Non-Opacified Dissolution Buffer Composition Formulations

Abstract

Disclosed herein are methods, compositions, and kits for use in detecting a target nucleic acid sequence in a sample. Provided are storage stable lysis buffers. In some embodiments, the lysis buffer comprises one or more surfactants and magnesium sulfate (MgSO4). In some embodiments, the lysis buffer contains a precipitate (e.g., MgSO4). 2+ and one or more surfactant complexes) is substantially inhibited over a period of time under at least one storage condition.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 307,092, filed February 5, 2022, the entire contents of which are incorporated herein by reference for all purposes. Sequence Listing Reference This application is filed with a sequence listing in electronic format. The sequence listing is provided in a file entitled 68EB-317336-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, identification of bacterial or viral nucleic acids in a sample can be useful for diagnosing certain types of infections. Other examples include identification of single nucleotide polymorphisms for disease control or forensics, and identification of 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 the polymerase chain reaction (PCR). This technique typically requires temperature cycling (i.e., thermal cycling) to proceed through the steps of denaturation (e.g., separation of strands of a double-stranded DNA (dsDNA) complex), annealing of oligonucleotide primers (short strands of complementary DNA sequences), and extension of the primers along their complementary targets by a polymerase. Such thermal cycling can be a time-consuming process that generally requires specialized machinery. Thus, 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. Furthermore, there is a need for such compositions and methods of nucleic acid detection where the lysis buffer used to lyse biological entities (e.g., viral particles, bacteria) is stable during storage (e.g., does not become opaque due to precipitation). There is a need for a lysis buffer that (i) lyses a sufficient percentage of organisms to maintain clinical performance; (ii) is stable for 18 months when stored at refrigerated or room temperature; (iii) does not destroy nucleic acids within the test sample during lysis; and (iv) is effective in the presence of clinical matrices. Summary of the Invention

[0003] Disclosed herein includes a dissolution buffer. In some embodiments, the dissolution buffer comprises one or more detergents; ammonium sulfate ((NH4)2SO4); and magnesium sulfate (MgSO4), wherein the dissolution buffer does not comprise one or more of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), and the formation of precipitate is substantially inhibited over a period of time under storage conditions. In some embodiments, the appearance of precipitate in the dissolution buffer does not occur over at least about 20 days under storage conditions.

[0004] The lysis buffer may include one or more surfactants; (NH4)2SO4 and MgSO4, where the formation of precipitates is substantially inhibited over a period of time under storage conditions, and the appearance of precipitates in the lysis buffer does not occur, or precipitates formed in the lysis buffer are undetectable over at least about 20 days under storage conditions. The lysis buffer may include one or more chelating agents (e.g., EDTA, EGTA). For example, the lysis buffer may include EDTA, EGTA, or other metal ion chelating agents that, in some embodiments, can form stronger complexes with calcium ions than with heavy metal ions or magnesium ions. In some embodiments, the chelating agents are present at a concentration 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, or any range or value between these values.

[0005] In some embodiments, the lysis buffer does not contain SDS, CTAB, or both. In some embodiments, the precipitate, the formation of which is substantially inhibited over a period of time under storage conditions in the methods and compositions disclosed herein, is formed by the addition of Mg. 2+and one or more surfactants. In some embodiments, the lysis buffer is a precipitation-stable aqueous composition (e.g., an aqueous composition in which precipitation is absent or inhibited over an extended period of time under storage conditions). In some embodiments, the lysis buffer is a precipitation-free aqueous composition. In some embodiments, the lysis buffer is a Mg 2+ There is no precipitation of complexes consisting of Mg and surfactant. 2+ In some embodiments, the precipitate of the complex of Mg and the surfactant is removed. 2+ In some embodiments, precipitation of the complexes of soluble Mg and surfactant is inhibited. 2+ does not decrease by more than about 1.1-fold compared to the beginning of the period. In some embodiments, the appearance of a precipitate in the lysis buffer does not occur during storage conditions for at least about 30 days, about 60 days, about 90 days, about 6 months, about 1 year, or about 2 years.

[0006] Storage conditions may include, for example, transportation of a lysis buffer. In some embodiments, storage conditions include temperature stress, one or more freeze-thaw cycles, sonication, shear forces, agitation, pressure changes, light exposure, or any combination thereof. In some embodiments, storage conditions include ambient conditions (e.g., in the range of about 20° C. to about 25° C.). In some embodiments, storage conditions include refrigerated conditions (e.g., about 4° C.). In some embodiments, storage conditions include 14° C. In some embodiments, the period of time is at least about 30 days, about 60 days, about 90 days, about 6 months, about 1 year, or about 2 years.

[0007] In some embodiments, the substantial inhibition of the formation of a precipitate comprises a lysis buffer that has no visible precipitate when assessed by visual inspection. In some embodiments, the absence of precipitate in the lysis buffer comprises a lysis buffer that has a visible precipitate when assessed by visual inspection. In some embodiments, the appearance of precipitate in the lysis buffer comprises a lysis buffer that has a visible precipitate when assessed by visual inspection. In some embodiments, the precipitation is monitored by using light scattering. In some embodiments, the precipitation is monitored using a turbidity sensor, a turbidimeter, or a nephelometer. In some embodiments, the precipitation is monitored using a spectrophotometer. In some embodiments, the substantial inhibition of the formation of a precipitate comprises a lysis buffer that has an absorbance at a wavelength below a threshold absorbance when determined by spectrophotometric analysis. In some embodiments, the appearance of precipitate in the lysis buffer comprises a lysis buffer that has an absorbance at a wavelength above a threshold absorbance when determined by spectrophotometric analysis. In some embodiments, the threshold absorbance is about 0.001 to about 6.0 absorbance units (AU). In some embodiments, the appearance of a precipitate in the lysis buffer comprises an absorbance at a wavelength greater than about 1.1 times the absorbance at the wavelength at the beginning of the period, as determined by spectrophotometric analysis. In some embodiments, the substantial inhibition of the formation of a precipitate comprises a lysis buffer having an absorbance at a wavelength less than about 1.1 times the absorbance at the wavelength at the beginning of the period, as determined by spectrophotometric analysis. In some embodiments, the spectrophotometric analysis is performed using a spectrophotometer at a wavelength of about 200 nm to about 900 nm. In some embodiments, the wavelength is the wavelength that maximizes the absorbance of the precipitate. In some embodiments, the absorbance value is due to light scattering of the precipitate. The spectrophotometer may be a UV-Vis spectrophotometer, an IR spectrophotometer, a Vis-Near Infrared spectrophotometer, a Raman spectrophotometer, or a combination thereof.

[0008] In some embodiments, the lysis buffer further comprises one or more alcohols. In some embodiments, the one or more alcohols have a carbon chain length of 1, 2, 3, 4, 5, or 6. In some embodiments, the one or more alcohols are selected from the group including ethanol, isopropanol, isobutyl alcohol, pentanol, and hexanol. In some embodiments, the one or more alcohols comprise about 0.001% (v / v) to about 20.0% (v / v) of the lysis buffer. In some embodiments, the lysis buffer comprises about 0.001% (v / v) to about 20.0% (v / v), for example, about 0.1% (v / v) to about 4.0% (v / v) of one or more alcohols. In some embodiments, MgSO4 is present at a concentration of about 0.1 mM to about 100 mM, e.g., about 4 mM. In some embodiments, (NH4)2SO4 is present at a concentration of about 0.1 mM to about 100 mM, e.g., about 5 mM. In some embodiments, (NH4)2SO4 is present at a concentration of about 10 mM, and the appearance of precipitate in the lysis buffer is delayed for at least about 10 days (under storage conditions described herein) when compared to an equivalent lysis buffer in which (NH4)2SO4 is present at a concentration of about 5 mM. The lysis buffer may include an acid. In some embodiments, the acid includes an organic acid, an inorganic acid, or a mixture thereof. In some embodiments, the inorganic acid is hydrogen chloride (HCl). In some embodiments, the acid is present at a concentration of about 8.8 mM. The lysis buffer may include a pH buffer. In some embodiments, the pH buffer includes glycine and HCl. In some embodiments, the pH buffer includes 10.0 mM glycine. In some embodiments, the pH of the lysis buffer is about 1.0 to about 6.0, for example, about 2.2.

[0009] In some embodiments, the one or more detergents can lyse the biological entity to release the sample nucleic acid contained therein. In some embodiments, the sample nucleic acid comprises a sample ribonucleic acid and / or a sample deoxyribonucleic acid. In some embodiments, the biological entity comprises a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof. In some embodiments, the biological entity comprises one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. In some embodiments, the one or more detergents comprise about 0.001% (w / v) to about 10.0% (w / v) of the lysis buffer. In some embodiments, the one or more detergents comprise one or more of a cationic detergent, an anionic detergent, a nonionic detergent, and an amphoteric detergent. In some embodiments, the lysis buffer further comprises a tween detergent. In some embodiments, the tween detergent is selected from the group consisting of Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85. In some embodiments, the tween detergent comprises about 0.01% to 0.2% (w / v) of the lysis buffer.

[0010] In some embodiments, the one or more detergents include CTAB. In some embodiments, the lysis buffer includes about 0.2% (w / v) CTAB and (NH4)2SO4 is present at a concentration of about 5 mM. In some embodiments, the one or more detergents include CTAC. In some embodiments, the lysis buffer includes about 0.2% (w / v) CTAC and (NH4)2SO4 is present at a concentration of about 5 mM. In some embodiments, the one or more detergents include SDS. In some embodiments, the lysis buffer includes about 0.4% (w / v) SDS and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the lysis buffer further includes Tween 80. In some embodiments, the one or more detergents include sodium decyl sulfate (SDeS). In some embodiments, the lysis buffer includes about 0.2% (w / v) SDeS and (NH4)2SO4 is present at a concentration of about 5 mM. In some embodiments, the one or more detergents include SDeS. In some embodiments, the lysis buffer comprises about 0.2% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the one or more detergents include SDeS. In some embodiments, the lysis buffer comprises about 0.4% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the one or more detergents include SDeS. In some embodiments, the lysis buffer comprises about 0.8% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the one or more detergents include sodium decyl sulfate (SDeS). In some embodiments, the lysis buffer comprises about 0.4% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the lysis buffer further comprises Tween 80. In some embodiments, the one or more detergents include CTAB. In some embodiments, the lysis buffer includes about 0.2% (w / v) CTAB, and (NH4)2SO4 is present at a concentration of about 5 mM. In some embodiments, the one or more detergents include CTAC.In some embodiments, the lysis buffer comprises about 0.2% (w / v) CTAC and (NH4)2SO4 is present at a concentration of about 5 mM. In some embodiments, the one or more detergents comprise sodium octyl sulfate (S Octyl S). In some embodiments, the lysis buffer comprises about 0.2% (w / v) S Octyl S and (NH4)2SO4 is present at a concentration of about 5 mM.

[0011] Disclosed herein are methods of processing a sample. In some embodiments, the method includes the steps of (a) contacting a sample containing biological entities with a lysis buffer provided herein to generate a processed sample, wherein the lysis buffer is capable of lysing the biological entities to release sample nucleic acids contained therein.

[0012] In some embodiments, the sample nucleic acid is suspected of containing a target nucleic acid sequence, and the method further comprises detecting the target nucleic acid sequence in the sample. In some embodiments, detecting the target nucleic acid sequence in the sample comprises: (b) contacting a reagent composition (e.g., wet composition, dry composition) with the process sample to generate an amplification reaction mixture, where the reagent composition comprises one or more amplification reagents; (c) amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product; and (d) detecting the nucleic acid amplification product, where the detecting step is performed less than about 20 minutes from the time the reagent composition contacts the process sample.

[0013] In some embodiments, the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid. In some embodiments, the sample ribonucleic acid comprises cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or any combination thereof. In some embodiments, the one or more amplification reagents comprise a reverse transcriptase and / or an enzyme having hyperthermophile polymerase activity. In some embodiments, the enzyme having hyperthermophile polymerase activity has reverse transcriptase activity. In some embodiments, contacting the reagent composition with the process sample comprises dissolving the reagent composition in the process sample. In some embodiments, the reagent composition comprises one or more of a reverse transcriptase, an enzyme having hyperthermophile polymerase activity, a first primer, a second primer, and a reverse transcription primer. In some embodiments, the amplification is performed under isothermal amplification conditions. In some embodiments, detecting the nucleic acid amplification products comprises using a real-time detection method.

[0014] In some embodiments, the reagent composition is lyophilized and / or heat dried (e.g., a dry composition) and includes one or more additives. In some embodiments, the one or more additives include an amino acid; a sugar or sugar alcohol (e.g., lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof); and / or a polymer (e.g., polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptide, collagen peptide, or any combination thereof).

[0015] In some embodiments, the sample nucleic acid comprises a nucleic acid that comprises a target nucleic acid sequence. In some embodiments, the target nucleic acid sequence comprises a first strand and a second strand that are complementary to each other. In some embodiments, amplifying a target nucleic acid sequence comprises amplifying a target nucleic acid sequence comprising a first strand and a second strand that are complementary to one another under isothermal amplification conditions, the amplification comprising contacting a nucleic acid comprising the target nucleic acid sequence with: i) a first primer and a second primer, where 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 hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, where the nucleic acid amplification product comprises (1) a sequence of the first primer and its reverse complement, (2) a sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by the (1) sequence of the first primer and its reverse complement and the (2) sequence of the second primer and its reverse complement, the spacer sequence being between 1 and 10 bases in length. In some embodiments, the amplification does not involve the use of an enzyme other than an enzyme having hyperthermophile polymerase activity. In some embodiments, the amplification does not involve thermal and / or enzymatic denaturation of the nucleic acid. In some embodiments, the method does not involve contacting the nucleic acid with a single-stranded DNA binding protein prior to or during step (c).

[0016] In some embodiments, the nucleic acid is double stranded DNA. In some embodiments, the nucleic acid is a product of a reverse transcription reaction. In some embodiments, the nucleic acid is a product of a reverse transcription reaction generated from a sample ribonucleic acid. In some embodiments, step (c) comprises generating the nucleic acid by a reverse transcription reaction. In some embodiments, the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to generate cDNA. In some embodiments, the amplification of the target nucleic acid sequence includes the steps of: (c1) contacting a sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), 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, the amplification including contacting the dsDNA with (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 (ii) an enzyme having hyperthermophile 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 1-10 bases in length.

[0017] In some embodiments, the method does not include the use of an enzyme other than the reverse transcriptase and the enzyme having hyperthermophile polymerase activity. In some embodiments, step (d) further comprises determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the enzyme having hyperthermophile polymerase activity has an amino acid sequence that is at least about 90% or at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. In some embodiments, the enzyme having hyperthermophile polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme having hyperthermophile polymerase activity has low or no exonuclease activity. In some embodiments, the amplification of the target nucleic acid sequence is performed at a constant temperature of about 55°C to about 75°C, e.g., about 65°C.

[0018] In some embodiments, the first primer, the second primer, and / or the reverse transcription primer are about 8-16 bases in length. In some embodiments, the first primer, the second primer, and / or the reverse transcription primer comprise one or more of a DNA base, a modified DNA base, or a combination thereof. In some embodiments, the nucleic acid amplification product is about 20-40 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 1-10 bases in length. The method may include contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product, the signal-generating oligonucleotide comprising a fluorophore, a quencher, or both. 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 is performed in a single reaction vessel.

[0019] In some embodiments, the biological entity comprises 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 comprises a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof. In some embodiments, the target nucleic acid sequence is a viral, bacterial, fungal, or protozoan nucleic acid sequence. In some embodiments, the sample nucleic acid is derived from a virus, a bacterium, a fungus, or a protozoa. In some embodiments, the virus comprises one or more of 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, herpes virus 6, herpes virus 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.In some embodiments, the bacteria 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, Mycoplasma sp., Legionella pneumophila, Legionella dumophila, and the like. dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, and Listeria monocytogenes. In some embodiments, the fungus comprises one or more of Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum.In some embodiments, the protozoan is Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., or Eimeria sp.

[0020] In some embodiments, 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. In some embodiments, the two or more target nucleic acid sequences are specific for two or more different organisms. In some embodiments, the two or more different organisms comprise one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C.

[0021] The amplification may and / or may not include one or more of the following: archaeal polymerase amplification (APA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), and isothermal multiple displacement amplification (IMDA). In some embodiments, the amplification does not include LAMP.

[0022] In some embodiments, the method does not include one or more of the following: (i) diluting the sample to be processed; (ii) diluting the amplification reaction mixture; (iii) heat denaturing the sample to be processed; (iv) sonicating the sample to be processed; (v) sonicating the amplification reaction mixture; (vi) adding a RNase inhibitor to the sample to be processed; (vii) adding a RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysing agents from the sample to be processed or the amplification reaction mixture; (xii) heat and / or enzymatic denaturation of the sample nucleic acid prior to and / or during amplification; and (xiii) adding RNase H to the sample to be processed or the amplification reaction mixture. [Brief description of the drawings]

[0023] [Figure 1A-1B] FIG. 1 shows a non-limiting, exemplary schematic diagram of an isothermal amplification reaction provided herein. [Diagram 2] FIG. 1 illustrates data on BioAssay colorimetric magnesium assay for fresh DNA Assay Lysis Solution (DALB) as produced and precipitated DNA Assay Lysis Solution after 7 days (no visible precipitate). [Figure 3A-3C] FIG. 3 illustrates data regarding the effect of CTAB substitution in lysis buffer on Group A Streptococcus assay performance. Assays using standard lysis buffer (FIG. 3A) or lysis solution containing 0.2% CTAB (FIGS. 3B-3C) are shown. Fluorescence versus time (min) is shown for assays using 50 cp / reaction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, similar symbols typically identify similar components unless the context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments can be used and other changes can 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 the disclosure herein. 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.

[0025] Disclosed herein is a dissolution buffer comprising one or more detergents; (NH4)2SO4; and MgSO4, and not comprising one or more of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), wherein the formation of precipitate is substantially inhibited over a period of time under storage conditions.In some embodiments, the appearance of precipitate in the dissolution buffer does not occur over at least about 20 days under storage conditions. Disclosed herein includes a dissolution buffer.In some embodiments, the dissolution buffer comprises one or more surfactants;(NH4)2SO4;and MgSO4, wherein the formation of precipitate is substantially inhibited over a period of time under storage conditions, and the appearance of precipitate in the dissolution buffer does not occur over at least about 20 days under storage conditions. Disclosed herein are methods of processing a sample. In some embodiments, the method includes contacting a sample containing biological entities with a lysis buffer provided herein to generate a processed sample, wherein the lysis buffer is capable of lysing the biological entities to release sample nucleic acids contained therein. definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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.

[0026] As used herein, the term "precipitation" shall be given its ordinary meaning and shall refer to the formation of solid or insoluble particles in a solution. Various forms of precipitation occur and exemplary precipitates are described herein. As used herein, the term "precipitation" shall be given its ordinary meaning and shall refer to the formation of solids (e.g., precipitates) in a solution. As used herein, the term "solution" shall be given its ordinary meaning and shall refer to a substantially homogeneous mixture containing two or more substances dissolved in a solvent. As used herein, the terms "substantially inhibit precipitation" and "inhibit precipitation" shall be given their ordinary meaning and shall be used to describe the inhibition of much or all visible precipitation to maintain homogeneity over a period ranging from at least one month to at least two years. As defined herein, the terms "precipitation", "precipitate", "particle formation", "turbidity" and "flocculation" may be used interchangeably and include, but are not limited to, Mg 2+ The term may refer to any physical interaction or chemical reaction that results in the "aggregation" of a complex of the surfactant with the surfactant.

[0027] Provided herein are methods and compositions for amplifying nucleic acids. Conventional nucleic acid amplification methods typically require a thermal cycling process, a protein (e.g., an enzyme) (e.g., a helicase, a recombinase) that promotes nucleic acid denaturation, strand unwinding, strand separation, and / or strand exchange, and / or an endonuclease agent (e.g., a restriction enzyme, a nicking enzyme), 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 a protein (e.g., an enzyme) that promotes strand unwinding, strand separation, and / or strand exchange, without the use of an endonuclease agent, and within a reaction time of about 10 to 15 minutes.

[0028] Non-opaque dissolution buffer formulation Provided herein include methods and compositions for direct pathogen lysis in clinical samples that allow 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. Virus particles, bacterial cells, or other pathogens still need to be lysed so that their DNA and 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 not compatible with enzyme function because they would also inactivate DNA polymerase or other enzymes. Therefore, an effective chemical lysis method that is compatible with enzyme function and does not require any nucleic acid purification step to remove the lysis reagent would be of significant value.

[0029] Provided herein are methods and compositions for rapid DNA / RNA amplification, including Archaeal Polymerase Amplification (APA). The workflow may include isothermal amplification at about 68° C. and does not include thermal cycling. The disclosed methods and compositions may allow for rapid sample preparation and may include chemical lysis without purification steps. The disclosed methods and compositions may include an integrated reagent-loaded consumable. The compositions and methods provided herein may use, for example, two-color fluorescent detection based on molecular beacons or dsRNA fluorescent dyes. The methods and compositions provided herein may be used, for example, on the NATDx platform. The workflow can provide a sample-to-result / time-to-result (TTR) of 10 minutes or less and may include (i) pre-heating the tube in the reader (3 minutes); (ii) removing the cap and adding the sample to 1 mL of lysis buffer (1 minute); (iii) closing the tube with a second cap that breaks the seal and measures about 100 μL of lysate into the pre-heated lyophilized reagent using a magnetic motor used for mixing (1 minute); and (iv) performing real-time detection at about 68° C. (5 minutes) with a goal of calling negative in 5 minutes and calling positive in less than 5 minutes. Lysis may take about 2 minutes in some embodiments at about 75-80° C. for DNA assays and about 65-70° C. for RNA assays.

[0030] The rapid RNA / DNA assays disclosed herein can use buffer solutions that chemically dissolve clinical or biological samples (e.g., harvested human samples) to allow sample analysis in 10 minutes or less. RNA and DNA assays may each have unique lysis buffer formulations that are effective for these different test samples. There are four fundamental performance qualities for each lysis buffer solution. The lysis buffers provided herein have one or more of the following attributes: (i) the solution dissolves a sufficient percentage of organisms to maintain clinical performance; (ii) the solution is stable for 18 months when stored at refrigerated or room temperature (room temperature storage is preferred in some embodiments for ease of use by users); (iii) the solution does not destroy nucleic acids in the test sample during lysis; and (iv) the solution is effective in the presence of clinical matrices. In some embodiments, all four properties are required for the methods and compositions provided herein. With regard to (ii), in some embodiments, room temperature storage is required, but in some embodiments, refrigerator storage stability is not required.

[0031] As described in Example 1, the DNA lysis buffer formulation can become opaque (contains precipitate) within as little as 2-3 days after preparation when stored at room temperature or 14°C. This performance is unacceptable because it does not meet the second essential performance quality above (i.e., the solution is stable for over 18 months when stored at refrigerated or room temperature). Furthermore, a lysis buffer solution with an opaque appearance may be perceived by the end user as damaged, non-homogeneous, or unstable. A need exists for methods and compositions that eliminate the formation of the observed opacity (precipitate). In some embodiments, techniques and compositions are provided that mitigate the formation of precipitates in lysis buffers. Disclosed herein are chemical compositions that, in some embodiments, provide a non-opaque lysis buffer formulation. Without being bound by any particular theory, several technical principles are associated with the non-opaque formulations described herein, including, for example, salt effects, Le Chatelier's principle, solvent polarity, general ionic effects, hydrophilic-lipophilic equilibrium, and colligative properties. In some embodiments, instructions for use are provided for the use of the lysis buffers provided herein, including the composition of the lysis buffer. In some embodiments provided herein, the lysis buffer shows stability during accelerated aging procedures.As described herein, several solution routes were initially considered and evaluated for DNA lysis buffer (DALB) reformulation.As described in the examples, several solution routes were tested to address the challenge of developing a storage stable lysis buffer.

[0032] Disclosed herein are compositions and methods for reducing precipitate formation that may or may not include one or more of the following approaches: (i) replacing anionic detergents with non-ionic detergents; (ii) adding additional metal ions to the lysis buffer; (iii) removing MgSO4 from the lysis buffer; (iv) adding PEG or glycerol to the lysis buffer; (v) increasing glycine in the lysis buffer; (vi) using alternative counter ions (e.g., exchanging MgSO4 for MgX); (vii) using alternative anionic detergents; (viii) increasing the (NH4)2SO4 concentration; (ix) using solvents (e.g., DMSO, alcohol); and (x) using alternative detergents to SDS (e.g., sodium decyl sulfate, sodium octyl sulfate).

[0033] In some embodiments, the methods and compositions provided herein do not include replacing anionic surfactants with non-ionic surfactants. Without being bound by any particular theory, this approach can avoid the charged interaction between Mg cations and SDS, but non-ionic surfactants are unlikely to have sufficient dissolving power and matrix tolerance. In some embodiments, the methods and compositions provided herein do not include the addition of additional metal ions to the lysis buffer. Without being bound by any particular theory, this approach can provide an alternative to Mg ions in SDS by selecting a metal that forms a complex with SDS (such as cobalt(II) sulfate) that is less prone to precipitation, based on data suggesting that a four-fold reduction in SDS is required to reduce precipitation, but it is expected that a significant amount of supplemental ions would be required, which would pose the risk of assay interference. In some embodiments, the methods and compositions provided herein do not include removing MgSO4 from the lysis buffer. Without being bound by any particular theory, this approach may thereby prevent Mg-SDS interactions, but there is a high risk of lyophilizing MgSO4 along with the enzyme.

[0034] In some embodiments, the methods and compositions provided herein do not include adding EGTA or EDTA to chelate Mg ions. In some embodiments, the lysis buffer further comprises a metal chelator (e.g., EDTA or EGTA) to chelate divalent and trivalent cations such as zinc, manganese, nickel, copper, and cobalt ions, which are cofactors for many enzymes, including nucleases and proteases. Without being bound by any particular theory, Mg ions must be available to the enzymes in the reaction, but other divalent and trivalent cations may have deleterious effects on APA, so chelation must be very well balanced as a function of pH and functionality of Mg ions for subsequent amplification.

[0035] In some embodiments, the methods and compositions provided herein do not include adding PEG or glycerol to the lysis buffer. Without being bound by any particular theory, the solvents can aid in dissolving SDS, but PEG brings viscosity concerns and glycerol brings hydrophobicity concerns. In some embodiments, the methods and compositions provided herein do not include increasing glycine in the lysis buffer. Without being bound by any particular theory, this approach can take advantage of the fact that glycine is a weak chelator for Mg at low pH.

[0036] 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 lysis buffers provided herein comprise one or more reducing agents described herein (e.g., dithiothreitol (DTT)), and / or the reagent compositions provided herein comprise one or more protecting agents described herein (e.g., cyclodextrin compounds). In some embodiments, the methods and compositions of the present disclosure allow for isothermal amplification and real-time detection of nucleic acids for direct pathogen lysis in clinical samples without the need to separate or purify samples for point-of-care molecular diagnostics. In some embodiments, a lysis buffer is provided that comprises a strong ionic detergent that can be used to lyse pathogens in clinical samples. The amplification reagents may contain a protectant for the lysis reagent, may be dried (e.g., lyophilized, heat dried) and can be used in a point-of-care setting to amplify the released nucleic acid.

[0037] The methods and compositions provided herein can be applied to other amplification methods that do not involve 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 methods or any nucleic acid (DNA or RNA) amplification or detection methods that require 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.

[0038] In some embodiments, a storage-stable lysis buffer is provided. As used herein, the term "storage-stable" shall be given its ordinary meaning and shall be used to describe a lysis buffer that has an acceptable shelf life of the product in the commercial distribution chain, for example, at least 12 months at a given temperature, preferably at least 24 months at a given temperature. The lysis buffer may be a precipitation-stable aqueous composition. Sufficient stability includes stability in storage such that after an extended period (e.g., 6 months, 12 months, 18 months, and 24 months), the lysis buffer can still be used in the DNA and / or RNA assays described herein without significant loss of signal compared to assays in which the same lysis buffer is freshly prepared. In some embodiments, the lysis buffer comprises one or more surfactants; (NH4)2SO4; and MgSO4, the lysis buffer does not comprise SDS, CTAB, or both, and the formation of precipitates is substantially inhibited over a period of time under storage conditions. In some embodiments, the appearance of precipitates in the lysis buffer does not occur over at least about 20 days during storage conditions. In some embodiments, the lysis buffer comprises one or more detergents; (NH4)2SO4; and MgSO4, where the formation of precipitates is substantially inhibited over a period of time under storage conditions, and the appearance of precipitates in the lysis buffer does not occur over at least about 20 days under storage conditions. In some embodiments, the lysis buffer does not comprise one or more of SDS and CTAB.

[0039] In some embodiments, the precipitate comprises Mg 2+ The lysis buffer may be a precipitation-stable aqueous composition. 2+ The precipitation of complexes consisting of Mg and surfactants can be suppressed. 2+and surfactant complex precipitates can be removed. In some embodiments, the appearance of precipitates in the lysis buffer does not occur during storage conditions for at least about 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 22 months, 28 months, 32 months, 36 months, or any value or range between any two of these values.

[0040] The storage conditions may include transportation of a lysis buffer. The storage conditions may include temperature stress, one or more freeze-thaw cycles, agitation, pressure changes, light exposure, or any combination thereof. The storage conditions may include ambient conditions (e.g., in the range of about 20°C to about 25°C). The storage conditions may include refrigerated conditions (e.g., about 4°C). The storage conditions may include 14°C. The period of time can be at least about 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 22 months, 28 months, 32 months, 36 months, or a value or range between any two of these values.

[0041] MgSO4 is approximately 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, MgSO4 may be present in the lysis buffer at a concentration of about 4 mM. In some embodiments, soluble ... 2+ The concentration of soluble Mg does not decrease by more than about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, or a value or range between any two of these values, compared to the beginning of the period. 2+ Methods for measuring concentration are known to those of skill in the art and include those described in Example 1.

[0042] The amount of precipitation or uniformity of the lysis buffer can be measured using various methods. In some embodiments, precipitation is monitored by using light scattering. In some embodiments, precipitation is monitored using a turbidity sensor, turbidimeter, or nephelometer. In some embodiments, precipitation is monitored using a spectrophotometer. For example, it can be quantitatively measured using light scattering by irradiating the lysis buffer with a spectrophotometer. Or alternatively, uniformity can be qualitatively measured by visually observing the visual clarity of the solution. Substantial inhibition of the formation of precipitates may include lysis buffers with no visible particles when assessed by visual inspection. The appearance of precipitates in the lysis buffer may include lysis buffers with visible particles when assessed by visual inspection.

[0043] The substantial inhibition of the formation of a precipitate may include a lysis buffer having an absorbance at a wavelength below a threshold absorbance as determined by spectrophotometric analysis. The appearance of a precipitate in the lysis buffer may include a lysis buffer having an absorbance at a wavelength above a threshold absorbance as determined by spectrophotometric analysis. The threshold absorbance may be about 0.001, about 0.005, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.4, about 0.6, about 0.8, about 1.0, about 2.0, about 4.0, about 6.0, or a number or range between any two of these values ​​in absorbance units (AU). The appearance of a precipitate in the lysis buffer, as determined by spectrophotometric analysis, may include an absorbance at a wavelength that is about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, or a value or range between any two of these values, greater than the absorbance at the wavelength at the start of the period of time. Substantial inhibition of precipitate formation may include a lysis buffer having an absorbance at a wavelength that is about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, or a value or range between any two of these values, less than the absorbance at that wavelength at the start of the period of time, as determined by spectrophotometric analysis.Spectrophotometric analysis was performed at about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, , about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, about 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm, about 750 nm, about 760 nm, about 770 nm, about 780 nm, about 790 nm, about 800 nm, about 810 nm, about 820 nm, about 830 nm, about 840 nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm, about 890 nm, about 900 nm, or a value or range (or wavelength range) between any two of these values. The wavelength may be the wavelength that maximizes the absorbance of the precipitate. In some embodiments, the spectrophotometric analysis is performed in a wavelength range that exhibits maximum absorbance for the precipitate. The absorbance value may be due to light scattering of the precipitate. The spectrophotometer may be selected from the group consisting of a UV-Vis spectrophotometer, an IR spectrophotometer, a Vis-Near Infrared spectrophotometer, and a Raman spectrophotometer.

[0044] The dissolution buffer may further comprise one or more alcohols. The alcohol present in the dissolution buffer provided herein may vary depending on the embodiment. For example, the carbon chain length of the alcohol may vary. For example, in some embodiments, the alcohol is at least or at most 1, 2, 3, 4, 5, or 6 carbons long. The alcohol is not particularly limited and may be cyclic or acyclic, or saturated or unsaturated. The one or more alcohols may increase the solubility of the surfactant. The one or more alcohols may be selected from the group including ethanol, isopropanol, isobutyl alcohol, pentanol, and hexanol. Alcohols contemplated for use with the lysis buffers provided herein include, but are not limited to, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-1-pentanol, 4-methyl-2-pentanol, 2-ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-methyl-2-pentanol, 2-ethyl-1-butanol ...hexanol, 2-hexanol, 3-methyl-2-pentanol, 2-ethyl-1-butanol, 1-hexanol, 2-hexanol, 3-methyl-2-pentanol, 2-ethyl-1-butanol, 1-hexanol, 2-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol, 3-hexanol -heptanol, 1-octanol, 2-octanol, 2-ethyl-1-hexanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, allyl alcohol, propargyl alcohol, benzyl alcohol, cyclohexanol, 1-methylcyclohexanol, 2-methylcyclohexanol, 3-methylcyclohexanol, 4-methylcyclohexanol, 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,5-pentanediol, and glycerol. The one or more alcohols may comprise the lysis buffer at about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, or any value or range (v / v) between any two of these values. The lysis buffer may further comprise a reducing agent.The reducing agent 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, 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, 89 mM, 90 mM, 91 mM, 92 mM The reducing agent may be present at a concentration of 7 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, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a value or range between any two of these values. In some embodiments, the reducing agent is or comprises cysteine.

[0045] (NH4)2SO4 is approximately 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, It may be present at a concentration of 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, or any value or range between any two of these values. (NH4)2SO4 may be present at a concentration of about 5 mM or about 10 mM, and the appearance of a precipitate in the lysis buffer is delayed by at least about 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 1 year, 2 years, or a value or range between any two of these values, compared to an equivalent lysis buffer in which (NH4)2SO4 is present at a concentration of about 5 mM.

[0046] The lysis buffer may include one or more acids. The acids present in the lysis buffers provided herein may vary depending on the embodiment. The acid may include an organic acid, an inorganic acid, or a mixture thereof. The inorganic acid may be hydrogen chloride (HCl). The inorganic acid may include one or more of hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, phosphinic acid, phosphonic acid, sulfonic acid, sulfuric acid, sulfurous acid, and boric acid. The organic acid may be acetic acid, CHCOOH, CHCOOH, CHCOOH, (COOH)2, 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, carboxylic acid, formic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, glucosamine sulfate, L-threonine acid, camphoric acid, gluconic acid, L-glutamic acid, D-glutamic acid, trifluoroacetic acid, or Ranelic acid. Acid is approximately 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM , 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, or a concentration of any value or range between any two of these values.

[0047] The lysis buffer may include a pH buffering agent (e.g., a buffering agent). The pH buffering agent may be 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, It may be present in the lysis buffer at a concentration of 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, or a value or range between any two of these values. Buffering agents present in the lysis buffers provided herein may vary depending on the embodiment and may include citrate buffer, maleate, phosphate, glycine, glycylglycine, malate, succinate, carbonate, ethanolamine, DIPSO, ADA, imidazole, hydrazine, HEPBS, MES, MOBS, PIPES, EPPS, TAPS, TABS, borate, taurine, N-(2-acetamido)-aminoethanesulfonic acid (ACES), salts of acetic acid (acetate), N-(2-acetamido)-iminodiacetic acid (ADA), 2-aminoethanesulfonic acid, taurine (AE), 2-aminoethanesulfonic acid, ... S), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (Amediol or AMPD), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic 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, Dimethylarsinic acid (Cacodylate), 3-(Cyclohexylamino)-propanesulfonic acid (CAPS), 3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), Sodium carbonate, Cyclohexylaminoethanesulfonic acid (CHES), Salt of citric acid (Citrate), 3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), Salt of formic acid (Formate Salt of formic acid) 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), imidazole, salt of malic acid, salt of maleic acid, 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), salt of phosphoric acid, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,Examples of suitable buffers include N'-bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, salts of succinic acid (succinate salts), 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. The pH buffer may include glycine. The pH buffer may include glycine-HCl. The pH buffer may include about 0.1 mM to 50 mM glycine. The pH buffer may include 10.0 mM glycine. The pH buffer may include about 0 mM to about 20 mM HCl. The pH of the lysis buffer may be from about 1.0 to about 6.0. The pH of the lysis buffer may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or higher. The pH of the lysis buffer may be as high as about 1, 2, 3, 4, or 5. The pH of the lysis buffer may be about 2.2. The concentration of glycine may determine the buffering capacity of the glycine-HCL, and the ratio of glycine / HCl may determine the pH of the buffer.

[0048] In some embodiments, the percentages of the lysis buffer components disclosed herein are provided as %w / w, %m / v, %v / v, %m / w, %w / v, or variations thereof. In some embodiments, the percentages (%w / w, %m / v, %v / v, %m / w, %w / v, or variations thereof) of the lysis buffer components disclosed herein (e.g., one or more surfactants, (NH4)2SO4, MgSO4, acid, alcohol, pH buffer, tween surfactant) in the lysis buffer are about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.01%, 0.02%, 0.0 3%, 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%,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%、1.68%、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.The percentage may be, or may be, 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%, or a number or range between any two of these values. In some embodiments, the percentages of the lysis buffer components disclosed herein (e.g., one or more detergents, (NH4)2SO4, MgSO4, acid, alcohol, pH buffer, tween detergent) are provided as %w / w, %m / v, %v / v, %m / w, %w / v, or variations thereof. In some embodiments, the percentages of the lysis buffer components disclosed herein are described with respect to their final concentration once the lysis buffer is contacted with a sample containing a biological entity. Additionally, in some embodiments, the lysis buffer components disclosed herein are described with respect to a standard concentration of the lysis buffer (e.g., 1x), although the present disclosure also contemplates concentrated versions of the disclosed lysis buffers (e.g., 2x lysis buffer).

[0049] The one or more detergents may comprise about 0.001% (w / v) to about 2.0% (w / v) of the lysis buffer. The one or more detergents may lyse the biological entity to release the sample nucleic acid contained therein. The sample nucleic acid may comprise a sample ribonucleic acid and / or a sample deoxyribonucleic acid. The biological entity may comprise a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof. The biological entity may comprise one or more of a prokaryotic cell, a eukaryotic cell, a virus particle, an exosome, a protoplast, and a microvesicle. The one or more detergents may comprise about 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a value or range (w / v) between any two of these values. The one or more surfactants disclosed herein may include one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The lysis buffer may further include a tween surfactant. The tween surfactant may be selected from the group consisting of Tween 20, Tween 40, Tween 45, Tween 60, Tween 65, Tween 80, Tween 81, and Tween 85. The surfactant may comprise about 0.01% (w / v) to about 1.0% (w / v) of the lysis buffer.

[0050] The one or more detergents may include CTAB. In some embodiments, the lysis buffer comprises about 0.2% (w / v) CTAB and (NH4)2SO4 is present at a concentration of about 5 mM. The one or more detergents may include CTAC. In some embodiments, the lysis buffer comprises about 0.2% (w / v) CTAC and (NH4)2SO4 is present at a concentration of about 5 mM. The one or more detergents may include SDS. In some embodiments, the lysis buffer comprises about 0.4% (w / v) SDS and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the lysis buffer further comprises Tween 80. The one or more detergents may include SDeS. In some embodiments, the lysis buffer comprises about 0.2% (w / v) SDeS and (NH4)2SO4 is present at a concentration of about 5 mM. The one or more detergents may include SDeS. In some embodiments, the lysis buffer comprises about 0.2% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the lysis buffer comprises about 0.4% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. The one or more surfactants may comprise SDeS. The lysis buffer may, for example, comprise about 0.8% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM, or about 0.4% (w / v) SDeS, and (NH4)2SO4 is present at a concentration of about 10 mM. In some embodiments, the lysis buffer further comprises Tween 80. In some embodiments, the lysis buffer comprises about 0.2% (w / v) CTAB, and (NH4)2SO4 is present at a concentration of about 5 mM. In some embodiments, the lysis buffer comprises about 0.2% (w / v) CTAC and (NH4)2SO4 is present at a concentration of about 5 mM. The one or more detergents may comprise sodium octyl sulfate (S Octyl S). In some embodiments, the lysis buffer comprises about 0.2% (w / v) S Octyl S and (NH4)2SO4 is present at a concentration of about 5 mM.

[0051] 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 biological entities with a lysis buffer provided herein to generate a processed sample, where the lysis buffer is capable of dissolving the biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence. The method may include (b) contacting a reagent composition (e.g., wet composition, dry composition) with the processed sample to generate an amplification reaction mixture, where the reagent composition includes one or more amplification reagents. The method may include (c) amplifying the target nucleic acid sequence in the amplification reaction mixture, thereby generating a nucleic acid amplification product. The method may include (d) detecting the nucleic acid amplification product, wherein the detection is performed less than about 20 minutes (e.g., about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 minutes, or any value or range between any two of such values) from the time the reagent composition is contacted with the process sample. In some embodiments, steps (b) and (c) are performed simultaneously (e.g., amplification begins once contact of the reagent composition and the process sample 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).

[0052] 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 hyperthermophile polymerase activity. In some embodiments, the enzyme having hyperthermophile polymerase activity has reverse transcriptase activity. Contacting the reagent composition with the process sample may include dissolving the reagent composition in the process sample. The reagent composition may include one or more of a reverse transcriptase, an enzyme having hyperthermophile polymerase activity, a first primer, a second primer, and a reverse transcription primer. The amplification may be performed under isothermal amplification conditions. Detecting the nucleic acid amplification product may include using a real-time detection method.

[0053] The one or more lysis reagents (e.g., one or more surfactants) may comprise about 0.001% (w / v) to about 1.0% (w / v) (e.g., about 0.2% (w / v)) of the treated sample. The sample nucleic acid may comprise 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.

[0054] 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, the amplification comprising contacting a nucleic acid comprising the target nucleic acid sequence with: i) a first primer and a second primer, where 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 hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, the nucleic acid amplification product comprising: (1) a sequence of the first primer and its reverse complement, (2) a sequence of the second primer and its reverse complement, and (3) a spacer sequence flanked by the (1) sequence of the first primer and its reverse complement and the (2) sequence of the second primer and its reverse complement, the spacer sequence being 1-10 bases in length. In some embodiments, the amplification does not include using an enzyme other than an enzyme having hyperthermophile polymerase activity, and the amplification step does not include denaturing the nucleic acid. In some embodiments, the method does not include contacting the nucleic acid with a single-stranded DNA binding protein prior to or during step (c). In some embodiments, the method does not include thermal or enzymatic denaturation of the sample nucleic acid.

[0055] The nucleic acid may be double-stranded DNA. The nucleic acid may be a product of a reverse transcription reaction. The nucleic acid may be a product of a reverse transcription reaction generated from a sample ribonucleic acid. Step (c) may include generating the nucleic acid by a reverse transcription reaction. The sample nucleic acid may comprise a sample ribonucleic acid. The method may comprise contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA. Amplifying the target nucleic acid sequence comprises the steps of: (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), 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, the amplification comprising: (i) amplifying the dsDNA with a first primer and a second primer, the first primer capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence and the second strand complementary to each other. and (ii) an enzyme having hyperthermophilic polymerase activity, thereby generating 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.

[0056] In some embodiments, the method does not include the use of enzymes other than reverse transcriptase and the enzyme with hyperthermophile polymerase activity. Step (d) may further comprise determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample. In some embodiments, the enzyme with hyperthermophile 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 hyperthermophile polymerase activity may be a polymerase comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the enzyme with hyperthermophile polymerase activity has low or no exonuclease activity. Amplification of the target nucleic acid sequence may 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 long. 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 long. The spacer sequence may contain a portion of the target nucleic acid sequence. The spacer sequence may be 1 to 10 bases long.

[0057] 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 contacted simultaneously with the reverse transcriptase and the enzyme having hyperthermophile polymerase activity. The sample ribonucleic acid may be contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, and the first and second primers. In some embodiments, the sample ribonucleic acid is contacted simultaneously with the reverse transcriptase, the enzyme having hyperthermophile polymerase activity, the first primer, the second primer, and the reverse transcription primer. Reverse transcription of the sample ribonucleic acid may occur by addition of 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 an endogenous poly(A)+ tail at the 3' end of the mRNA. The random hexanucleotide primer can bind to various complementary sites of 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 are reverse transcription primers.

[0058] 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 said two or more target nucleic acid sequences. The two or more target nucleic acid sequences may be specific for two or more different organisms. The two or more different organisms may comprise one or more of SARS-CoV-2, influenza A, influenza B, and / or influenza C.

[0059] The lysis buffer provided herein can be used upstream of various amplification reactions, such as, for example, isothermal amplification reactions. In some embodiments, the amplification comprises one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplification does not comprise one or more of the following amplification methods: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA. In some embodiments, the amplification does not comprise LAMP.

[0060] In some embodiments, the method does not include or does not include one or more of the following: (i) diluting the sample to be processed; (ii) diluting the amplification reaction mixture; (iii) heat denaturing the sample to be processed; (iv) sonicating the sample to be processed; (v) sonicating the amplification reaction mixture; (vi) adding an RNase inhibitor to the sample to be processed; (vii) adding an RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing one or more lysis agents from the sample to be processed or the amplification reaction mixture; (xii) heat and / or enzymatic denaturation of the sample nucleic acid before or during amplification; and (xiii) adding RNase H to the sample to be processed or the amplification reaction mixture. In some embodiments, the sample is held at the amplification temperature (e.g., 67° C.). In some embodiments, a 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.

[0061] In some embodiments, step (a), step (b), step (c), and / or step (d) are performed for a period of about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 2.5 minutes, or about 1 minute. In some embodiments, step (a), step (b), step (c), and / or step (d) comprise sonication, osmotic shock, chemical treatment, heating, or any combination thereof.

[0062] 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. Isothermal amplification compositions and methods are described in WO2017176404, the contents of which are incorporated herein by reference in their entirety.

[0063] Disclosed herein includes a method for amplifying a nucleic acid. In some embodiments, the method includes contacting a sample nucleic acid under isothermal amplification conditions with components including 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 a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the method includes contacting a 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 to a hyperthermophile polymerase, thereby generating a nucleic acid amplification product. In some embodiments, the method includes contacting a 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 activity consisting of i) a hyperthermophile polymerase activity and optionally ii) a reverse transcriptase activity, thereby generating a nucleic acid amplification product.

[0064] Disclosed herein is a method for processing nucleic acids. In some embodiments, the method comprises amplifying a nucleic acid, the amplification essentially consisting of 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 a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product. In some embodiments, the method for processing a nucleic acid comprises amplifying a nucleic acid, the amplification essentially consisting of contacting a sample nucleic acid under isothermal amplification conditions with a) non-enzymatic component comprising at least one oligonucleotide comprising a polynucleotide complementary to a target sequence in the sample nucleic acid, and b) enzymatic component comprising a hyperthermophile polymerase or a polymerase comprising an amino acid sequence at least about 90% identical to a hyperthermophile polymerase, thereby generating a nucleic acid amplification product. In some embodiments, the method for processing nucleic acids comprises amplifying the nucleic acid, the amplification consisting essentially of 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 activity consisting of i) a hyperthermophile polymerase activity and, optionally, ii) a reverse transcriptase activity, thereby generating a nucleic acid amplification product. In some embodiments, the enzymatic activity consists of i) a hyperthermophile polymerase activity, and ii) a reverse transcriptase activity. In some embodiments, the method comprises amplifying the nucleic acid, the amplification consisting essentially of 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 a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product.In some embodiments, the method for processing nucleic acids comprises amplifying nucleic acids, the amplification comprising contacting sample nucleic acids under isothermal amplification conditions with non-enzymatic components comprising a) 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 to a hyperthermophile polymerase, thereby generating nucleic acid amplification products. In some embodiments, the method for processing nucleic acids comprises amplifying nucleic acids, the amplification comprising contacting sample nucleic acids under isothermal amplification conditions with a) non-enzymatic components consisting of 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 generating nucleic acid amplification products.

[0065] Disclosed herein are methods for determining the presence, absence, or amount of a target nucleic acid in a sample nucleic acid. In some embodiments, the method includes the steps of 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, and the amplification comprises 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 being a product of: 1) the first oligonucleotide and the second 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 a nucleic acid amplification product, wherein the detection of the nucleic acid amplification product comprises the use of a real-time detection method and is performed 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.

[0066] Disclosed herein is a kit for determining the presence, absence, or amount of a target nucleic acid in a sample nucleic acid. In some embodiments, the kit comprises: a) components for amplifying a target sequence in a sample nucleic acid under helicase-free isothermal amplification conditions, including i) a first oligonucleotide comprising or consisting of a first polynucleotide that is sequentially complementary to a sequence in a first strand of the target sequence and a second oligonucleotide comprising or consisting of a second polynucleotide that is sequentially complementary to a sequence in a second strand of the target sequence, wherein the first strand and the second strand of the target sequence are complementary to each other; and ii) at least one component that provides a hyperthermophile polymerase activity; and b) at least one component that provides a real-time detection activity for nucleic acid amplification products.

[0067] The enzymatic activity may comprise or consist of one or more of the following: i) hyperthermophile polymerase activity, ii) reverse transcriptase activity, and iii) 3'-5' exonuclease 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 not modified before amplification. In some embodiments, the unmodified sample nucleic acid is from disrupted cells. In some embodiments, the sample nucleic acid comprises DNA. In some embodiments, the sample nucleic acid is 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 may comprise single-stranded nucleic acid, double-stranded nucleic acid, or both. For example, the double-stranded nucleic acid may comprise a first strand and a second strand. In some embodiments, the at least one oligonucleotide comprises a first oligonucleotide and a second oligonucleotide. In some embodiments, the at least one oligonucleotide 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 a first polynucleotide complementary to a target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide complementary to a target sequence in the second strand of the sample nucleic acid. In some embodiments, the first oligonucleotide comprises a first polynucleotide sequentially complementary to a target sequence in the first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide sequentially complementary to a target sequence in the second strand of the sample nucleic acid.In some embodiments, the first oligonucleotide consists of a first polynucleotide that is sequentially complementary to a target sequence in a first strand of the sample nucleic acid, and the second oligonucleotide consists of a second polynucleotide that is sequentially complementary to a target sequence in a second strand of the sample nucleic acid. In some embodiments, the sample nucleic acid is obtained from a subject before amplification. In some embodiments, an unpurified sample nucleic acid is amplified. In some embodiments, a purified sample nucleic acid is amplified. In some embodiments, the method further comprises purifying the sample nucleic acid before amplification.

[0068] In some embodiments, the hyperthermophile polymerase activity is provided by a hyperthermophile polymerase or a functional fragment thereof, or 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 a hyperthermophilic archaeal polymerase or a functional fragment thereof. In some embodiments, the hyperthermophile polymerase activity is provided by a polymerase comprising an 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 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 having low exonuclease activity. In some embodiments, the hyperthermophile polymerase activity is provided by a polymerase having no exonuclease activity. In some embodiments, amplification is carried out at a constant temperature of about 55°C to about 75°C, for example, at a constant temperature of about 55°C to about 65°C or at a constant temperature of about 65°C or at a constant temperature of about 60°C.

[0069] In some embodiments, the nucleic acid amplification product is detectable in 10 minutes or less. In some embodiments, the nucleic acid amplification product comprises a polynucleotide that is contiguous complementary or substantially identical to a target sequence in the sample nucleic acid. In some embodiments, the nucleic acid amplification product consists of a polynucleotide that is contiguous complementary or substantially identical to a target sequence in the sample nucleic acid. In some embodiments, the nucleic acid amplification product is about 20-40 bases in length. In some embodiments, the nucleic acid amplification product comprises i) a first nucleotide sequence that is contiguous complementary or substantially identical to a first polynucleotide of the first oligonucleotide, ii) a second nucleotide sequence that is contiguous complementary or substantially identical to a second polynucleotide of the second oligonucleotide, and iii) a spacer sequence, where the spacer sequence is flanked by the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the nucleic acid amplification product consists of i) a first nucleotide sequence that is contiguous complementary or substantially identical to a first polynucleotide of the first oligonucleotide, ii) a second nucleotide sequence that is contiguous complementary or substantially identical to a second polynucleotide of the second oligonucleotide, and iii) a spacer sequence, where the spacer sequence is flanked by the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the spacer sequence comprises 1-10 bases. In some embodiments, the spacer sequence comprises 1-5 bases. In some embodiments, the spacer sequence is not complementary or identical to the first polynucleotide of the first oligonucleotide and is not complementary or identical to the second polynucleotide of the second oligonucleotide. In some embodiments, the spacer sequence is contiguous complementary or substantially identical to a portion of a target sequence in the sample nucleic acid.

[0070] In some embodiments, the method further comprises detecting the nucleic acid amplification product. In some embodiments, the detection of the nucleic acid amplification product is performed 10 minutes or less from the time the sample nucleic acid is contacted with the component providing a hyperthermophile polymerase activity and the at least one oligonucleotide. In some embodiments, the detection of the nucleic acid amplification product comprises using a real-time detection method. In some embodiments, the detection of 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 (e.g., the detection step) 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, where 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.

[0071] In some embodiments, the enzymatic activity consists of i) hyperthermophile polymerase activity, and ii) reverse transcriptase activity. In some embodiments, the first oligonucleotide comprises a first polynucleotide complementary to a target sequence in a first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide complementary to a target sequence in a second strand of the sample nucleic acid. In some embodiments, the first oligonucleotide comprises a first polynucleotide contiguously complementary to a target sequence in a first strand of the sample nucleic acid, and the second oligonucleotide comprises a second polynucleotide contiguously complementary to a target sequence in a second strand of the sample nucleic acid. In some embodiments, the first oligonucleotide consists of a first polynucleotide contiguously complementary to a target sequence in a first strand of the sample nucleic acid, and the second oligonucleotide consists of a second polynucleotide contiguously complementary to a target sequence in a second strand of the sample nucleic acid.

[0072] In some embodiments, the first oligonucleotide consists essentially of a first polynucleotide that is contiguous to a sequence in the first strand and the second oligonucleotide consists essentially of a second polynucleotide that is contiguous to a sequence in the second strand; and / or the nucleic acid amplification product consists essentially of 1) a first nucleotide sequence that is contiguous complementary to or substantially identical to a first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is contiguous complementary to or substantially identical to a second polynucleotide of the second oligonucleotide, and 3) a spacer sequence comprising 1-10 bases.

[0073] In some embodiments, the first oligonucleotide consists of a first polynucleotide that is contiguous to a sequence in the first strand and the second oligonucleotide consists of a second polynucleotide that is contiguous to a sequence in the second strand; and / or the nucleic acid amplification product consists of 1) a first nucleotide sequence that is contiguous complementary to or substantially identical to a first polynucleotide of the first oligonucleotide, 2) a second nucleotide sequence that is contiguous complementary to or substantially identical to a second polynucleotide of the second oligonucleotide, and 3) a spacer sequence comprising 1 to 10 bases.

[0074] In some embodiments, amplifying comprises contacting the sample nucleic acid under helicase-free and recombinase-free isothermal amplification conditions. In some embodiments, the at least one component providing a hyperthermophile polymerase activity comprises a hyperthermophile polymerase or a functional fragment thereof, or 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 at least one component providing a hyperthermophile polymerase activity consists of a hyperthermophile polymerase or a functional fragment thereof, or a polymerase comprising 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 a reverse transcriptase activity. In some embodiments, the at least one component providing a hyperthermophile polymerase activity further provides a reverse transcriptase activity.

[0075] In some embodiments, the first oligonucleotide consists essentially of a first polynucleotide that is contiguous to a sequence in the first strand of the target sequence, and the second oligonucleotide consists essentially of a second polynucleotide that is contiguous to a sequence in the second strand of the target sequence.In some embodiments, the first oligonucleotide consists essentially of a first polynucleotide that is contiguous to a sequence in the first strand of the target sequence, and the second oligonucleotide consists essentially of a second polynucleotide that is contiguous to a sequence in the second strand of the target sequence. 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 use to carry out the methods provided herein for determining the presence, absence or amount of a target sequence in a sample nucleic acid.

[0076] Some embodiments of the methods and compositions provided herein do not include agents and / or conditions that denature nucleic acids (e.g., promote strand separation and / or promote unwinding) other than acid and / or low pH conditions. Compositions, kits, and methods for nucleic acid detection in which nucleic acid strands are dissociated under low pH conditions (e.g., by contact with an acidic lysis buffer) to facilitate subsequent rapid amplification and detection are described in U.S. Provisional Patent Application No. 63 / 307,085, filed February 5, 2022, and entitled "METHOD FOR SEPARATING GENOMIC DNA FOR SEPARATING GENOMIC DNA FOR AMPLIFICATION OF SHORT NUCLEIC ACID TARGETS," the contents of which are incorporated herein by reference in their entirety.

[0077] 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" can be used interchangeably herein. The terms refer to nucleic acids of any composition, such as 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., including base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in single-stranded or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Nucleic acids may be present 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 a mixed-base and / or deoxyinosine residue. The term nucleic acid can be used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.The term may also include nucleotide analogs, single-stranded polynucleotides ("sense" or "antisense", "plus" or "minus" strands, "forward" or "reverse" reading frames, "forward" or "reverse" strands) 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 the regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product and the regulation of transcription / translation, as well as the 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 the case of RNA, the base thymine is replaced by uracil. The length or size of a nucleic acid may be expressed as the number of bases.

[0078] 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. When 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 target sequence that is single-stranded. When the target nucleic acid is double-stranded, the target nucleic acid generally includes a first strand and a second strand. The first strand and the second strand may be referred to as a forward strand and a reverse strand, and are generally complementary to each other. When the target nucleic acid is single-stranded, the complementary strand may be generated, for example, by polymerization and / or reverse transcription, making the target nucleic acid double-stranded and having a first / forward strand and a second / reverse strand.

[0079] A target sequence may refer to either the sense or antisense strand of a nucleic acid sequence, or to a sequence present in a target nucleic acid, an amplified copy of the original target sequence, or an amplification product. A target sequence may be a subsequence within a larger polynucleotide. The larger polynucleotide may be at least about 2 times (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 10000 times, 1000000 times, 10000000 times, 10000000 times, or any value or range between any of these values) larger in length than the target sequence. For example, a target sequence may be a short sequence (e.g., about 30 bases) within a nucleic acid fragment, chromosome, or plasmid that is targeted for amplification. In some embodiments, a target sequence may refer to a sequence in a target nucleic acid that is complementary to an oligonucleotide (e.g., a primer) used to amplify the nucleic acid. Thus, a target sequence may refer to the entire sequence targeted for amplification, or to a subsequence in a target nucleic acid to which an oligonucleotide binds. An amplification product may be a larger molecule that includes the target sequence as well as at least one other sequence or other nucleotide. In some embodiments, an amplification product is approximately the same length as the target sequence, or exactly the same length as the target sequence. In some embodiments, an amplification product comprises the target sequence. In some embodiments, an amplification product consists of the target sequence.

[0080] 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. Target sequences may include, for example, one or more types of repetitive elements (e.g., multiple repeat sequences, inverted repeat sequences, palindromic sequences, tandem repeats, microsatellites, and minisatellites, etc.). Target nucleic acids may include microRNAs, miRNAs, small interfering RNAs (siRNAs), and small temporal RNAs (stRNAs). In some embodiments, genomic target nucleic acids may be present within a particular genomic locus or multiple genomic loci. Genomic loci 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.

[0081] The nucleic acids used in the methods described herein can be obtained from any suitable biological specimen or sample, and are often isolated from a sample obtained from a subject. The subject may 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), porcines (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 of any age (eg, embryo, fetus, infant, child, adult).

[0082] As used herein, the term "sample" shall be given its ordinary meaning and shall include both biological samples containing nucleic acids and environmental samples. Environmental samples may be obtained from food samples, beverage samples, paper surfaces, fabric surfaces, metal surfaces, wood surfaces, plastic surfaces, soil samples, fresh water samples, wastewater samples, salt water samples, exposure to air or other gas samples, cultures thereof, or any combination thereof. A sample may be any specimen isolated or obtained from a subject or a part thereof (or a culture thereof). Non-limiting examples of specimens include fluids or tissues from a subject, including, but not limited to, blood or blood products (such as, for example, 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), biopsy samples, intestinal puncture samples, 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 tissues (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. The term sample also includes samples that have been manipulated in some way after their procurement, such as by treatment with a reagent, culturing, washing, and / or enriching for a particular cell population (such as cancer cells). A sample may include any sample that contains RNA and / or DNA (e.g., to determine whether a target DNA and / or target RNA is present in a population of RNA and / or DNA). A sample may be derived from any source (e.g., a sample may be a synthetic combination of purified DNA and / or RNA). In some embodiments, a sample is a cell-free liquid sample. In some embodiments, a sample is a liquid sample that may contain cells. A sample may be derived from a patient (e.g., for diagnostic purposes). A sample may be derived from permeabilized cells, crosslinked cells, and / or tissue sections. A sample may be derived from tissue that has been prepared by crosslinking followed by delipidation and adjustment to a uniform refractive index.

[0083] The sample may include nucleic acid from spores, viruses, cells, prokaryotes or eukaryotes, and / or samples containing any 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.

[0084] Nucleic acids can be derived (e.g., isolated, extracted, purified) from one or more samples by methods known in the art. Any suitable method for isolating, extracting, and / or purifying nucleic acids from a biological sample can be used. Nucleic acids can be provided for performing the methods described herein without processing a sample containing nucleic acids. For example, in some embodiments, nucleic acids are provided for performing the amplification methods described herein without prior nucleic acid purification. In some embodiments, target sequences are directly amplified from a sample (e.g., without any nucleic acid extraction, isolation, purification, and / or partial purification steps). In some embodiments, nucleic acids are provided for performing the methods described herein after processing a sample containing nucleic acids. For example, nucleic acids can be extracted, isolated, purified, or partially purified from a sample. The term "isolated" generally refers to a nucleic acid that has been removed from its original environment (e.g., the natural environment if naturally occurring, or a host cell if exogenously expressed) and thus has been altered from its original environment by human intervention (e.g., "by the hand of man"). The term "isolated nucleic acid" can refer to a nucleic acid that has been removed from a subject (e.g., a human subject). It is possible to provide isolated nucleic acids having less non-nucleic acid components (e.g., proteins, lipids, carbohydrates) than the amount of components present in the source sample. A composition comprising an isolated nucleic acid may contain no more than about 50%-99% of non-nucleic acid components. A composition comprising an isolated nucleic acid may contain no more than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or no more than 99% of non-nucleic acid components. The term "purified" generally refers to a nucleic acid when it 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 that contains purified nucleic acid may contain no 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.

[0085] The disclosed methods for detecting a target nucleic acid sequence (e.g., gDNA, dsDNA, dsRNA, etc.) in a sample can detect a target nucleic acid sequence (e.g., DNA or RNA) with high sensitivity. In some embodiments, the disclosed methods can be used to detect a 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 / DNAs are 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 There are one or more copies per non-target DNA / RNA. As used herein, the terms "RNA / DNA" and "RNAs / DNAs" shall be given their ordinary meanings and shall refer to DNA, or RNA, or a combination of DNA and RNA.

[0086] In some embodiments, the detection threshold for the subject method of detecting target RNA / DNA in a sample is 10 nM or less. The term "detection threshold" or "detection limit" shall be given its ordinary meaning and shall describe the minimum amount of target RNA / DNA that must be present in a sample to perform detection. Thus, 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 have a detection threshold of less than or equal to 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, 0.001 nM, 0.0005 nM, 0.0001 nM, 0.00005 nM, 0.00001 nM, 10 pM, 1 pM, 500 fM, 250 fM, 100 fM, 50 fM, 500 aM (attomolar), 250 aM, 100 aM, 50 aM, 10 aM, or 1 aM. In some embodiments, a composition or method of the present disclosure exhibits attomolar (aM), femtomolar (fM), picomolar (pM), and / or nanomolar (nM) detection sensitivity.

[0087] The disclosed samples include 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 includes two or more (e.g., 3, 5, 10, 20, 50, 100, 500, 1000, 5000, or more) sample nucleic acids (e.g., DNA / RNA). The disclosed method can be used as a highly sensitive method 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 includes 5, 10, 20, 25, 50, 100, 500, 10, 20, 25, 30 ... 3 Seeds, 5x10 3 seeds, 10 4 Seeds, 5x10 4 seeds, 10 5 Seeds, 5x10 5 seeds, 10 6 Seeds or 10 7A "sample" may include a target nucleic acid (e.g., a 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.

[0088] The source of the sample may be a diseased (or suspected) cell, fluid, tissue, or organ, or may be a normal (non-diseased) cell, fluid, tissue, or organ. In some embodiments, the source of the sample is a cell, tissue, or organ infected (or suspected) with a pathogen. For example, the source of the sample may be an individual, which may 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, fecal 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. "Helminths" include roundworms, heartworms, and plant-eating nematodes (Nematoda), trematodes (Tematoda), thorny head worms, and cestodes (Taenia). Protozoan infections include infections with 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, Candida albicans, and the like.albicans). Pathogenic viruses include, but are not limited to, immunodeficiency viruses (e.g., HIV), influenza viruses, dengue fever, West Nile virus, herpes viruses, yellow fever viruses, hepatitis C viruses, hepatitis A viruses, hepatitis B viruses, and papilloma viruses. Pathogenic viruses include, but are not limited to, papovaviruses (e.g., HPV, polyomaviruses); 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, pityriasis rosea, and the like. 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, Haemophilus influenzae type B, and the like.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.

[0089] amplification Provided herein are methods for amplifying nucleic acids. In some embodiments, the nucleic acids are amplified using a suitable amplification process. Nucleic acid amplification typically involves enzymatic synthesis of nucleic acid amplicons (copies) that contain sequences complementary to the nucleotide sequence being amplified. In some embodiments, the amplification method is carried out in a single vessel, a single chamber, and / or a single volume (i.e., a continuous volume). In some embodiments, the amplification method and the detection method (e.g., the detection methods described herein) are carried out in a single vessel, a single chamber, and / or a single volume (i.e., a continuous volume).

[0090] 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 one-time, 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 some amplification, but stops the amplification before the exponential phase, typically producing about 500 copies of the desired nucleotide sequence. Preamplification can be used to limit the inaccuracies associated with reactant exhaustion in a particular amplification reaction and can also reduce amplification bias due to target nucleotide sequence or species abundance. In some embodiments, a one-time primer extension can be performed as a pre-step to linear or exponential amplification.

[0091] 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, the 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, hybridized primer-target, or primer-target duplex. The term "near" or "adjacent" when referring to a nucleotide sequence of interest refers 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 is within 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., the 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.

[0092] 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 the like), and a suitable buffer (e.g., a buffer containing a detergent, a reducing agent, monovalent ions, and divalent ions). The amplification reaction may further include one or more of a reverse transcriptase, a reverse transcription primer, and one or more detection agents.

[0093] 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 adenylic acid, guanylic acid, cytidylic acid, thymidylic acid, or uridylic acid. 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, U. Deoxynucleoside triphosphate substrates are referred to as dNTPs, where N can be A, G, C, T, or U. Monomeric nucleotide subunits may be represented herein as A, G, C, T, or U, without specific reference to DNA or RNA. In some embodiments, non-naturally occurring nucleotides or nucleotide analogs can be used, such as analogs that include a detectable label (e.g., a fluorescent or colorimetric label). For example, nucleic acid amplification can be performed using labeled dNTPs, such as dNTPs, where N can be A, G, C, T, or U. 32 P, 33 P, 125 I, or 35It 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, antigens, haptens, or fluorescent dyes. In some embodiments, nucleic acid amplification can be performed in the presence of modified dNTPs, e.g., heat-activated dNTPs (e.g., CleanAmp™ dNTPs from TriLink).

[0094] 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 polymerases or consist of polymerases and reverse transcriptases. 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.

[0095] In some embodiments, the amplification conditions include an enzymatic activity (e.g., an enzymatic activity provided by a polymerase, or an enzymatic activity provided by a polymerase and a reverse transcriptase). In some embodiments, the enzymatic activity does not include an enzymatic activity provided by an enzyme other than a polymerase and / or a reverse transcriptase, such as, for example, a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, and a recombinase. The polymerase activity and the 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). The amplification of nucleic acids may include non-thermal cycling type PCR. In some embodiments, the amplification of nucleic acids includes isothermal amplification processes, such as isothermal polymerase chain reaction (iPCR). Isothermal amplification is generally an amplification process carried out at a constant temperature. Terms such as isothermal conditions, isothermally, and constant temperature generally refer to reaction conditions in which the reaction temperature is kept essentially constant during the course of 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 kept at an essentially constant temperature, meaning that the temperature does not have to be maintained at exactly one temperature. For example, isothermal amplification processes may experience small variations in temperature (e.g., ±1-5°C, etc.) due to environmental or equipment-based variables, etc. In many cases, the entire reaction volume is kept at an essentially constant temperature, and isothermal reactions herein generally do not include amplification conditions that rely on temperature cycling based on temperature gradients and / or convection generated within the reaction vessel.

[0096] 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 about these values, or a value or range between any two of these values. In some embodiments, the temperature element (e.g., heat source) is maintained at an essentially constant temperature, for example, about 75°C or less, about 70 degrees Celsius or less, about 65°C or less, or about 60°C or less.

[0097] 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 such values. In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that denature the nucleic acid. In some embodiments, the nucleic acid target can be amplified without exposure to agents or conditions that promote strand separation during the amplification step (and / or other steps). In some embodiments, the nucleic acid target 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 the nucleic acid 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), etc.

[0098] In some embodiments, the methods disclosed herein do not include heat denaturation (e.g., heating a solution containing nucleic acid to an elevated temperature, such as, for example, 75°C, 80°C, 90°C, or 95°C, or higher) or protein-based (e.g., enzymatic) denaturation of nucleic acid. Protein-based (e.g., enzymatic) denaturation may include contacting the nucleic acid with one or more of a helicase, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, a recombinase, an 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, a topoisomerase, a ligase, an exonuclease, an endonuclease, a restriction enzyme, a nicking enzyme, a recombinase, an 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, dimethylsulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods of the disclosure do not include contacting the nucleic acid with a denaturing agent (e.g., formamide). In some embodiments, the amplification step does not include agents and / or conditions that denature the nucleic acid (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 the nucleic acid (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophile polymerase). In some embodiments, the methods and compositions provided herein do not include agents and / or conditions that denature the nucleic acid (e.g., promote strand separation and / or promote unwinding) other than a polymerase (e.g., a hyperthermophile polymerase) and / or low pH conditions (e.g., contact with acid).

[0099] 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-stranded 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 a helicase are helicase-free amplification conditions.

[0100] The nucleic acid target can be amplified without exposure to a recombinase, including but not limited to Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, T4 uvsX. In some embodiments, the nucleic acid target is amplified without exposure to a recombinase accessory protein, such as a recombinase loading factor (e.g., T4 uvsY). The nucleic acid target can be amplified without exposure to a nucleic acid binding protein (e.g., single-stranded binding protein or single-stranded DNA binding protein (SSB)), such as T4 gp32. In some embodiments, the nucleic acid target is amplified without exposure to a topoisomerase. The nucleic acid target can be amplified with or without exposure to agents or conditions that destabilize the nucleic acid. 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, 17 pages). Destabilization generally does not refer to 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, dimethylsulfoxide (DMSO), or N,N,N-trimethylglycine (betaine). In some embodiments, the methods provided herein include the use of one or more destabilizing agents. In some embodiments, the methods provided herein exclude the use of destabilizing agents. In some embodiments, the nucleic acid target is amplified without exposure to a ligase and / or an RNA replicase.

[0101] In some embodiments, the nucleic acid target can be amplified without cleavage or digestion. For example, the nucleic acid target can be amplified without prior exposure to one or more cleavage agents, and the intact nucleic acid is amplified. In some embodiments, the nucleic acid target is amplified without exposure to one or more cleavage agents during amplification. In some embodiments, the nucleic acid target is 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, sometimes a chemical or an enzyme, that can cleave a nucleic acid at one or more specific or non-specific sites. A specific cleavage agent often specifically cleaves a specific site according to a specific nucleotide sequence. Cleavage agents can include endonucleases (such as, for example, 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.

[0102] 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., RNAseH), 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, hi some embodiments, the nucleic acid is amplified without RNAseH treatment.

[0103] 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 the sample nucleic acid (e.g., the 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.

[0104] 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 a nucleotide sequence of a first strand, for example, in which each base is paired in sequence (e.g., reading from 5' to 3') with a correspondingly ordered base of a second strand, and there are no gaps, additional sequences, or unpaired bases within the sequence considered to be contiguously complementary. In other words, contiguously complementary generally refers to every contiguous base of the nucleotide sequence of the first strand being complementary to the corresponding contiguous base of 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 to be contiguously complementary to a second strand having the sequence 5'-GCATGCATGCAT-3' (SEQ ID NO: 4), and every contiguous base of the first strand is complementary to every corresponding contiguous base of 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 A's) in the middle of the first strand would not pair with the bases of the second strand. A contiguously complementary sequence is 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 comprises a polynucleotide that is contiguously complementary 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 sequences (e.g., at the 5' and / or 3' ends or within the product) that are not contiguous complementary 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, the amplification product will not include products in the form of tandem repeats unless the target sequence includes tandem repeats.

[0105] The nucleic acid amplification product may comprise sequences complementary 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 complementary or identical to a first primer sequence, and a second nucleotide sequence that is contiguous complementary or identical to a second primer sequence.

[0106] The nucleic acid amplification product may include a spacer sequence. As described herein, a spacer sequence in an amplification product is a sequence (one or more bases) that is consecutively complementary 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 or substantially identical to one or more primers used in the amplification reaction. A spacer sequence flanked by sequences in an amplification product is generally located between a first sequence (complementary or substantially identical to a first primer) and a second sequence (complementary or substantially identical to a second primer). Thus, 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 primer. The spacer sequence may be or include about 1 to 10 bases, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. In some embodiments, the nucleic acid amplification product consists or consists essentially of a first nucleotide sequence that is contiguous complementary or identical to the first primer sequence, a second nucleotide sequence that is contiguous complementary or identical to the second primer sequence, and a spacer sequence. In some embodiments, the nucleic acid amplification product does not include any additional sequence that is not contiguous complementary 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 additional sequence incorporated into the amplification product by other mechanisms. In some embodiments, the nucleic acid amplification product generally does not include any additional sequence that is not contiguous complementary 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 additional sequence incorporated into the amplification product by other mechanisms.However, in such embodiments, the nucleic acid amplification product may contain some mismatched (i.e., non-complementary) bases or one extra base (e.g., at the 5' and / or 3' end, or within the product) introduced into the product, for example, by errors or promiscuity in the amplification process.

[0107] The nucleic acid amplification products may be up to 50 bases long, including 10, 15, 20, 25, 30, 35, 40, 45, 50, or any number or range of base lengths between any two of these values. In some embodiments, the nucleic acid amplification products of a given target sequence have the same length or substantially the same length (e.g., within 1-10 bases). Thus, the nucleic acid amplification products of a given target sequence can produce a single signal (e.g., a band in an electrophoretic gel) and generally do not produce multiple signals indicating multiple lengths (e.g., ladders or smears in an electrophoretic gel). In a multiplex reaction, the nucleic acid amplification products of different target sequences may have different lengths.

[0108] 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 amplifying multiple sequences from the same sample, or amplifying one of several sequences in a sample. Multiplex amplification can also refer to the simultaneous or stepwise amplification of one or more sequences present in multiple samples. For example, multiplex amplification can be used to amplify at least two target sequences that are amplifiable (e.g., the amplification reaction includes suitable primers and enzymes to amplify at least two target sequences). In some embodiments, the amplification reaction is arranged 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 suitable primers and enzymes can result in the amplification of one, some, or all of the target sequences. In some embodiments, the amplification reaction is set up to detect two sequences using one 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 different spacer bases or sequences than the target). In some embodiments, the amplification reaction is set up to detect multiple sets of sequences using corresponding primer pairs, each set including a target sequence and a control sequence.

[0109] Primer Nucleic acid amplification is generally carried out in the presence of one or more primers. A primer is generally characterized as an oligonucleotide that comprises 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., a target sequence). A primer can, for example, allow for the specific determination of the nucleotide sequence of a target nucleic acid or the detection of a target nucleic acid or a feature 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 to 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 the substantially lesser recognition, contact, or complex formation of either of those two molecules with the other molecule. 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.

[0110] Primers can be designed and synthesized using any suitable process and may be of any length suitable for hybridizing to a target sequence and performing the amplification process 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 long, 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 long. 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 modifiers), alkynes (e.g., 5' hexynyl, 5-octadiynyl dU), biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, desthiobiotin-TEG), thiol modification (e.g., thiol modifier C3S-S, dithiol, thiol modifier C6S-S)); fluorophores (e.g., Freedom™ dyes, Alexa Fluor® dyes, LI-COR IRDyes®, ATTO™ dyes, rhodamine dyes, WellRED dyes, 6-FAM (azide), Texas Red®-X (NHS ester), Lightcycler® 640 (NHS ester), Dy750 (NHS ester); Iowa Black® dark quencher modifications (e.g., Iowa Black® FQ, Iowa Black® RQ); dark quencher modifications (e.g., Black Hole Quencher®-1, Black Hole Quencher®-2, Dabcyl);Spacers (C3 Spacer, PC Spacer, Hexanediol, Spacer 9, Spacer 18, 1',2'-dideoxyribose (dSpacer); modified bases (e.g., 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, SuperT®, SuperG®, locked nucleic acid (LNA), 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, UNA unlocked nucleic acids (e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dC, Iso-dG, Fluoro-C, Fluoro-U, Fluoro-A, Fluoro-G); phosphorothioate (PS) bond modifications (e.g., phosphorothioated DNA bases, phosphorothioated RNA bases, phosphorothioated 2'O-methyl bases, phosphorothioated LNA bases);and click chemistry modifications. In some embodiments, the modifications and modified bases include uracil bases, ribonucleotide bases, O-methyl RNA bases, PS linkages, 3' phosphate groups, spacer bases (such as C3 spacer or other spacer bases). For example, the primers may include 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 the RNA:RNA duplex and provide stability in the presence of single-stranded ribonucleases and DNases. 2'-O-methyl RNA bases can be included in the primers to, for example, increase stability and binding affinity with the target sequence. In some embodiments, the primers may include one or more phosphorothioate (PS) linkages (e.g., PS linkage modifications). PS linkages replace non-bridging oxygens in the phosphate backbone of the primer with sulfur atoms. This modification typically renders the internucleotide bond resistant to nuclease degradation. PS bonds can be introduced at the 5' or 3' end of the primer, between about the last 3-5 nucleotides, for example, to inhibit exonuclease degradation. In some embodiments, PS bonds included throughout the primer can help reduce attack by endonucleases. The primer can include, for example, a 3' phosphate group. 3' phosphorylation can inhibit degradation by certain 3'-exonucleases, and in certain cases can be used to block extension by DNA polymerase. In some embodiments, the primer includes one or more spacer bases (e.g., one or more C3 spacers). C3 spacer phosphoramidites can be incorporated internally or at the 5' end of the primer. Multiple C3 spacers can be added to either end of the primer, for example, to introduce long chain hydrophilic spacer arms for attachment of fluorophores or other pendant groups.;

[0111] A primer may comprise DNA bases, RNA bases, or both, where one or more of the DNA and RNA bases are 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 an RNA base. In some embodiments, a primer does not comprise a DNA base. 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).

[0112] 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 the hybridization conditions can be varied to allow for various amounts of sequence mismatch. The target sequence and the 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.

[0113] 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 the 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, the amplification of the target sequence is performed using a pair of primers, and the amplification of the target sequence does not include additional primers or oligonucleotides (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, and 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 detection processes that are not considered part of the amplification. In some embodiments, the 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.

[0114] The amplification reaction components may include or consist of a first primer (first oligonucleotide) that is complementary to a target sequence of a first strand (e.g., sense strand, forward strand) of a sample nucleic acid, and a second primer (second oligonucleotide) that is complementary to a target sequence of 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 sequentially complementary to a target sequence of a first strand of a sample nucleic acid, and the second primer (second oligonucleotide) comprises a second polynucleotide that is sequentially complementary to a target sequence of a second strand of a sample nucleic acid. Continuously complementary with respect to primer-target generally refers to a nucleotide sequence of a primer in which each base pairs sequentially with a corresponding sequential base in the target sequence, with no gaps, additional sequences, or unpaired bases in the sequence considered to be continuously 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 tail primers, loop primers, primers capable of forming step-loop structures, hairpin structures, and / or additional sequence that provides a cleavage agent recognition site (e.g., a nicking enzyme recognition site).

[0115] 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 characteristics 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, and the like).

[0116] Polymerase Amplification reaction components (e.g., one or more amplification reagents) may include one or more polymerases. A polymerase is a protein capable of catalyzing the specific incorporation of nucleotides that extend the 3' hydroxyl end of a primer molecule, such as an amplification primer described herein, to 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 is capable of incorporating 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 between any two of these values ​​in a single synthesis.

[0117] 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™ Gamma DNA polymerase; Bst DNA polymerase; Bst DNA polymerase (large fragment); Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I, large (Klenow) fragment; Klenow fragment (3'-5' 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.

[0118] 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.

[0119] In some embodiments, the amplification reaction components include 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 of 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, for example, a detectable nucleic acid amplification method described herein. In some embodiments, the amplification reaction components include a hyperthermophile DNA polymerase that includes 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 that comprises an amino acid sequence that is at least about 90% identical to a Hyperthermophile polymerase or a functional fragment thereof, hi some embodiments, an amplification reaction component comprises a polymerase that comprises an amino acid sequence that is 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.

[0120] The polymerase may have reverse transcriptase ability. In such an embodiment, the amplification reaction may, 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 ability 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 may 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% 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).

[0121] Detection and Quantification The methods described herein may include detecting and / or quantifying the nucleic acid amplification products. The amplification products may be detected and / or quantified by any suitable detection and / or quantification method, including, for example, any of the detection or quantification methods 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's molecular inversion probe (MIP) technology, restriction fragment length polymorphism (RFLP analysis), allele-specific oligonucleotide (ASO) analysis, molecular quantification (MIP) analysis, and other methods. Chilled 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 minisequencing, 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 includes 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 includes 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 directly incorporated into the target sequence or that are incorporated 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 creating (or referencing) a standard curve and / or look-up table for quantification of nucleic acid amplification products.

[0122] The detection of nucleic acid amplification products may include 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 allowing the molecule to function as a specific informative signal. Non-limiting examples of detectable properties include optical properties (e.g., fluorescence), electrical properties, magnetic properties, chemical properties, and time or speed of passage through an aperture of known size. Molecular beacons for detecting nucleic acid molecules may be, for example, hairpin-shaped oligonucleotides 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 of complementary arm sequences located on either side of the probe sequence. The fluorophore and the 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 the quenching molecule are in close proximity to each other, preventing fluorescence resonance energy transfer (FRET). When the molecular beacon encounters a target molecule (e.g., a nucleic acid amplification product), hybridization can occur, and the loop structure is converted into a stable, more rigid conformation, causing the separation of the fluorophore molecule and the quencher molecule, resulting in fluorescence. Because the probe is specific, the generation of fluorescence is generally only due to 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 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 not identical or 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).

[0123] Molecular beacons are highly specific and can distinguish single nucleotide polymorphisms. Molecular beacons can also be synthesized with different color 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 a quantitative amplification process, molecular beacons can specifically bind to the amplified target after each cycle of amplification, and since unhybridized molecular beacons are dark in color, it is not necessary to isolate the probe-target hybrid to quantitatively determine the amount of amplified product. The signal obtained is proportional to the amount of amplified product. Detection using molecular beacons can be performed in real time or as an end-point detection method.

[0124] Detection of nucleic acid amplification products may include using FRET, using surface capture, using 5'-3' exonuclease hydrolysis probes (e.g., TAQMAN), using intercalating and / or binding dyes, and / or using absorbance methods (e.g., colorimetry, turbidity). In some embodiments, detection of nucleic acid amplification products includes using colorimetric detection methods. Any suitable colorimetric detection can be used, non-limiting examples include assays using nanoparticles (e.g., metal nanoparticles, modified nanoparticles, unmodified nanoparticles) and / or peptide nucleic acid (PNA) probes. FRET can be useful, for example, to quantify the molecular dynamics of DNA-DNA interactions, as described for molecular beacons. To monitor the production of a specific product, one end of the probe can be labeled with a donor molecule and the other end with an acceptor molecule. Probe-target hybridization changes the distance or orientation of the donor and acceptor, and a FRET change is observed. Detection of nucleic acid amplification products by using surface capture can be achieved by immobilizing specific oligonucleotides on a surface to create a biosensor that is both highly sensitive and selective. Exemplary surfaces that can be used include gold and carbon, and surface capture methods may use several covalent or non-covalent coupling methods to attach the probe to the surface. Subsequent detection of the target nucleic acid can be monitored by a variety of methods. In some embodiments, detection of nucleic acid amplification products involves the use of dyes that specifically stain nucleic acids. For example, intercalating dyes exhibit enhanced fluorescence when bound to DNA or RNA. Dyes include DNA or RNA intercalating fluorophores, and may include, for example, 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). Dyes provide an opportunity to increase the sensitivity of nucleic acid detection when used with various detection methods.

[0125] In some embodiments, detection of the nucleic acid amplification products includes the use of electrophoresis (e.g., gel electrophoresis, capillary electrophoresis), the use of mass spectrometry, the use of nucleic acid sequencing, and / or the use of digital amplification (e.g., digital PCR). Mass spectrometry methods include, for example, MALDI, MADLI-TOF, or electrospray ionization (ESI-MS). Such methods can be combined with gas chromatography (GC / MS) and liquid chromatography (LC / MS). Mass spectrometry methods (e.g., matrix-assisted laser desorption / ionization mass spectrometry (MALDI MS)) can achieve high throughput due to fast signal acquisition from solid surfaces and automated analysis. The entire or partial sequence of the amplification products can be determined, and the determined nucleotide sequences may be referred to as reads. For example, linear amplification products can be directly analyzed in some embodiments without further amplification (e.g., by using single molecule sequencing methods). In some embodiments, linear amplification products are subjected to further amplification and then analyzed (e.g., using ligation or sequencing by pyrosequencing methods). Any suitable sequencing method can be used to detect and, in some cases, determine the amount of detectable products generated by the amplification methods described herein. Capillary gel electrophoresis (CGE) is a combination of traditional gel electrophoresis and liquid chromatography that uses a medium such as polyacrylamide in a narrow-bore capillary to provide rapid, highly efficient separation of nucleic acid molecules with up to single-base resolution. CGE can be combined with laser-induced fluorescence (LIF) detection, which can detect as few as six molecules of stained DNA. CGE / LIF detection generally involves the use of fluorescent DNA intercalating dyes, including ethidium bromide, YOYO, and SYBR® Green 1, and may also involve the use of fluorescent DNA derivatives in which the fluorescent dye is covalently attached to the DNA. Simultaneous identification of several different target sequences (e.g., products derived from multiplex reactions) can be performed using this method.

[0126] Lysis buffer Solubilizer The lysis buffer provided herein may 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, as a counter ion, I - , Br - , Cl - , or SO4 -2 The lysis buffer may further include EDTA, EGTA, or others as metal ion chelators that form stronger complexes with calcium ions than with heavy metal ions or magnesium ions.

[0127] The anionic surfactant may be selected from 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 its salts, cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, alkyl sulfonates, aryl sulfonates, alkyl phosphates, alkyl sulfonates, stearic acid and its salts, calcium stearate, phosphates, sodium carboxymethylcellulose, dioctyl sulfosuccinate, dialkyl esters of sodium sulfosuccinate, phospholipids, and calcium carboxymethylcellulose.

[0128] Cationic surfactants include, for example, quaternary ammonium compounds, benzalkonium chloride, cetyltrimethylammonium bromide, chitonate, lauryldimethylbenzylammonium chloride, acylcarnitine hydrochloride, alkylpyridinium halide, 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, coconut trimethylammonium chloride, coconut trimethylammonium bromide, coconut methyldihydroxyethylammonium chloride, coconut methyldihydroxyethylammonium bromide, decyltriethylammonium chloride, decyldimethylhydroxyethylammonium chloride, decyldimethylhydroxyethylammonium chloride bromide, C 12-15 -Dimethylhydroxyethylammonium chloride, C 12-15 -Dimethylhydroxyethylammonium chloride bromide, coconut dimethylhydroxyethylammonium chloride, coconut dimethylhydroxyethylammonium bromide, myristyltrimethylammonium methylsulfate, lauryldimethylbenzylammonium chloride, lauryldimethylbenzylammonium bromide, lauryldimethyl(ethenoxy)4 ammonium chloride, lauryldimethyl(ethenoxy)4 ammonium bromide, N-alkyl(C 12-18 ) Dimethylbenzylammonium chloride, N-alkyl(C 14-18 ) Dimethyl-benzyl ammonium chloride, N-tetradecyl dimethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl(C 12-14) dimethyl 1-naphthylmethyl ammonium chloride, trimethyl ammonium halide alkyl-trimethyl ammonium salt, dialkyl-dimethyl ammonium salt, lauryl trimethyl ammonium chloride, ethoxylated alkyl amide alkyl dialkyl ammonium salt, ethoxylated trialkyl ammonium salt, dialkyl benzene dialkyl ammonium chloride, N-didecyl dimethyl ammonium chloride, N-tetradecyl dimethyl benzyl ammonium chloride monohydrate, N-alkyl (C 12-14 ) Dimethyl 1-naphthylmethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, dialkyl benzene alkyl ammonium chloride, lauryl trimethyl ammonium chloride, alkyl benzyl methyl ammonium chloride, alkyl benzyl dimethyl ammonium bromide, C 8-16 Trimethylammonium bromide, C 8-16 Trimethylammonium chloride, C 15 Trimethylammonium bromide, C 17 trimethylammonium bromide, dodecylbenzyltriethylammonium 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.

[0129] The non-ionic surfactant may be, for example, a polyoxyethylene fatty alcohol ether, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene fatty acid ester, a polyoxyethylene alkyl ether, a polyoxyethylene castor oil derivative, a sorbitan ester, a glyceryl ester, glycerol monostearate, a polyethylene glycol, a polypropylene glycol, a polypropylene glycol ester, a cetyl alcohol, a cetostearyl alcohol, a stearyl alcohol, an aryl alkyl polyether alcohol, a polyoxyethylene polyoxypropylene copolymer, a poloxamer, a poloxamine, a methylcellulose, a hydroxycellulose, a hydroxymethylcellulose, a hydroxyethylcellulose, a hydroxypropylcellulose, a hydroxypropylmethylcellulose, a hydroxypropylmethylcellulose phthalate, a non-crystalline cellulose, a polysaccharide, a starch, a starch derivative, a hydroxyethyl starch, a polyvinyl alcohol, a triethanolamine stearate, an amine oxide, a dextran, a glycerol, acacia gum, a cholesterol, a tragacanth, or a polyvinylpyrrolidone.

[0130] The nonionic surfactant may be, for example, an alkyl sulfate, an alkyl sulfonate, a fatty acid soap, a hydroxy fatty acid, a hydroperoxy fatty acid, a polyhydroxy fatty acid, a salt of an epoxy fatty acid, a salt of a mono- and polycarboxylic acid, a prostanoic acid and a prostaglandin, a leukotriene and a lipoxin, an alkyl phosphate, an alkyl phosphonate, a sodium dialkyl sulfosuccinate, an n-alkyl ethoxylated sulfate, a bile salt of a cholate and a deoxycholate, a perfluorocarboxylic acid, a fluoroacliphatic phosphonate, or a fluoroaliphatic sulfate.

[0131] 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 proteins. The strength of a denaturing agent or denaturant will be determined by both the properties and the concentration of the particular denaturing agent or denaturant. A suitable denaturing agent or denaturant may be a chaotrope, a detergent, an organic solvent, a water-miscible solvent, a phospholipid, 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 strong detergents such as SDS or polyoxyethylene ethers (e.g., Tween or Triton detergents), sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(dioleoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents, including, but not limited to, sulfobetaine (Zwittergent), 3-(3-chloramidopropyl)dimethylammonio-1-propanesulfate (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.

[0132] Suitable surfactant levels may be from about 0.1% to about 25%, from about 0.25% to about 10%, or from about 0.5% to about 5% by weight of the total composition. In some embodiments, the surfactant is an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant, and mixtures thereof. In some embodiments, it may be advantageous to use anionic, amphoteric, nonionic, and zwitterionic surfactants (and mixtures thereof). Anionic surfactants useful herein include the water-soluble salts of alkyl sulfates and alkyl ether sulfates having from 10 to 18 carbon atoms in the alkyl radical, and the water-soluble salts of sulfonated monoglycerides of fatty acids having from 10 to 18 carbon atoms. Sodium lauryl sulfate and sodium coconut monoglyceride sulfonate are examples of this type of anionic surfactant. 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 germicides in the compositions disclosed herein.

[0133] Suitable nonionic surfactants that can be used in the compositions, methods, and kits of the present disclosure can be broadly defined as compounds that are produced by 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 materials. Such materials are useful for stabilizing air bubbles without contributing to excessive viscosity increase of consumer product compositions.

[0134] 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, one of the aliphatic substituents contains from about 8 to 18 carbon atoms, and one contains an anionic water-solubilizing group, such as carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of anionic short chain surfactants include alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, saturated or unsaturated fatty acids, and their salts.The moiety containing a polar head group 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 alkyl trimethylammonium halides, alkyl trimethylammonium tosylates, and N-alkyl pyridinium halides.

[0135] Alkyl sulfates can include sodium octyl sulfate, sodium decyl sulfate (SDeS), SDS, and sodium tetra-decyl sulfate. Alkyl sulfonates can include sodium octyl sulfonate, sodium decyl sulfonate, and sodium dodecyl sulfonate. Alkyl benzene sulfonates can include sodium octyl benzene sulfonate, sodium decyl benzene sulfonate, and sodium dodecyl benzene sulfonate. Fatty acid salts can include sodium octanoate, sodium decanoate, sodium dodecanoate, and the sodium salt of oleic acid. The alkyl trimethyl ammonium halides can include octyl trimethyl ammonium bromide, decyl trimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, and CTAB. The alkyl trimethyl ammonium tosylates can include octyl trimethyl ammonium tosylates, decyl trimethyl ammonium tosylates, dodecyl trimethyl ammonium tosylates, myristyl trimethyl ammonium tosylates, and cetyl trimethyl ammonium tosylates. For example, the N-alkyl pyridinium halides can include decyl pyridinium chloride, dodecyl pyridinium chloride, cetyl pyridinium chloride, decyl pyridinium bromide, dodecyl pyridinium bromide, cetyl pyridinium bromide, decyl pyridinium iodide, dodecyl pyridinium iodide, and cetyl pyridinium iodide.

[0136] The cationic surfactant may comprise at least one compound selected from the following: 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, cetri ... ammonium halides, primary polyethoxylated fatty 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, and amine oxides.

[0137] The anionic surfactant may comprise at least one compound selected from 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 alkylalkoxylated)sulfates, mono(C1-C30 alkyl)(ether)phosphates, di(C6-C30 alkyl)(ether)phosphates, (C6-C 30 alkyl) sarcosinate, 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 sulfatesodium 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 tridecyl sulfate, sodium tridecyl sulfate, ammonium tridecyl 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, and SDS.

[0138] Nonionic surfactants include, for example, C6-C18 alkyl alcohols, (C6-C18 alkyl)phenols, (C6-C18 alkyl)ethoxylates, (C6-C18 alkyl)phenols (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)alkylalkanolamides, (C6-C30 alkyl)polysaccharides, sorbitan fatty acid esters, polyoxyethylene ... Bitol esters, polyoxyethylene nonylphenyl ether, polyoxyethylene acid, polyoxyethylene alcohol, coco monoethanolamide, coco diethanolamide, coco diglycoside, (C8-C30 alkyl) polyglycosides, cocamidopropyl, 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(CH2CH2O). 20 (CH2CH(CH3)O) 70 (CH2CH2O) 20 It may contain H.

[0139] Reagent Composition The reagent compositions (e.g., dry compositions) described herein can be provided in a "dry form" or in 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 may 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.

[0140] The dry composition may be frozen or lyophilized or spray dried. The dry composition may be heat dried. The dry composition may include one or more additives (e.g., polymers, sugars, or sugar alcohols). 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, polypeptides, collagen peptides, 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 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 or lyophilized or spray-dried or heat-dried composition, or the aqueous composition for preparing the frozen or lyophilized or spray-dried composition, may include one or more of the following: (i) non-aqueous solvents such as ethylene glycol, glycerol, dimethylsulfoxide, 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.

[0141] kit Disclosed herein includes a kit for detecting a target nucleic acid sequence in a sample. In some embodiments, the kit includes a lysis buffer comprising one or more lysis agents (e.g., one or more detergents), the one or more lysis agents being capable of lysing biological entities to release sample nucleic acids contained therein, the sample nucleic acids being suspected of containing a target nucleic acid sequence. The lysis buffer may include one or more of detergents, (NH4)2SO4, MgSO4, chelating agents, acids, alcohols, pH buffers, and / or tween detergents. The kit may include a reagent composition (e.g., wet composition, dry composition) comprising one or more amplification reagents, the one or more amplification reagents comprising one or more components for amplifying a target nucleic acid sequence under isothermal amplification conditions. In some embodiments, the components include (i) a first primer and a second primer, where the first primer is capable of hybridizing to a sequence of a first strand of a 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 hyperthermophile polymerase activity capable of generating a nucleic acid amplification product.

[0142] The kit may include at least one component that provides real-time detection activity of nucleic acid amplification products. The real-time detection activity may be provided by a molecular beacon. The reagent composition may include a reverse transcriptase and / or a reverse transcription primer.

[0143] The kit may include one or more polymerases and one or more primers, and optionally one or more reverse transcriptases and / or reverse transcription primers, for example as described herein. If one target is being amplified, a pair of primers (forward and reverse) may be included in the kit. If multiple target sequences are being amplified, multiple primer pairs may be included in the kit. The kit may include a control polynucleotide, and if multiple target sequences are being amplified, multiple control polynucleotides may be included in the kit. In some embodiments, the enzyme with hyperthermophile 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 hyperthermophile polymerase activity may be a polymerase that includes the amino acid sequence of SEQ ID NO:1. The nucleic acid amplification product may be about 20 to 40 bases in length. The nucleic acid amplification product may include (1) a first primer sequence and its reverse complement, (2) a second primer sequence and its reverse complement, and (3) a spacer sequence flanked by (1) the first primer sequence and its reverse complement and (2) the second primer sequence and its reverse complement, the spacer sequence being 1 to 10 bases in length.

[0144] 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 viral, bacterial, fungal, or protozoan nucleic acid sequence. The sample nucleic acid may be derived from a virus, a bacterium, a fungus, or a protozoan. The kit may also include 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 one container. In some embodiments, the enzymes (e.g., polymerase and / or reverse transcriptase) may be provided in a separate container 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 the primers are present in a single container in lyophilized or heat-dried form.

[0145] The kit may further include, for example, dNTPs used in the reaction, 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, for example, be 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. EXAMPLES

[0146] 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.

[0147] Example 1 Lysis buffer stability test This example provides the results of a stability test of lysis buffers for use with the disclosed nucleic acid detection methods. The compositions of the initial DNA assay lysis buffer (DALB; Table 2) and RNA assay lysis buffer (RALB; Table 3) are shown below. [Table 1]

[0148] [Table 2] Stability testing and opacity (precipitation) screening was performed at several temperatures. In addition to testing at room temperature (RT), formulations were also aged at 4°C (accelerated temperature) in addition to 14°C (intermediate accelerated temperature) to accelerate screening of candidate formulations. Upon visual inspection for opacity / precipitation, it was found that initial batches of DNA Assay Lysis Buffer experienced rapid precipitation (over several days) when stored at RT and 4°C. A series of test mixtures were prepared and evaluated after mixing to identify the root cause for the observed opacity (precipitation). Without being bound by theory, it is believed that the precipitate is Mg 2+The possibility that the precipitate is a complex of MgSO4 with anionic SDS was explored. Based on the results shown in Table 4, it was determined that the opacity (precipitate) is caused by the interaction of MgSO4 with SDS. These results suggest that the precipitate is Mg 2+ and SDS complex, confirming that precipitation occurs regardless of pH.

[0149] [Table 3] FIG. 2 illustrates the data for the BioAssay colorimetric magnesium assay for fresh DNA assay lysis solution (DALB) as produced and for precipitated DNA assay lysis solution after 7 days (no alarming precipitate). 2+ A significant decrease in the concentration of MgSO4 was noted in the precipitated DNA assay lysis solution only after 7 days. Since MgSO4 is a cofactor for the polymerase, the DNA assay is sensitive to it and this component is not considered an adjustable parameter for stability. As seen in Table 5, a 4-fold reduction in SDS did not improve stability (all other components are at nominal levels). It was therefore reasoned that "small" concentration changes are unlikely to result in significant improvements.

[0150] [Table 4] Without being bound by any particular theory, the relationship between Mg and SDS concentration and precipitation was explored. Dilution of the lysis buffer indicates that a greater than 4-fold dilution is required to maintain opacity (Table 6). It should be noted that this data differs from the experiments in Table 5 in that the complete buffer (not just SDS) is being diluted. This means that a significant reduction in the concentration of Mg / SDS is required for a non-opaque lysis buffer formulation.

[0151] [Table 5]

[0152] Example 2 Evaluation of a first set of techniques for mitigating sedimentation This example provides the results of various procedures and compositions that were tested for reducing precipitate formation in lysis buffer. The effect of varying the order of reagent addition on precipitate formation was tested, with MgSO4 being added last to see if this would alleviate precipitation, however, the permuted protocol was found not to improve stability. The effect of replacing the cationic detergent with SDS was examined. Without being bound by theory, it was explored that this approach may avoid altering the interaction of Mg with SDS. As seen in Table 7, the lysis solution containing CTAC showed good stability across all temperatures, with CTAB not precipitating at 4°C, but better than SDS at 14°C and RT.

[0153] [Table 6] Despite the observed improvement in lysis buffer stability with the use of CTAB, DNA assay performance was found to be negatively affected by CTAB. Substitution of CTAB in the lysis buffer negatively affected Group A Streptococcus assay performance (Figures 3A-3C). Figures 3A-3C illustrate data regarding the effect of CTAB substitution in the lysis buffer on Group A Streptococcus assay performance. Assays using standard lysis buffer (Figure 3A) or lysis solution containing 0.2% CTAB (Figures 3B-3C) are shown. Fluorescence (FAM) versus time (min) is illustrated for assays using 50 cp / reaction. Without being bound by theory, cationic detergents may stabilize double-stranded DNA, and thus targets are less likely to be denatured to allow for an initial priming event.

[0154] The effect of using alternative counter ions (e.g., replacing MgSO4 with MgX) on precipitate formation was tested. Literature suggests that anions affect salting out or salting out of SDS as follows: SO42->OH->F->Cl->NO3->I->SCN-De. However, no significant benefit was observed for MgCl2 or MgI2, as neither improved stability at low temperature (Table 8). MgI2 had to be filtered to remove particles. Note that Mg was assayed and adjusted low for MgI2 after formulation.

[0155] [Table 7] The effect of using an alternative anionic detergent was tested. Literature suggests that sodium 1-dodecanesulfonate complexes less with Mg than SDS and may also bind weaker to Mg ions than SDS. However, 0.2% sodium 1-dodecanesulfonate quickly became turbid when MgSO4 was added and the final lysis buffer quickly precipitated at RT. Thus, sodium 1-dodecanesulfonate is not a preferred option. Next, the use of lithium dodecyl sulfate was considered because it is stable at low temperatures and its use as a lysis agent was suggested by some literature. However, low temperature storage was recommended for LDS in solution and this alternative detergent was not pursued based on the recommendation to store the solution at -20°C.

[0156] Example 3 Evaluation of a second set of techniques for mitigating sedimentation This example provides techniques and compositions that reduce the formation of precipitates in lysis buffers. Increase in ammonium sulfate concentration The effect of increasing (NH4)2SO4 concentration on the stability of the lysis buffer was investigated. Without being bound by any particular theory, this approach may improve the stability of the lysis buffer by the effect of salt based on the principle of Le Chatelier's equilibrium. 5 mM is the nominal (NH4)2SO4 concentration, and increasing (NH4)2SO4 was found to have an effect on opacity at 14° C. and room temperature (Table 9). Examination of the impact of the assay found a benefit of increasing the (NH4)2SO4 concentration. In some embodiments provided herein, increasing (NH4)2SO4 is combined with one or more of the other approaches described herein (e.g., alcohol addition) to increase the stability of the lysis buffer. In some embodiments, such combination-based approaches result in a synergistic improvement in the stability of the lysis buffer.

[0157] [Table 8]

[0158] Solvents that improve SDS solubility The effect of adding solvent (to improve SDS solubility) on the stability of the lysis buffer was investigated. The addition of DMSO resulted in a gradual improvement in reducing precipitate but no stability at 4° C. (Table 10).

[0159] [Table 9]

[0160] Various alcohols were tested for their effect on increasing lipid dissolving power and thus improving SDS solubility. In some embodiments, the optimal assay temperature was not affected by the inclusion of alcohol in the lysis buffer. In some embodiments, the inclusion of alcohol has little or no effect on archaeal polymerase amplification (APA). Addition of ethanol and isopropanol resulted in improved stability of the lysis buffer, although precipitate formation occurred at 4° C. even at high concentrations (Tables 10 and 11, respectively).

[0161] [Table 10]

[0162] [Table 11] Given the slightly better performance observed for isopropanol compared to ethanol (compare Tables 11 and 12), longer chain alcohols were next tested. Inclusion of 3% isobutyl alcohol (IBA) in the lysis buffer significantly improved stability, with lysis buffers containing 4% IBA forming no precipitate under any conditions tested (Table 13). The addition of pentanol was also found to improve the stability of the lysis buffer (Table 14).

[0163] [Table 12]

[0164] [Table 13] Hexanol was found to have difficulty entering the dissolution solution even at low concentrations. Based on the studies described herein, the improvement in alcohol solubility and SDS solubility in water appears to correlate with carbon chain length (e.g., ethanol (2C) < isopropanol (3C) < isobutyl alcohol (4C) < pentanol (5C) < hexanol (6C)), showing increased solubility in water with decreasing carbon chain length, and increased lipid (SDS) solubility with increasing carbon chain length.

[0165] The effect of combining IBA addition with increased (NH4)2SO4 on lysis buffer stability was next investigated. Improved stability for IBA combined with increased (NH4)2SO4 (Table 16) compared to inclusion of IBA alone (Table 15). In some embodiments, the combination of IBA and increased (NH4)2SO4 did not affect downstream assay performance. In some embodiments, increased (NH4)2SO4 allows for increased stability of the lysis buffer at lower alcohol concentrations.

[0166] [Table 14]

[0167] [Table 15] Alternative Surfactants The use of sodium decyl sulfate (SDeS) and sodium octyl sulfate (SOctylS) as alternative surfactants was also tested. Although literature suggests that SDeS and sodium octyl sulfate are not as soluble as SDS, these surfactants were tested at different pH and temperature than the nucleic acid detection methods and compositions described herein. Both of these alternative surfactants resulted in significant improvement in the stability of the lysis buffer compared to SDS (Table 17). In some embodiments, these alternative surfactants did not substantially affect lysis and assay performance compared to SDS.

[0168] [Table 16]

[0169] Example 4 Stable DNA and RNA Assay Lysis Buffer Formulations The compositions of and stability data for the DNA and RNA assay lysis buffer formulations provided herein are shown in Tables 18-19. Substitution of cetyltrimethylammonium chloride (CTAC) with CTAB enhanced the stability of the RNA assay lysis buffer. Substitution of SDeS with SDS and increasing the concentration of (NH4)2SO4 greatly improved the stability of the DNA assay lysis buffer. In some embodiments, Tween 80 (Tw80) was added to enhance assay performance.

[0170] [Table 17]

[0171] [Table 18]

[0172] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0173] With respect to the use of substantially any plural and / or singular terminology herein, one 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 herein and in 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 otherwise indicated.

[0174] 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.). Moreover, those skilled in the art will understand that if a specific number is intended in the introduced claim language, such intent will be expressly recited in the claim, and that in the absence of such recitation, no such intent 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 the claim language. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim containing such an introduced claim recitation to an embodiment containing only one such recitation, even if the same claim 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. In addition, even if a specific number is explicitly recited in an introduced claim recitation, a person skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the basic 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 one 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, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). 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 one 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, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). 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 such terms, or both terms.

[0175] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of every individual member or subgroup of members of the Markush group. As will be appreciated by those of skill in the art, 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 the 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. Also, as will be appreciated by those of skill in the art, all terms such as "up to," "at least," "greater than," and "less than" refer to ranges that are inclusive of the numerical values ​​recited and that can be subsequently divided into subranges as discussed above. Finally, as will be appreciated by those of skill in the art, ranges include each individual member. Thus, for example, a group containing 1-3 items refers to a group containing 1, 2, or 3 items. Similarly, a group containing 1-5 items refers to a group containing 1, 2, 3, 4, or 5 items, etc.

[0176] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are intended to be illustrative and not limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. One or more surfactants, including cetyltrimethylammonium bromide (CTAB); Ammonium sulfate ((NH 4 ) 2 SO 4 ); and Magnesium sulfate (MgSO 4 ) A lysis buffer comprising: The lysis buffer does not contain sodium dodecyl sulfate (SDS), the formation of a precipitate in the lysis buffer is substantially inhibited over a period of time under storage conditions; Optionally, the lysis buffer may contain about 0.2% (w / v) CTAB, and the (NH 4 ) 2 SO 4 may be present at a concentration of about 5 mM.

2. One or more surfactants, including cetyltrimethylammonium chloride (CTAC); Ammonium sulfate ((NH 4 ) 2 SO 4 ); and Magnesium sulfate (MgSO 4 ) A lysis buffer comprising: the lysis buffer does not contain sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), or both; the formation of a precipitate in the lysis buffer is substantially inhibited over a period of time under storage conditions; Optionally, the lysis buffer may contain about 0.2% (w / v) CTAC, and the (NH 4 ) 2 SO 4 may be present at a concentration of about 5 mM.

3. One or more surfactants, including sodium dodecyl sulfate (SDS); Ammonium sulfate ((NH 4 ) 2 SO 4 ); and Magnesium sulfate (MgSO 4 ) A lysis buffer comprising: The lysis buffer does not contain cetyltrimethylammonium bromide (CTAB), the formation of a precipitate in the lysis buffer is substantially inhibited over a period of time under storage conditions; Optionally, the lysis buffer may contain about 0.4% (w / v) SDS, and the (NH 4 ) 2 SO 4 may be present at a concentration of about 10 mM, and optionally, the lysis buffer may further comprise Tween 80.

4. One or more surfactants comprising sodium decyl sulfate (SDeS); Ammonium sulfate ((NH 4 ) 2 SO 4 ); and Magnesium sulfate (MgSO 4 ) A lysis buffer comprising: the lysis buffer does not contain sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), or both; The lysis buffer, wherein the formation of a precipitate in the lysis buffer is substantially inhibited over a period of time under storage conditions.

5. The lysis buffer of claim 4, (a) the lysis buffer contains about 0.2% (w / v) SDeS, and the (NH 4 ) 2 SO 4 is present at a concentration of about 5 mM, (b) the lysis buffer contains about 0.2% (w / v) SDeS, and the (NH 4 ) 2 SO 4 is present at a concentration of about 10 mM, (c) the lysis buffer contains about 0.8% (w / v) SDeS, and the (NH 4 ) 2 SO 4 is present at a concentration of about 10 mM, Or, (d) the lysis buffer contains about 0.4% (w / v) SDeS, and the (NH 4 ) 2 SO 4 is present at a concentration of about 10 mM, and optionally, the lysis buffer may further comprise Tween 80; Lysis buffer as above.

6. One or more surfactants including sodium octyl sulfate (S Octyl S); Ammonium sulfate ((NH 4 ) 2 SO 4 ); and Magnesium sulfate (MgSO 4 ) A lysis buffer comprising: the lysis buffer does not contain sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), or both; the formation of a precipitate in the lysis buffer is substantially inhibited over a period of time under storage conditions; Optionally, the lysis buffer may contain about 0.2% (w / v) S Octyl S, and the (NH 4 ) 2 SO 4 may be present at a concentration of about 5 mM.

7. The lysis buffer according to any one of claims 1 to 6, (a) the appearance of said precipitate in said lysis buffer does not occur during said storage conditions for at least about 20 days; (b) Mg 2+ and the precipitation of the complex consisting of the surfactant is inhibited, and optionally, soluble Mg 2+ may not decrease by more than about 1.1-fold compared to the start of said period of time; (c) the appearance of the precipitate in the lysis buffer does not occur during the storage conditions for at least about 30 days, about 60 days, about 90 days, about 6 months, about 1 year, or about 18 months; (d) the storage conditions include shipping the lysis buffer; (e) the storage conditions are: (i) temperature stress, one or more freeze-thaw cycles, agitation, pressure changes, light exposure, or any combination thereof; (ii) ambient conditions, which may optionally range from about 20°C to about 25°C; (iii) refrigerated conditions, which may optionally be at a temperature of about 4°C; and / or (iv) comprising a temperature of 14°C; (f) the period of time is at least about 30 days, about 60 days, about 90 days, about 6 months, about 1 year, or about 18 months; (g) the substantial inhibition of precipitate formation comprises the lysis buffer having no visible particles, as assessed by visual inspection. and / or (h) the absence of precipitate in the lysis buffer comprises the lysis buffer as assessed by visual inspection; Lysis buffer as above.

8. The lysis buffer according to any one of claims 1 to 6, (a) the lysis buffer further comprises one or more alcohols; Optionally, (i) the one or more alcohols may have a carbon chain length ranging from 1 to 6, and optionally the one or more alcohols may be selected from the group consisting of ethanol, isopropanol, isobutyl alcohol, pentanol, and hexanol; and / or (ii) the lysis buffer may comprise from about 0.001% (v / v) to about 5.0% (v / v) of the one or more alcohols, and optionally from about 0.1% (v / v) to about 4.0% (v / v) of the one or more alcohols; (b) the MgSO 4 may be present at a concentration of about 0.1 mM to about 10 mM, optionally 4 mM; (c) Said (NH 4 ) 2 SO 4 is present at a concentration of about 0.1 mM to about 20 mM, and optionally, the (NH 4 ) 2 SO 4 may be present at a concentration of about 5 mM, (d) Said (NH 4 ) 2 SO 4 is present at a concentration of about 10 mM, and the appearance of the precipitate in the lysis buffer is indicative of the (NH 4 ) 2 SO 4 is present at a concentration of about 5 mM, the lysis buffer is delayed for at least about 10 days. (e) the lysis buffer further comprises an acid at a concentration of about 0.1 mM to about 20 mM, optionally wherein the acid comprises an organic acid, an inorganic acid, or a mixture thereof, and further optionally wherein the inorganic acid is hydrogen chloride (HCl), and optionally wherein the acid is present at a concentration of about 8.8 mM; (f) the lysis buffer further comprises a pH buffer; Optionally, (i) the pH buffer may comprise glycine and an acid, and optionally may comprise 10.0 mM glycine and HCl, and optionally may comprise 8.8 mM glycine and HCl; and / or (ii) the pH of the lysis buffer may be from about 1.0 to about 4.0, and optionally, the pH of the lysis buffer may be about 2.2; (g) the one or more surfactants are capable of dissolving biological entities to release sample nucleic acids contained therein; Optionally, (i) the sample nucleic acid may include sample ribonucleic acid and / or sample deoxyribonucleic acid; (ii) the biological entity may include a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof; and / or (iii) the biological entity may comprise one or more of a prokaryotic cell, a eukaryotic cell, a viral particle, an exosome, a protoplast, and a microvesicle; (h) the one or more surfactants comprise from about 0.001% (w / v) to about 2.0% (w / v) of the lysis buffer; (i) the one or more surfactants comprise a cationic surfactant, an anionic surfactant, a nonionic surfactant, or an amphoteric surfactant; (J) the lysis buffer further comprises a non-ionic detergent, 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 non-ionic detergent may constitute about 0.01% (w / v) of the lysis buffer; and / or (k) the lysis buffer further comprises a reducing agent, and optionally, the reducing agent may be present at a concentration of about 0.1 mM to about 100 mM, and optionally, the reducing agent may be or comprise cysteine; Lysis buffer as above.

9. 1. A method of processing a subject, comprising: (a) contacting a sample containing biological entities with a lysis buffer according to any one of claims 1 to 6 to produce a treated sample, thereby lysing the biological entities and releasing sample nucleic acids contained therein. The above method, comprising:

10. The method according to claim 9, the sample nucleic acid is suspected of containing a target nucleic acid sequence, the method further comprising detecting the target nucleic acid sequence in the sample; Optionally, (a) the target nucleic acid sequence may comprise a first strand and a second strand that are complementary to each other; and / or (b) the target nucleic acid sequence may be a viral, bacterial, fungal, or protozoan nucleic acid sequence, and optionally the sample nucleic acid may be derived from a viral, bacterial, fungal, or protozoan; The above method.

11. The method according to claim 10, Detecting the target nucleic acid sequence in the sample includes: (a) contacting a reagent composition with the process sample to form an amplification reaction mixture, the reagent composition comprising one or more amplification reagents; (b) amplifying a target nucleic acid sequence in the amplification reaction mixture, thereby producing a nucleic acid amplification product; and (c) detecting the nucleic acid amplification products, the step being carried out in less than about 20 minutes from the time the reagent composition contacts the processing sample. The above method, comprising:

12. The method of claim 9, (a) the sample nucleic acid comprises sample ribonucleic acid and / or sample deoxyribonucleic acid, and optionally the sample ribonucleic acid may comprise cellular RNA, mRNA, microRNA, bacterial RNA, viral RNA, or any combination thereof; (b) the sample nucleic acid comprises a nucleic acid comprising the target nucleic acid sequence; Optionally, (i) the nucleic acid may be double-stranded DNA; and / or (ii) the nucleic acid may be the product of a reverse transcription reaction, optionally the nucleic acid may be the product of a reverse transcription reaction generated from a sample ribonucleic acid, and further optionally, step (c) may comprise generating the nucleic acid by a reverse transcription reaction; (c) the sample nucleic acid comprises a sample ribonucleic acid, and the method comprises contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (d) the method is carried out in a single reaction vessel; (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; (f) the biological entity comprises a virus, a bacterium, a fungus, a protozoa, a part thereof, or any combination thereof; and / or (g) the method does not include one or more of: (i) diluting the process sample; (ii) diluting the amplification reaction mixture; (iii) heat-denaturing the process sample; (iv) sonicating the process sample; (v) sonicating the amplification reaction mixture; (vi) adding an RNase inhibitor to the process sample; (vii) adding an RNase inhibitor to the amplification reaction mixture; (viii) purifying the sample; (ix) purifying the sample nucleic acid; (x) purifying the nucleic acid amplification product; (xi) removing the one or more lysing agents from the process sample or the amplification reaction mixture; (xii) heat-denaturing and / or enzymatically denaturing the sample nucleic acid before and / or during amplification; and (xiii) adding RNase H to the process sample or the amplification reaction mixture. The above method.

13. The method of claim 12, comprising: (a) the one or more amplification reagents comprise a reverse transcriptase and / or an enzyme with hyperthermophilic polymerase activity, and optionally, the enzyme with hyperthermophilic polymerase activity may also have reverse transcriptase activity; (b) contacting the reagent composition with the process sample comprises dissolving the reagent composition in the process sample; (c) the reagent composition comprises one or more of a reverse transcriptase, an enzyme having hyperthermophilic polymerase activity, a first primer, a second primer, and a reverse transcription primer; (d) the amplifying step is carried out under isothermal amplification conditions; (e) detecting the nucleic acid amplification products comprises using a real-time detection method; (f) the reagent composition is lyophilized and / or heat-dried and comprises one or more additives, wherein the one or more additives are: amino acid; 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 a polymer, wherein optionally the polymer may comprise polyethylene glycol, dextran, polyvinyl alcohol, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethylcellulose, ficoll, albumin, polypeptides, collagen peptides, or any combination thereof; Including, (g) amplifying the target nucleic acid sequence, The method comprises 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 amplifying step comprises contacting a nucleic acid comprising the target nucleic acid sequence with: i) and ii) a nucleic acid having: 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 the following (1), (2), and (3): (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) a sequence of a first primer and its reverse complement and (2) a sequence of a second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length, and optionally, the spacer sequence may include a portion of the target nucleic acid sequence; (h) the amplifying step does not include using an enzyme other than an enzyme having hyperthermophilic polymerase activity, and optionally the amplifying step may not include thermal and / or enzymatic denaturation of the nucleic acid, and further optionally the method may not include contacting the nucleic acid with a single-stranded DNA binding protein prior to or during step (c); (i) amplifying the target nucleic acid sequence, (c1) contacting the sample ribonucleic acid with a reverse transcriptase and / or a reverse transcription primer to produce cDNA; (c2) contacting the cDNA with an enzyme having hyperthermophile polymerase activity to produce double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, the target nucleic acid sequence comprising a first strand and a second strand that are complementary to each other; (c3) amplifying the target nucleic acid sequence under isothermal amplification conditions, wherein said amplifying comprises contacting the dsDNA with: (i) and (ii) a nucleic acid sequence selected from the group consisting of: (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 the following (1), (2), and (3): (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) a sequence of a first primer and its reverse complement and (2) a sequence of a second primer and its reverse complement, wherein the spacer sequence is 1 to 10 bases in length, and optionally, the spacer sequence may include a portion of the target nucleic acid sequence; Including, Optionally, the method may not include using enzymes other than the reverse transcriptase and the enzyme with hyperthermophile polymerase activity. (j) step (d) further comprises determining the amount of dsDNA and / or nucleic acid comprising the target nucleic acid sequence in the sample; (k) 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; (l) the enzyme having hyperthermophilic polymerase activity has low or no exonuclease activity; (m) the step of amplifying the target nucleic acid sequence is carried out at a constant temperature of about 55°C to about 75°C, and optionally, the step of amplifying the target nucleic acid sequence may be carried out at a constant temperature of about 65°C; (n) the nucleic acid amplification product is about 20 to 40 bases in length; (o) the method further comprises contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product, wherein the signal-generating oligonucleotide comprises a fluorophore, a quencher, or both; (p) detecting the nucleic acid amplification product comprises detecting a fluorescent signal, and optionally, the fluorescent signal may be from a molecular beacon; (q) the amplifying step comprises multiplex amplification of two or more target nucleic acid sequences, and the detecting step comprises multiplex detection of two or more nucleic acid amplification products derived from the two or more target nucleic acid sequences, optionally the two or more target nucleic acid sequences may be specific to two or more different organisms, and optionally the two or more different organisms may comprise one or more of SARS-CoV-2, influenza A, influenza B, and influenza C; (r) 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); and / or (s) the amplifying step does not include one or more of the following: APA, LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA, MDA, RAM, cHDA, SPIA, SMART, 3SR, GEAR, and IMDA; optionally, the amplifying step does not include LAMP; The above method.

14. The method of claim 13, the first primer, the second primer, and / or the reverse transcription primer are about 8 to 16 bases in length, and optionally, the first primer, the second primer, and / or the reverse transcription primer may comprise one or more of DNA bases, modified DNA bases, or a combination thereof.

15. The method of claim 14, the virus is SARS-CoV-2, human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex, herpesvirus 6, herpesvirus 7, Epstein-Barr virus, respiratory syncytial virus (RSV), cytomegalovirus, varicella-zoster virus, JC virus, parvovirus B19, influenza A, influenza B, influenza C, rotavirus, human adenovirus, rubella virus, human enterovirus, genital human papillomavirus (HPV), or hantavirus; The bacteria include Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and the like. pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumoniae pneumonia, S. agalactiae, or Listeria monocytogenes; the fungus is Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, or Trichophyton rubrum; and / or The method of any one of the preceding claims, wherein the protozoan is Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., or Eimeria sp.

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