acidic composition (e.g., lysis buffer). The one or more surfactants can be capable of lysing biological entities to release sample nucleic acids contained therein. The one or more surfactants can comprise about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a number or range (wt / vol) between any two of these values. The one or more surfactants disclosed herein can include one or more of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants. The acidic composition can further include a tween surfactant. The tween detergent may be selected from Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85. The tween detergent may comprise about 0.01% (w / v) to about 1.0% (w / v) of the acidic composition. In some embodiments, the acidic composition comprises about 0.2% (w / v) SDS, and (NH4)2SO4 is at a concentration of less than about 5 mM. In some embodiments, the acidic composition further comprises MgSO4. The one or more surfactants can include Triton X-100. The Triton X-100 surfactant can comprise about 0.01% (w / v) to about 1.0% (w / v) of the acidic composition. In some embodiments, the acidic composition includes about 0.1% Triton X-100. In some embodiments, the acidic composition includes about 0.1% Triton X-100 and less than about 5 mM ammonium sulfate ((NH4)2SO4) and / or magnesium sulfate (MgSO4). In some embodiments, the acidic composition includes about 0.1% Triton X-100, about 5 mM ammonium sulfate ((NH4)2SO4), and about 4 mM magnesium sulfate (MgSO4).

[0143] Chelators and reducing agents In some embodiments, the acidic composition includes a chelating agent, a reducing agent, or both. In some embodiments, the acidic composition does not include a reducing agent, a chelating agent, or both. The chelating agent can be ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), Tris, or a combination thereof. Examples of divalent ion (e.g., magnesium ion) chelating agents include, but are not limited to, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, EDTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), citrate-containing buffers, N,N-bis(2-(bis-(carboxymethyl)amino)ethyl)-glycine (DTPA), NTA, buffers that precipitate calcium ions from a sample (e.g., phosphate buffers, carbonate buffers, and bicarbonate buffers), citric acid and its salts, gluconic acid and its salts, alkali metal pyrophosphates, alkali metal polyphosphates, sodium hexametaphosphate, triethylenetetramine, diethylenetriamine, o-phenanthroline, oxalic acid, or any combination thereof. The reducing agent can be 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythriol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, salts of sulfite, thioglycolic acid, and hydroxyethyl disulfide (HED), or any combination thereof.

[0144] The chelating agent and / or reducing agent can be present in the acidic composition at a concentration in the range of about 0.1 mM to about 14 mM, e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mM, or a number or range between any two of these values.

[0145] Reagent Composition The reagent compositions (e.g., dry compositions) described herein can be provided in a "dry form," or a form not suspended in a liquid medium. A "dry form" of a composition can include a dry powder, a lyophilized composition, a spray-dried composition, or a precipitated composition. A "dry form" composition can include one or more lyoprotectants, such as sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine and histidine; lyotropic salts, such as MgSO4; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or polypropylene glycol; and combinations thereof. Additional exemplary lyoprotectants include gelatin, dextrin, modified starch, and carboxymethylcellulose. As used herein, the terms "lyophilization," "lyophilized," and "freeze-drying" refer to a process in which the material to be dried is first frozen and then the ice or freezing solvent is removed by sublimation in a vacuum environment. "Lyophilisate" refers to a material that has been freeze-dried. As disclosed herein, a dry composition can include one or more additives and one or more amplification reagents. The compositions described herein (e.g., wet compositions) can be provided in a "wet form" or suspended in a liquid medium.

[0146] The dried composition may be frozen, lyophilized, or spray-dried. The dried composition may be heat-dried. The dried composition may include one or more additives (e.g., a polymer, a sugar, or a sugar alcohol). The sugar or sugar alcohol may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The polymer may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof. The one or more additives may include one or more amino acids. The one or more additives may include Tween 80, Tween 20, and / or Triton X-100. In some embodiments, the one or more additives aid in the lyophilization of the reaction composition and / or the dissolution of the dried pellet. The one or more additives may include a non-ionic detergent at a concentration of about 0.01% in the dry composition (eg, dry pellets). The frozen, lyophilized, spray-dried, or heat-dried composition, or the aqueous composition for preparing the frozen, lyophilized, or spray-dried composition, may contain one or more of the following: (i) non-aqueous solvents such as ethylene glycol, glycerol, dimethyl sulfoxide, and dimethylformamide; (ii) surfactants such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic F127 (polyoxyethylene-polyoxypropylene copolymer), also known as poloxamer, poloxamine, and SDS; (iii) disaccharides such as trehalose, sucrose, lactose, and maltose; (iv) polymers (which may have different MW) such as polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, and albumin. (v) Amino acids such as glycine, proline, 4-hydroxyproline, L-serine, glutamic acid, alanine, lysine, sarcosine, and gamma-aminobutyric acid.

[0147] The reagent composition (e.g., dry composition, wet composition) can include one or more amplification reagents. In some embodiments, the reagent composition can include a reducing agent, a chelating agent, or both. In some embodiments, the reagent composition does not include a reducing agent, a chelating agent, or both. The chelating agent can be EDTA, EGTA, NTA, Tris, or any combination thereof. The reducing agent can be DTT, TCEP, DTE, reduced glutathione, cysteamine, TBP, dithioerythriol, THPP, 2-mercaptoethylamine-HCl, DTBA, cysteine, cysteine-thioglycolate, a salt of sulfite, thioglycolic acid, HED, or any combination thereof.

[0148] kit Provided herein are kits for isothermal amplification and detection of genomic DNA in biological samples. In some embodiments, the kits contain an acidic composition (e.g., an elution / lysis solution) comprising a solubility reagent, magnesium, and either an acid or a low pH buffer, such as glycine / HCl at pH 2.2. In some embodiments, the kits also contain reagent reaction components, including primers, enzymes, dNTPs, detection probes, and a buffer with sufficient capacity to maintain an optimal pH for amplification after mixing with the acidic composition (e.g., the elution / lysis solution). Disclosed herein is a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit includes (a) an acidic composition provided herein, the acidic composition being capable of dissolving biological entities to release sample nucleic acids contained therein, the sample nucleic acid comprising dsDNA suspected of comprising a target nucleic acid sequence, and the target nucleic acid sequence being 100 nucleotides or less in length. In some embodiments, the kit includes (b) a reagent composition (e.g., a dry composition, a wet composition) comprising a buffer and one or more amplification reagents for amplifying the target nucleic acid sequence under isothermal amplification conditions, the one or more amplification reagents comprising: (i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a first strand sequence of the target nucleic acid sequence and the second primer is capable of hybridizing to a second strand sequence of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophile polymerase activity capable of producing a nucleic acid amplification product.

[0149] The buffer can be MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium buffer, or any combination thereof. The kit can also include at least one component that provides real-time detection of nucleic acid amplification products. The real-time detection can be provided by a molecular beacon. The kit can also include a control polynucleotide, and if multiple target sequences are being amplified, multiple control polynucleotides can be included in the kit. In some embodiments, the enzyme with hyperthermophilic polymerase activity has an amino acid sequence that is at least about 90% or 95% identical to the amino acid sequence of SEQ ID NO: 1, or a functional fragment thereof. The enzyme with hyperthermophilic polymerase activity can be a polymerase comprising the amino acid sequence of SEQ ID NO: 1.

[0150] The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may include (1) the sequence of the first primer and its reverse complement, (2) the sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by (1) the sequence of the first primer and its reverse complement and (2) the sequence of the second primer and its reverse complement, the spacer sequence being 1 to 10 bases in length. The biological entity may include one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. The biological entity may include a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof. The target nucleic acid sequence may be a nucleic acid sequence of a virus, a bacterium, a fungus, or a protozoan. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoan disclosed herein. The first primer and / or the second primer can be about 8 to 16 bases in length. The first primer and / or the second primer can include one or more of DNA bases, modified DNA bases, or a combination thereof.

[0151] The reagent composition (e.g., dry composition) may be freeze-dried and / or heat-dried and may contain one or more additives. The one or more additives may include an amino acid; a sugar or a sugar alcohol. The sugar or sugar alcohol may include sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof. The one or more additives may include a polymer. The polymer may include polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof. The mixture of the acidic composition and the reagent composition can have a pH of about 7 to about 9, for example, a pH of about 8.8. In some embodiments, the mixture of the sample, the acidic composition, and the reagent composition has a pH of about 7 to about 9, for example, a pH of about 8.8. The buffer can include Tris. The mixture of the sample, the acidic composition, and the reagent composition can include Tris at a concentration in the range of about 30 mM Tris to about 50 mM Tris. The kit can include a sterile container that contains the acidic composition and the reagent composition.

[0152] The kit may also contain one or more of the components in any number of separate vessels, chambers, containers, packets, tubes, vials, microtiter plates, and the like, or the components may be combined in various combinations in such containers. The components of the kit may, for example, be present in one or more containers. In some embodiments, all of the components are provided in a single container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a container separate from the primers. The components may, for example, be lyophilized, heat-dried, freeze-dried, or present in a stable buffer. In some embodiments, the polymerase and / or reverse transcriptase are present in a single container in lyophilized or heat-dried form, and the primers are either lyophilized, heat-dried, freeze-dried, or present in a buffer in a different container. In some embodiments, the polymerase and / or reverse transcriptase and primers are present in a single container in lyophilized or heat-dried form.

[0153] The kit may further include dNTPs or modified nucleotides used in the reaction, vessels, cuvettes, or other containers, or vials of water or buffer for rehydrating lyophilized or heat-dried components. The buffer used may be, for example, suitable for both polymerase activity and primer annealing activity. The kits may also include instructions for practicing one or more of the methods described herein and / or instructions for one or more of the components described herein. The instructions and / or instructions may be in printed form or may be included in a kit insert. The kits may also include a written description of an internet location that provides such instructions or instructions. The kit may further include reagents used in detection methods, such as reagents used in FRET, lateral flow devices, dipsticks, fluorescent dyes, colloidal gold particles, latex particles, molecular beacons, or polystyrene beads. [Example]

[0154] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Example 1 Study of DNA dissociation in acidic solutions This example demonstrates dsDNA separation in acidic compositions provided herein for use with the amplification and detection methods disclosed herein. The use of a low pH solution for dissociation of gDNA for APA amplification was investigated. Figure 1A shows the results of an experiment in which target genomic DNA (gDNA) was preincubated in TE solution (10 mM Tris, pH 8.0, 0.1 mM EDTA) at 78°C for 2 minutes, then cooled and added to the master mix. In the absence of acidic components, only 5 / 6 curves showed amplification at a target input of 200 copies (cps) per reaction, and only 2 / 6 curves obtained amplification for 50 copies per reaction. Figure 1B shows the results of an experiment in which target gDNA was preincubated in acidic solution (5 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Tx-100, and 10 mM HCl) at 78°C for 2 minutes, then cooled and added to the master mix. This method was found to yield excellent detection down to 5 copies.

[0155] The effect of preincubation temperature on N. gonorrhoeae (Ng) gDNA detection was investigated (Figure 2). Tests compared the incubation of 50 copies of Ng gDNA in an acidic solution (5 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Triton X-100, and 10 mM HCl) for 2 minutes with or without heating before amplification. Results indicate that initial preheating is not necessary for DNA dissociation in the acidic solution. Indeed, simply preincubating the gDNA at room temperature was found to be sufficient to determine the effect. Next, we investigated the effect of elution buffer pH on N. gonorrhoeae (Ng) gDNA detection in wet reactions (Figures 3A-B) and before mixing with dry reaction components (Figures 4A-B). N. gonorrhoeae (Ng) gDNA was diluted into either an acidic solution (5 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Tx-100, and 10 mM HCl; Figure 3B) or a control solution (TE; pH 8; 10 mM Tris, pH 8.0, 0.1 mM EDTA; Figure 3A) before archaeal polymerase amplification (APA) in wet reactions at 68 °C for 10 min. N. gonorrhoeae (Ng) gDNA was diluted in a neutral pH elution buffer (5 mM (NH4)2SO4, 4 mM MgSO4, and 0.2% SDS; Figure 4A) or an acidic elution buffer (5 mM (NH4)2SO4, 4 mM MgSO4, 10 mM glycine / 8.8 mM HCl (glycine / HCl buffer), and 0.2% SDS; Figure 4B) at room temperature before being mixed with dried reaction components containing Tris buffer at pH 8.8 for amplification at 68°C for 10 min.

[0156] The effects of temperature and pH on N. gonorrhoeae (Ng) gDNA stability were also investigated. 5000 cps of Ng gDNA per reaction was incubated in acidic solution (5 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Tx-100, and 10 mM HCl) or TE solution (pH 8; 10 mM Tris, pH 8.0, 0.1 mM EDTA) for 2 min vs. 1 h at 78°C (Figure 5B) or room temperature (Figure 5A) before being mixed with amplification components containing Tris buffer at pH 8.8 for 15 min of amplification at 68°C. Next, we investigated the effects of temperature and pH on N. gonorrhoeae (Ng) gDNA stability in a 10% urine matrix. Five thousand copies of Ng gDNA per reaction were incubated in an acidic solution (5 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Tx-100, and 8.8 mM HCl) or TE solution (10 mM Tris, pH 8.0, 0.1 mM EDTA) containing 10% urine for 2 min or 1 h at 78°C (Figure 6A) or room temperature (RT; Figure 6B) before mixing with amplification components containing Tris buffer (pH 8.8) for 15 min of amplification at 68°C.

[0157] The effects of temperature and pH on N. gonorrhoeae (Ng) gDNA stability in a dry reaction format were also investigated (Figure 7). Five thousand copies of Ng gDNA per reaction were incubated in an acidic solution (5 mM (NH4)2SO4, 4 mM MgSO4, 10 mM glycine / 8.8 mM HCl (glycine / HCl buffer), and 0.2% SDS) for 2 min vs. 1 h at 78°C or room temperature before being mixed with the dry reaction components for amplification at 68°C for 15 min. The lyophilized (lyo) pellet contained Tris buffer at pH 8.8. Taken together, these results yield the surprising finding that exposure of genomic DNA to a low pH solution as an initial step in archaeal polymerase amplification does not adversely affect amplification, as commonly believed. Thus, the methods, compositions, and kits provided herein can advantageously provide point-of-care molecular diagnostics for DNA pathogens in clinical samples. After pathogen lysis, genomic DNA strands can be separated without the need for a separate thermal or enzymatic step.

[0158] Example 2 Improvements in assay LOD for gDNA detection This example provides an approach for improving the limit of detection (LOD) of an assay for the detection of gDNA using the disclosed methods and compositions. In some embodiments, the current limit of detection (LOD) in the dried format is >50 copies of gDNA for C. trachomatis / N. gonorrhoeae (Ct / Ng) lyophilized with standard dNTPs. Without being bound by any particular theory, achieving a low LOD may involve destabilization of gDNA in the resuspension buffer; embodiments include using Mg 2+ and limiting the effect of monovalent salts, and increasing the pH to >9.5 or decreasing the pH to <4. Some embodiments of the methods and compositions provided herein address resuspension buffer stability, Mg precipitation when in high pH solutions, and the ability to reduce Mg precipitation when in a resuspension buffer (e.g., 5 mM (NH)SO, 4 mM MgSO, 0.2% SDS (7 mM Na) + , 7mM DS - )) can overcome various problems, such as the fact that low salt may already be present in the Factors identified to affect DNA duplex stability include, in some embodiments, %GC, dsDNA length, sequence dependence, the effect of magnesium and monovalent cations (e.g., Mg over monovalent cations on Tm under typical PCR conditions). 2+ Predominantly, 1M Na + Equivalent to 10mM Mg 2+ , 5mM Na + Equivalent to 10 mM Tris), containing extreme pH, as well as additives.

[0159] As can be seen from Figure 8 (adapted from Biophys J. 2001 Feb; 80(2): 874-881. Effect of pH on the overstretching transition of double-stranded DNA: evidence of force-induced DNA melting), which shows a non-limiting and exemplary melting curve of λDNA (48502bp) as a function of pH, a significant decrease in Tm is observed at extreme pH values (>pH9.7 and <pH3.9). In some embodiments provided herein, low pH is more effective than high pH for λDNA melting. In some embodiments, the LOD can be improved by reducing the pH in a resuspension buffer, such as resuspension buffer RBS (5 mM (NH4)2SO4, 4 mM MgSO4, 0.2% SDS, pH approximately 7), compared to low pH RBS (RBS / 10 mM HCl, pH 2-3). No apparent precipitation of RBS / 10 mM HCl was observed over 3 days at room temperature. Embodiments of the compositions and methods provided herein address possible issues such as competition between protons and Mg 2+ (e.g., Mg 2+ that inhibits DNA dissociation in low pH solutions) and the speed and extent of DNA depurination in low pH solutions.

[0160] In at least some of the foregoing embodiments, one or more elements used in an embodiment can be used interchangeably in another embodiment, provided such replacement is not technically infeasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims. With respect to the use of substantially any plural and / or singular terminology herein, those of ordinary skill in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless stated otherwise.

[0161] Those skilled in the art will understand that the terms used in this specification, generally, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Furthermore, those skilled in the art will understand that where a specific number is intended in an introduced claim recitation, such intention will be expressly recited in the claim; otherwise, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means limiting any particular claim containing such an introduced claim recitation to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Additionally, even if a particular number is explicitly recited in an introduced claim recitation, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a base recitation of "two recitations" means at least two recitations, or two or more recitations, in the absence of other modifiers).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that a person of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, a system having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that a person of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Moreover, those skilled in the art will appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either of those terms, or both terms.

[0162] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is also thereby described in terms of every individual member or subgroup of members of the Markush group. As one of ordinary skill in the art would understand, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as being capable of dividing that same range into at least two, three, four, five, ten, etc., as fully described. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Furthermore, as one of ordinary skill in the art would understand, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the recited numerical values ​​and that can be subsequently divided into subranges as discussed above. Finally, as one of ordinary skill in the art would understand, a range includes each individual member. Thus, for example, a group containing 1 to 3 items refers to groups containing 1, 2, or 3 items. Similarly, a group containing 1 to 5 items refers to groups containing 1, 2, 3, 4, or 5 items, etc.

[0163] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and not limitation, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for amplifying a target nucleic acid sequence in a sample, comprising: (a) contacting a sample containing a biological entity with an acidic composition to produce an acidic mixture; wherein the acidic composition is capable of dissolving the biological entities to release sample nucleic acids contained therein, the sample nucleic acids including double-stranded DNA (dsDNA) suspected of containing a target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length; the acidic composition has a pH of less than 4 and comprises (i) a monovalent salt and / or a divalent salt, (ii) one or more surfactants, and (iii) an acidic agent, which denatures the dsDNA to produce single-stranded DNA (ssDNA); (b) contacting a reagent composition comprising a buffer with the acidic mixture to produce a neutral mixture, wherein the neutral mixture comprises the ssDNA and the reagent composition comprises one or more amplification reagents, wherein the one or more amplification reagents comprise an enzyme having hyperthermophile polymerase activity; and (c) amplifying the target nucleic acid sequence in the neutral mixture under isothermal amplification conditions, thereby generating a nucleic acid amplification product; The above method, comprising:

2. A method according to claim 1, (a) amplifying the target nucleic acid sequence comprises producing the nucleic acid amplification product at a detectable level within about 20 minutes; (b) the acidic composition has a pH of about 1 to about 6.9, optionally about 1 to about 3.9, and further optionally about 2.2; and / or (c) the neutral mixture has a pH of about 7 to about 9, and optionally, the neutral mixture has a pH of about 8.

8.

3. A method according to claim 1, The method further comprises detecting the target nucleic acid sequence in the sample, wherein detecting the target nucleic acid sequence in the sample comprises: (d) detecting the nucleic acid amplification product, wherein the detecting step is performed less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes from the time the reagent composition contacts the acidic mixture. The above method, comprising:

4. The method of claim 1, (a) contacting the reagent composition with the acidic mixture comprises dissolving the reagent composition in the acidic mixture; (b) the one or more amplification reagents comprise one or more of a first primer, a second primer, and a dNTP, and optionally the first primer and / or the second primer may be about 8-16 bases in length, and optionally the first primer and / or the second primer may comprise one or more of DNA bases, modified DNA bases, or combinations thereof; (c) the reagent composition is a dry composition or a wet composition; (d) the reagent composition is lyophilized and / or heat-dried and comprises one or more additives, wherein the one or more additives are: (i) an amino acid; (ii) a sugar or sugar alcohol, optionally wherein the sugar or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or (iii) a polymer, optionally wherein the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof. Including, and / or (e) the dsDNA suspected of containing the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other.

5. The method of claim 1, The step of amplifying the target nucleic acid sequence comprises: amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the amplifying comprises subjecting the ssDNA to one of the following i) and ii): i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of the second strand of the target nucleic acid sequence; and ii) the enzyme having hyperthermophilic polymerase activity, thereby producing a nucleic acid amplification product, wherein the nucleic acid amplification product is (1) the sequence of the first primer and its reverse complement; (2) the sequence of the second primer and its reverse complement; and (3) (1) the sequence of the first primer and its reverse complement, and (2) a spacer sequence flanked by the sequence of the second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length. including contacting The above method.

6. The method according to claim 5, (a) the first primer and / or the second primer are about 8 to 16 bases in length, and optionally, the first primer and / or the second primer may comprise one or more of DNA bases, modified DNA bases, or a combination thereof; and / or (b) the spacer sequence comprises a portion of the target nucleic acid sequence, and optionally, the spacer sequence may be 1 to 10 bases in length.

7. The method of claim 1, (a) the enzyme having hyperthermophilic polymerase activity is (i) having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof, and optionally, the enzyme with hyperthermophile polymerase activity can have an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO:1, and further optionally, the enzyme with hyperthermophile polymerase activity can be a polymerase comprising the amino acid sequence of SEQ ID NO:1; and / or (ii) low or no exonuclease activity; (b) the nucleic acid amplification product is about 20 to 40 bases in length; (c) the dsDNA comprises genomic DNA (gDNA), plasmid DNA, or both; (d) the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle; (e) the biological entity comprises a virus, a bacterium, a fungus, a protozoan, a part thereof, or any combination thereof; (f) the target nucleic acid sequence is (i) a nucleic acid sequence of a virus, optionally the virus can be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus, and optionally The sample nucleic acid may be derived from a virus, optionally the virus may be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus. (ii) a nucleic acid sequence of a bacterium, optionally the bacterium being selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae gonorrhoeae), Streptococcus pneumonia, S. and optionally, the sample nucleic acid may be derived from a bacterium, optionally the bacterium being selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, S. agalactiae, and Listeria monocytogenes.pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes; (iii) a nucleic acid sequence of a fungus, optionally the fungus being selected from the group consisting of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum. and optionally the sample nucleic acid may be derived from a fungus, and optionally the fungus may comprise one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; or (iv) a nucleic acid sequence of a protozoan, optionally the protozoan may comprise one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp., and optionally the sample nucleic acid is derived from a protozoan, and optionally the protozoan may be Trypanosoma cruzi. cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; (g) the sample is a biological sample or an environmental sample; the environmental sample is or is derived from a food sample, a beverage sample, a paper surface, a textile surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saltwater sample, a sample of exposure to air or other gases, a culture thereof, or any combination thereof; and / or the biological sample is or is derived from a tissue sample, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, a swab of skin or a mucosal surface, a culture thereof, or any combination thereof; (h) the acidic agent is (i) comprising an organic acid, an inorganic acid, or both; (ii) selected from the group consisting of hydrochloric acid, glycine hydrochloride, sulfuric acid, acetic acid, citric acid, and phosphoric acid; (iii) is present in the acidic composition at a concentration of less than about 100 mM, optionally at a concentration in the range of about 1 mM to about 100 mM, and further optionally at a concentration of about 10 mM; (j) the monovalent salt and / or the divalent salt comprises a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or any combination thereof; (k) the monovalent salt is selected from the group consisting of ammonium sulfate, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, potassium fluoride, potassium chloride, potassium bromide, and potassium iodide; (l) the divalent salt is selected from the group consisting of magnesium sulfate, calcium chloride, magnesium chloride, copper (II) chloride, zinc chloride, calcium oxide, magnesium oxide, barium oxide, sodium sulfate, calcium sulfate, copper (II) sulfate, potassium carbonate, and sodium carbonate; (m) the monovalent salt and / or the divalent salt are present in the acidic composition at a concentration in the range of about 1 mM to about 14 mM, and optionally at a concentration of about 5 mM or about 4 mM; (n) the one or more surfactants are (i) comprising one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; and / or (ii) is present in the acidic composition at a concentration in the range of about 0.01% to about 2% by weight to volume (% wt / vol) of the acidic composition, and optionally, the one or more surfactants are present in the acidic composition at a concentration of about 0.1% by weight to volume (% wt / vol) of the acidic composition; and / or (o) the acidic composition further comprises a chelating agent, optionally selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), and Tris, and optionally present in the acidic composition at a concentration in the range of about 0.1 mM to about 14 mM.

8. The method of claim 1, (a) contacting the sample with the acidic composition comprises: (i) is carried out at a temperature in the range of about 18°C ​​to about 99°C, and optionally, contacting the sample with the acidic composition may be carried out at a temperature of about 78°C; (ii) carried out at a temperature within the range of about 18°C ​​to about 25°C; and / or (iii) conducted for a period of about 5 seconds to about 60 minutes; (b) the buffer comprises MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium buffer, or any combination thereof; (c) the neutral mixture has a pH in the range of about 7 to about 9, and optionally, the neutral mixture has a pH of about 8.8; (d) the target nucleic acid sequence comprises a length of about 90 nucleotides or less, and optionally, the target nucleic acid sequence comprises a length of about 30 nucleotides; (e) the amplifying step is carried out under isothermal amplification conditions, optionally comprising a constant temperature of about 30°C to about 72°C, and further optionally comprising a constant temperature of about 67°C; and / or (f) the amplifying step (i) is carried out for a period of about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes, optionally, the amplifying step may be carried out for a period of about 15 minutes; and / or (ii) The above method, which is carried out under helicase-free, single-stranded binding protein-free, cleavage agent-free and recombinase-free isothermal amplification conditions.

9. The method of claim 3, (a) step (d) further comprises determining the amount of the dsDNA comprising the target nucleic acid sequence in the sample; (b) detecting the nucleic acid amplification products comprises using a real-time detection method; (c) detecting the nucleic acid amplification product comprises contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the nucleic acid amplification product, wherein the signal-generating oligonucleotide comprises a fluorophore, a quencher, or both; and / or (d) the step of detecting the nucleic acid amplification product comprises detecting a fluorescent signal, and optionally, the fluorescent signal may be from a molecular beacon.

10. The method of claim 1, (a) carried out in a single reaction vessel; (b) the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences, and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally wherein the two or more target nucleic acid sequences may be specific to two or more different organisms; (c) the amplifying step does not include the use of any enzyme other than the enzyme having hyperthermophile polymerase activity; (d) the amplifying step comprises one or more of the following: Archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal-mediated RNA amplification technology (SMART), self-sustained sequence replication (3SR), genomic exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA); (e) the amplifying step does not include one or more of the following: Archaeal Polymerase Amplification (APA), LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA, and optionally the amplifying step does not include LAMP; and / or (f) (i) diluting the acidic mixture; (ii) diluting the neutral mixture; (iii) heat denaturing the acidic mixture; (iv) sonicating the acidic mixture; (v) sonicating the neutral mixture; (vi) adding an RNase inhibitor to the acidic mixture; (vii) adding an RNase inhibitor to the neutral mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing the one or more detergents from the acidic mixture or the neutral mixture; (xii) denaturing the sample nucleic acid before and / or during amplification. (xiii) adding RNase H to the acidic mixture or the neutral mixture; (xiv) contacting the sample, the acidic mixture or the neutral mixture with a helicase, a single-stranded binding protein, a nicking enzyme, a restriction enzyme or a recombinase; (xv) contacting the sample, the acidic mixture or the neutral mixture with glycerol, formamide or urea; (xvi) purifying the ssDNA from the acidic mixture or the neutral mixture; (xvii) a heat denaturation step; and (xviii) enriching, purifying and / or isolating the dsDNA.

11. The method of claim 1, (a) the acidic composition and / or the reagent composition does not include a reducing agent, a chelating agent, or both; Optionally, (i) the chelating agent may be selected from the group including ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), Tris, or any combination thereof; and / or (ii) the reducing agent may be selected from the group consisting of 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythriol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, salts of sulfurous acid, thioglycolic acid, and hydroxyethyl disulfide (HED), or any combination thereof; (b) the acidic composition is free of both anionic surfactants and cationic surfactants; and / or (c) the acidic composition further comprises a tween surfactant, optionally selected from the group consisting of Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85, and further optionally, the tween surfactant comprises about 0.01% (wt / vol) of the acidic composition.

12. 1. An acidic composition for dissolving biological entities and denaturing double-stranded DNA (dsDNA) contained therein, comprising: a monovalent salt at a concentration of less than 30 mM in said acidic composition and / or a divalent salt at a concentration of less than 15 mM in said acidic composition; one or more surfactants; and an acidic agent present in said acidic composition at a concentration of less than 100 mM and having a pH of less than 4.

13. The acidic composition of claim 12, (a) does not contain a reducing agent, a chelating agent, or both; Optionally, (i) the chelating agent may be selected from the group including ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), Tris, or any combination thereof; and / or (ii) the reducing agent may be selected from the group consisting of 2-mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), reduced glutathione, cysteamine, tri-n-butylphosphine (TBP), dithioerythriol, tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine-HCl, dithiobutylamine (DTBA), cysteine, cysteine-thioglycolate, salts of sulfurous acid, thioglycolic acid, and hydroxyethyl disulfide (HED), or any combination thereof; (b) does not contain both anionic surfactants and cationic surfactants; (c) does not contain glycerol, formamide, or urea; (d) having a pH in the range of about 1 to about 3.9, and optionally a pH of about 2; (e) the acidic agent includes an organic acid and / or an inorganic acid; (f) the acidic agent is selected from the group consisting of hydrochloric acid, glycine hydrochloride, acetic acid, citric acid, and phosphoric acid; (g) the acidic agent is present in the acidic composition at a concentration in the range of about 1 mM to about 100 mM, and optionally, the acidic agent may be present in the acidic composition at a concentration of about 10 mM; (h) the monovalent salt and / or the divalent salt comprises a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or any combination thereof; (i) the monovalent salt is selected from the group consisting of ammonium sulfate, ammonium chloride, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, potassium fluoride, potassium chloride, potassium bromide, and potassium iodide; (j) the divalent salt is selected from the group consisting of magnesium sulfate, calcium chloride, magnesium chloride, copper (II) chloride, zinc chloride, calcium oxide, magnesium oxide, barium oxide, sodium sulfate, calcium sulfate, copper (II) sulfate, potassium carbonate, and sodium carbonate; (k) the monovalent salt and / or the divalent salt is present in the acidic composition at a concentration in the range of about 1 mM to about 14 mM, and optionally, the monovalent salt and / or the divalent salt may be present in the acidic composition at a concentration of about 5 mM or about 4 mM; (l) the one or more surfactants include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant; (m) the one or more surfactants are present in the acidic composition at a concentration in the range of about 0.01% to about 2% by weight to volume (% wt / vol) of the acidic composition, and optionally, the one or more surfactants may be present in the acidic composition at a concentration of about 0.1% by weight to volume (% wt / vol) of the acidic composition; (n) the acidic composition further comprises a tween surfactant, optionally selected from the group consisting of Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85, and further optionally, comprising from about 0.01% to about 1% (weight / volume) of the acidic composition; and / or (o) The acidic composition further comprises a chelating agent, optionally selected from the group comprising ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(aminoethyl) N,N'-tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), Tris, or any combination thereof, and further optionally, the chelating agent may be present in the acidic composition at a concentration in the range of about 0.1 mM to about 14 mM.

14. 1. A kit for detecting a target nucleic acid sequence in a sample, comprising: (a) an acidic composition according to any one of claims 12 to 13, wherein the acidic composition is capable of dissolving biological entities to release sample nucleic acids contained therein, the sample nucleic acids comprising double-stranded DNA (dsDNA) suspected of containing a target nucleic acid sequence, the target nucleic acid sequence being 100 nucleotides or less in length; and (b) a reagent composition comprising a buffer and one or more amplification reagents for amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein the one or more amplification reagents are selected from the following: (i) and (ii): (i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of a first strand of the target nucleic acid sequence and the second primer is capable of hybridizing to a sequence of a second strand of the target nucleic acid sequence; and (ii) an enzyme having hyperthermophilic polymerase activity capable of producing nucleic acid amplification products; a reagent composition comprising: The above kit.

15. The kit of claim 14, (a) the buffering agent is selected from the group comprising MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium buffer, or any combination thereof; (b) the kit further comprises at least one component providing real-time detection activity of nucleic acid amplification products, optionally wherein the real-time detection activity may be provided by a molecular beacon; (c) the enzyme with hyperthermophilic polymerase activity has an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof, and optionally, the enzyme with hyperthermophilic polymerase activity can have an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO:1, and further optionally, the enzyme with hyperthermophilic polymerase activity can be a polymerase comprising the amino acid sequence of SEQ ID NO:1; (d) the nucleic acid amplification product is about 20 to 40 bases in length, and the nucleic acid amplification product is (1) the sequence of the first primer and its reverse complement; (2) the sequence of the second primer and its reverse complement; and (3) a spacer sequence flanked by (1) the sequence of a first primer and its reverse complement and (2) the sequence of a second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length. Including, (e) the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle, and optionally, the biological entity may comprise a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof; (f) the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid may be derived from: (i) a virus, optionally the virus can be SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), and hantavirus; (ii) a bacterium, optionally the bacterium is selected from the group consisting of Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae pneumonia, S. agalactiae, and Listeria monocytogenes; (iii) a fungus, optionally comprising one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and / or (iv) protozoa, optionally the protozoa may comprise one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.; (g) the first primer and / or the second primer are about 8 to 16 bases in length, and optionally the first primer and / or the second primer may comprise one or more of DNA bases, modified DNA bases, or a combination thereof; (h) the reagent composition is lyophilized and / or heat-dried and comprises one or more additives, wherein the one or more additives are: (i) an amino acid; (ii) a sugar or sugar alcohol, optionally wherein the sugar or sugar alcohol may comprise sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof; and / or (iii) a polymer, optionally wherein the polymer comprises polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof; Including, (j) the mixture of the acidic composition and the reagent composition may have a pH of about 7 to about 9, optionally a pH of about 8.8; (k) the mixture of the sample, the acidic composition, and the reagent composition may have a pH of about 7 to about 9, optionally a pH of about 8.8; (l) the buffer comprises Tris, and the mixture of the sample, the acidic composition, and the reagent composition comprises Tris at a concentration in the range of about 30 mM Tris to about 50 mM Tris; and / or (m) the above kit, comprising a sterile container containing the acidic composition and the reagent composition.

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