Methods and compositions for sample processing

Abstract

Provided herein are methods and compositions for processing a target nucleic acid sequence. The methods and compositions provided herein comprise a detergent, a solubilizer, and a cyclodextrin configured to stabilize enzymes and nucleic acids. The processed target nucleic acid sequence can be further contacted with a reaction mixture used in nucleic acid amplification (e.g., isothermal amplification). The processed target nucleic acid sample can be contacted with a stabilization buffer configured to stabilize one or more enzymes in nucleic acid amplification. The processed target nucleic acid sample can be further contacted with an amplification buffer configured to increase the rate of amplification during nucleic acid amplification (e.g., isothermal amplification).

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 580,018, filed September 1, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0002] Nucleic acid amplification techniques, such as polymerase chain reaction (PCR) and various isothermal amplification techniques, have become an indispensable part of nucleic acid-based diagnostics and research. Samples containing target nucleic acid molecules need to be processed before amplification in nucleic acid amplification assays. For example, the target nucleic acid molecules need to be extracted. Optimization of sample processing and preparation can improve the efficiency, accuracy, and yield of nucleic acid amplification techniques. Summary of the Invention

[0003] This article recognizes the need for improved methods and compositions for processing and preparing samples for nucleic acid amplification technologies. The methods and compositions described herein can be used to process samples for downstream applications, such as isothermal amplification, and improve accuracy, efficiency, and / or yield of target nucleic acid molecular products. The sample processing, stabilization, and amplification methods and compositions provided herein can reduce result turnaround time to less than 30 minutes.

[0004] In some aspects, this disclosure provides compositions for sample processing comprising: a detergent, a solubilizer, and a cyclodextrin, wherein the compositions are configured to stabilize enzymes during nucleic acid amplification, and wherein the compositions are configured to reduce and / or eliminate the activity of nucleases that degrade nucleases.

[0005] In some embodiments, the composition is configured to stabilize nucleic acids during the nucleic acid amplification. In some embodiments, the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof. In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

[0006] In some embodiments, the solubilizer is a nonionic surfactant. In some embodiments, the solubilizer is polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, or secondary alcohol polyoxyethylene ether. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or functional variants thereof. In some embodiments, the detergent is part of a pyrolysis buffer. In some embodiments, the solubilizer and the cyclodextrin are part of a recovery buffer. In some embodiments, the pyrolysis buffer and the recovery buffer are in the same mixture.

[0007] In some aspects, this disclosure provides compositions for sample treatment containing buffers, the compositions comprising: a detergent, a solubilizer, and a cyclodextrin, wherein the compositions are configured to stabilize an enzyme during nucleic acid amplification, and wherein the compositions are configured to inactivate a degrading nuclease.

[0008] In some embodiments, the composition is configured to stabilize nucleic acids during the nucleic acid amplification. In some embodiments, the enzyme is a polymerase, endonuclease, reverse transcriptase, or any combination thereof. In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof. In some embodiments, the solubilizer and the cyclodextrin are configured to shorten the cycle threshold or result time in nucleic acid amplification compared to the cycle threshold or result time in nucleic acid amplification of samples with the same other aspects treated by SDS, polysorbate 80, or cyclodextrin alone.

[0009] In some embodiments, the cycle threshold is at most 40 or the result time is at most 15 minutes. In some embodiments, the solubilizer and the cyclodextrin are configured to reduce the coefficient of variation. In some embodiments, the solubilizer and the cyclodextrin are configured to lower the detection limit. In some embodiments, the degrading nuclease is a ribonuclease. In some embodiments, the lysis buffer has a pH of 2 to 9. In some embodiments, the lysis buffer further comprises a chelating agent. In some embodiments, the chelating agent is deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, or N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).

[0010] In some embodiments, the pyrolysis buffer further comprises a reducing agent. In some embodiments, the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP). In some embodiments, the lysis buffer comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP). In some embodiments, the final concentration of EGTA in the lysis buffer in the presence of the sample is from about 0.1 mmol (mM) to 10 mM, the final concentration of EDTA in the lysis buffer in the presence of the sample is from about 0.1 mM to 5 mM, the final concentration of TCEP in the lysis buffer in the presence of the sample is from about 1 mM to 20 mM, or the final concentration of Tris in the lysis buffer in the presence of the sample is from about 1 mM to 60 mM.

[0011] In some embodiments, the composition further comprises an agent capable of reducing disulfide bonds. In some embodiments, the agent capable of reducing the disulfide bonds includes dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME).

[0012] In some embodiments, the detergent is present in the composition mixed with the sample at a final concentration that effectively lyses cells. In some embodiments, the cyclodextrin is present in the composition mixed with the sample at a final concentration that effectively isolates the detergent within the composition. In some embodiments, the detergent is configured to form a complex with the solubilizer and / or the cyclodextrin to stabilize the enzyme. In some embodiments, the cyclodextrin is configured to increase the efficiency of complex formation. In some embodiments, the cyclodextrin has a higher binding affinity for the detergent than the solubilizer. In some embodiments, the final concentration of the detergent is from about 0.1% to 10% w / v (g solute / 100 mL solution). In some embodiments, the final concentration of the cyclodextrin is from about 0.1 mM to 70 mM. In some embodiments, the cyclodextrin includes hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, anionic cyclodextrin, or any combination thereof.

[0013] In some embodiments, the solubilizer is present in the composition mixed with the sample at a final concentration of about 0.1% to 50% w / v. In some embodiments, the final concentration of the solubilizer effectively forms micelles containing the detergent. In some embodiments, the recovery buffer contains a salt. In some embodiments, the recovery buffer does not contain a salt. In some embodiments, the recovery buffer contains a pH buffer. In some embodiments, the recovery buffer does not contain a pH buffer. In some embodiments, the lysis buffer is lyophilized. In some embodiments, the recovery buffer is lyophilized.

[0014] In some embodiments, the composition further comprises a sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample comprises a target nucleic acid molecule for sample processing.

[0015] In some embodiments, the composition further comprises a reaction mixture for nucleic acid amplification. In some embodiments, the reaction mixture is lyophilized. In some embodiments, the reaction mixture comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, or probes. In some embodiments, the composition is configured to stabilize the enzymatic activity of the thermostable enzyme for use during nucleic acid amplification. In some embodiments, the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and any mutants thereof. In some embodiments, the dNTPs include dATP, dCTP, dGTP, dTTP, or dUTP. In some embodiments, the concentration of the dNTPs in the reaction mixture is from about 40 micromoles (µM) to 5000 µM. In some implementations, the primer is at least 4 nucleotides in length.

[0016] In some aspects, this disclosure provides a method for processing a sample, the method comprising mixing the sample with a lysis buffer described herein.

[0017] In some embodiments, the method further includes mixing the sample with the recovery buffer described herein.

[0018] In some aspects, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a lysis buffer containing a detergent; and (b) contacting the sample with a recovery buffer containing a solubilizer and a cyclodextrin, thereby processing the sample to produce a treated sample in a mixture containing the detergent, the solubilizer and the cyclodextrin.

[0019] In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

[0020] In some embodiments, the sample is a biological sample. In some embodiments, the sample is a purified sample. In some embodiments, (a) and (b) occur simultaneously. In some embodiments, the contact of the samples in (a) and (b) is carried out simultaneously in the same mixture. In some embodiments, the method further includes incubating the sample at room temperature for a sustained period of time. In some embodiments, the method further includes heating the sample at a constant temperature for a period of time. In some embodiments, the method further includes heating the sample at a cyclic temperature for a period of time. In some embodiments, the method further includes sonicating the sample. In some embodiments, sonicating the sample occurs before, after, or simultaneously with heating the sample.

[0021] In some aspects, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a pyrolysis buffer comprising a detergent; (b) incubating the sample at a first temperature or temperature range for a first time period; (c) heating the sample at a second temperature or temperature range for a second time period; and (d) contacting the sample with a recovery buffer comprising a solubilizer and a cyclodextrin, thereby processing the sample to generate a treated sample in a mixture comprising the detergent, the solubilizer, and the cyclodextrin.

[0022] In some embodiments, heating the sample in (c) further comprises heating the sample to the second temperature, cooling the sample, and then heating the sample to the second temperature after cooling. In some embodiments, the method further comprises sonicating the sample. In some embodiments, sonicating the sample is performed before, after, or simultaneously with heating the sample. In some embodiments, the method further comprises bead beating the sample. In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

[0023] In some embodiments, the sample is a biological sample. In some embodiments, the sample is a purified sample. In some embodiments, the first temperature or temperature range is about 2°C to 25°C. In some embodiments, the second temperature is about 60°C to 100°C. In some embodiments, the first time period is from at least about 1 minute to at least about 48 hours. In some embodiments, the second time period is from at least about 1 minute to at least about 10 minutes.

[0024] In some embodiments, the lysis buffer further comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

[0025] In some embodiments, the method further includes contacting the treated sample with a reaction mixture after contacting the sample with the recovery buffer. In some embodiments, the reaction mixture contains a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, or probes. In some embodiments, the reaction mixture stabilizes the enzymatic activity of the thermostable enzyme for use during nucleic acid amplification. In some embodiments, the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SDDNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and any mutants thereof. In some embodiments, the dNTPs include dATP, dCTP, dGTP, dTTP, or dUTP. In some embodiments, the concentration of the dNTPs in the reaction mixture is from about 40 µM to 5000 µM. In some embodiments, the primers are at least 4 nucleotides in length.

[0026] In some embodiments, the method further includes performing nucleic acid amplification on the treated sample mixed with the reaction mixture. In some embodiments, the nucleic acid amplification includes polymerase chain reaction (PCR) or isothermal amplification. In some embodiments, the nucleic acid amplification includes thermal cycling of the treated sample. In some embodiments, the nucleic acid amplification includes maintaining the treated sample at a constant temperature for amplification. In some embodiments, the method further includes obtaining the sample from the subject prior to (a). In some embodiments, the subject has or is suspected of having a disease, symptom, or infection. In some embodiments, the sample contains one or more different target nucleic acid molecules.

[0027] In some embodiments, the samples include blood samples, swab samples, saliva samples, urine samples, cerebrospinal fluid samples, pleural fluid samples, rectal samples, vaginal samples, fecal samples, sputum samples, lymph samples, raw emulsions, pasteurized and / or homogenized emulsions, pasteurized and / or treated emulsions, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue samples, cell cultures, purified nucleic acid samples, environmental samples, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof.

[0028] In some embodiments, the time from obtaining the sample to generating the treated sample is equal to or less than about 30 min, 25 min, 20 min, 15 min, 10 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less. In some embodiments, the concentration of one or more different target nucleic acid molecules is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more, compared to the concentration of one or more different target nucleic acid molecules in other aspects of the same sample treated alone with SDS, polysorbate 80, or cyclodextrin.

[0029] In some aspects, this disclosure provides a kit for sample processing, the kit comprising a lysis buffer containing detergent, a recovery buffer containing solubilizer and cyclodextrin, and instructions for use.

[0030] In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof. In some embodiments, the kit further comprises reagents for nucleic acid amplification, including thermostable enzymes, deoxyribonucleoside triphosphates (dNTPs), or primers. In some embodiments, the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and any mutants thereof. In some embodiments, the dNTPs include dATP, dCTP, dGTP, dTTP, or dUTP. In some embodiments, the concentration of the dNTPs in the reaction mixture is from about 40 µM to 5000 µM. In some embodiments, the primers are at least 4 nucleotides in length. In some embodiments, the kit further comprises a probe for detecting the amplification products generated using the kit. In some embodiments, the lysis buffer, the recovery buffer, or the reagent is lyophilized. In some embodiments, the recovery buffer further comprises cucurbituril. In some embodiments, the cucurbituril is cucurbit[n]urea, where n is an integer of 5, 6, 7, 8, or 10.

[0031] In some embodiments, the reaction mixture comprises an excipient. In some embodiments, the excipient comprises one or more agents selected from Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, nonylphenyl alcohol ether-9, trehalose, dextran, polysucrose 400, and cyclodextrin. In some embodiments, the cyclodextrin includes hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, anionic cyclodextrin, or any combination thereof. In some embodiments, the final concentration of Tris in the excipients in the presence of the sample is from about 0.001 mol (M) to 1.0 M; the final concentration of sodium chloride and / or potassium chloride in the presence of the sample is from about 0.0001 M to 0.25 M; the final concentration of EDTA in the excipients in the presence of the sample is from about 0.00001 M to 0.1 M; the final concentration of nonoxynol-9 in the excipients in the presence of the sample is from about 0.01% v / v to 2.0% v / v; the final concentration of trehalose in the excipients in the presence of the sample is from about 0.001 M to 2.0 M; the final concentration of dextran in the excipients in the presence of the sample is from about 0.1% w / v to 10% w / v; and the final concentration of sucrose 400 in the excipients in the presence of the sample is from about 0.01% w / v to 5.0%. w / v; and / or, in the presence of the sample, the final concentration of the cyclodextrin in the excipient is from about 0.001 M to 5.0 M.

[0032] In some embodiments, the excipient further comprises an additional reagent. In some embodiments, the additional reagent includes a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof. In some embodiments, the additional reagent is configured to stabilize the enzyme. In some embodiments, the additional reagent is configured to reduce the Cq value of nucleic acid amplification.

[0033] In some embodiments, the composition further comprises a sample stabilizing buffer. In some embodiments, the sample stabilizing buffer comprises one or more agents selected from collapse modifiers, protein stabilizers, and glass transition modifiers. In some embodiments, the sample stabilizing buffer comprises at least one salt. In some embodiments, the sample stabilizing buffer comprises cyclodextrin. In some embodiments, the sample stabilizing buffer is configured to reconstruct a lyophilized sample. In some embodiments, the stabilizing buffer comprises at least one reducing agent. In some embodiments, the at least one reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP), or any combination thereof.

[0034] In some embodiments, the total time for performing (a) and (b) is at most 1 min, at most 50 seconds, at most 40 seconds, or at most 20 seconds. In some embodiments, the time for processing the sample is the period from the contact in (a) to contacting the treated sample with the reaction mixture, wherein the period is at most 20 seconds. In some embodiments, the nucleic acid amplification generates an amplified treated sample. In some embodiments, the time period for the nucleic acid amplification to generate the amplified treated sample is at most 5 minutes. In some embodiments, the method of processing the sample does not include heating the sample.

[0035] In some aspects, this disclosure provides compositions for sample amplification comprising: a nonionic surfactant, cyclodextrin, and a sucrose / epicochlorohydrin polymer, wherein the compositions are configured to increase the amplification rate during nucleic acid amplification.

[0036] In some embodiments, the composition is configured to stabilize an enzyme during nucleic acid amplification. In some embodiments, the enzyme is a polymerase, endonuclease, reverse transcriptase, or any combination thereof. In some embodiments, the reverse transcriptase is avian myeloid leukemia virus (AMV) reverse transcriptase or murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the nonionic surfactant is nonoxynol-9. In some embodiments, the final concentration of the cyclodextrin in the composition, in the presence of a sample, is from 0.01% v / v to 2.0% v / v. In some embodiments, the cyclodextrin comprises hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof. In some embodiments, the final concentration of the cyclodextrin in the composition, in the presence of a sample, is from about 0.001 M to 10 M.

[0037] In some embodiments, the sucrose / epicochlorohydrin polymer is polysucrose 400. In some embodiments, the final concentration of the sucrose / epicochlorohydrin polymer in the composition in the presence of a sample is about 0.001% to 5% w / v (g solute / 100 mL solution). In some embodiments, the composition further comprises at least one salt. In some embodiments, the final concentration of the at least one salt in the composition in the presence of a sample is about 0.001 mol (M) to 10 M. In some embodiments, the at least one salt is sodium chloride, potassium phosphate, potassium chloride, or any combination thereof. In some embodiments, the composition comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP). In some embodiments, the final concentration of EDTA in the composition in the presence of a sample is from about 0.01 mmol (mM) to 10 mM, and / or the final concentration of Tris in the composition in the presence of a sample is from about 0.1 mM to 25 mM.

[0038] In some embodiments, the composition further comprises an agent capable of reducing disulfide bonds. In some embodiments, the agent capable of reducing the disulfide bonds includes dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME).

[0039] In some embodiments, the composition further comprises at least one sugar and / or sugar alcohol. In some embodiments, the at least one sugar and / or sugar alcohol includes sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the final concentration of the at least one sugar and / or sugar alcohol is about 0.001 M to 10 M or about 0.1% to 10% w / v (g solute / 100 mL solution).

[0040] In some embodiments, the composition further comprises additional reagents. In some embodiments, the additional reagents include a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof. In some embodiments, the composition is lyophilized.

[0041] In some embodiments, the composition further comprises a sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample comprises a target nucleic acid molecule for sample processing.

[0042] In some embodiments, the composition further comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, probes, or any combination thereof. In some embodiments, the composition is configured to stabilize the enzymatic activity of the thermostable enzyme for use during the nucleic acid amplification. In some embodiments, the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and IsoFast. TM Bst and any mutants thereof. In some embodiments, the dNTPs include dATP, dCTP, dGTP, dTTP, or dUTP. In some embodiments, the concentration of the dNTPs in the composition is from about 40 micromoles (µM) to 5000 µM. In some embodiments, the primers are at least 4 nucleotides long. In some embodiments, the probes are at least 15 nucleotides long.

[0043] In some aspects, this disclosure provides compositions comprising: a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose / epicochlorohydrin polymer; and a sample stabilizing buffer configured to stabilize enzymes during nucleic acid amplification.

[0044] In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the solubilizer is a nonionic surfactant. In some embodiments, the solubilizer is polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, or secondary alcohol polyoxyethylene ether. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof. In some embodiments, the detergent is part of a lysis buffer. In some embodiments, the solubilizer and the cyclodextrin are part of a recovery buffer. In some embodiments, the lysis buffer and the recovery buffer are a single mixture in the sample treatment buffer. In some embodiments, the solubilizer and the cyclodextrin are configured to shorten the cycle threshold or result time in nucleic acid amplification compared to the cycle threshold or result time in nucleic acid amplification of samples otherwise identical to those treated with SDS, polysorbate 80, or cyclodextrin alone. In some embodiments, the cycle threshold is up to 40 minutes or the result time is up to 15 minutes.

[0045] In some embodiments, the solubilizer and the cyclodextrin are configured to reduce the coefficient of variation. In some embodiments, the solubilizer and the cyclodextrin are configured to lower the limit of detection.

[0046] In some embodiments, the lysis buffer further comprises a chelating agent. In some embodiments, the chelating agent is deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, or N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN). In some embodiments, the lysis buffer further comprises a reducing agent. In some embodiments, the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP). In some embodiments, the lysis buffer comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

[0047] In some embodiments, the compositions described herein contain EGTA in a lysis buffer at a final concentration of about 0.1 mmol (mM) to 10 mM in the presence of the sample, EDTA in a lysis buffer at a final concentration of about 0.1 mM to 5 mM in the presence of the sample, TCEP in a lysis buffer at a final concentration of about 1 mM to 20 mM in the presence of the sample, or Tris in a lysis buffer at a final concentration of about 1 mM to 60 mM in the presence of the sample.

[0048] In some embodiments, the sample processing buffer further comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME). In some embodiments, the detergent is present in the sample processing buffer mixed with the sample at a final concentration that effectively lyses cells. In some embodiments, the cyclodextrin is present in the sample processing buffer mixed with the sample at a final concentration that effectively isolates the detergent within the composition. In some embodiments, the detergent is configured to form a complex with the solubilizer and / or cyclodextrin to stabilize the enzyme. In some embodiments, the cyclodextrin is configured to increase the efficiency of complex formation. In some embodiments, the final concentration of the detergent is about 0.1% to 10% w / v (g solute / 100 mL solution). In some embodiments, the final concentration of the cyclodextrin is about 0.1 mM to 70 mM.

[0049] In some embodiments, the cyclodextrin includes hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof.

[0050] In some embodiments, the solubilizer is present in the composition mixed with the sample at a final concentration of about 0.1% to 50% w / v. In some embodiments, the final concentration of the solubilizer effectively forms micelles containing the detergent. In some embodiments, the recovery buffer contains a salt. In some embodiments, the recovery buffer contains a pH buffer. In some embodiments, the recovery buffer does not contain a pH buffer. In some embodiments, the sample processing buffer is lyophilized. In some embodiments, the nonionic surfactant of the sample amplification buffer is nonoxynol-9. In some embodiments, the final concentration of the cyclodextrin in the sample amplification buffer in the presence of the sample is from 0.01% v / v to 2.0% v / v. In some embodiments, the cyclodextrin includes hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof. In some embodiments, the final concentration of the cyclodextrin in the sample amplification buffer in the presence of the sample is from about 0.001 M to 10 M.

[0051] In some embodiments, the sucrose / epicochlorohydrin polymer is polysucrose 400. In some embodiments, the final concentration of the sucrose / epicochlorohydrin polymer in the composition in the presence of a sample is about 0.001% to 5% w / v (g solute / 100 mL solution). In some embodiments, the sample amplification buffer further comprises at least one salt. In some embodiments, the final concentration of the at least one salt in the composition in the presence of a sample is about 0.001 mol (M) to 10 M. In some embodiments, the at least one salt is sodium chloride, potassium phosphate, potassium chloride, or any combination thereof.

[0052] In some embodiments, the sample amplification buffer further comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformin, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP). In some embodiments, the final concentration of EDTA in the composition in the presence of the sample is from about 0.01 mmol (mM) to 10 mM, and / or the final concentration of Tris in the composition in the presence of the sample is from about 0.1 mM to 60 mM. In some embodiments, the sample amplification buffer further comprises an agent capable of reducing disulfide bonds. In some embodiments, the agent capable of reducing the disulfide bonds includes dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME). In some embodiments, the sample amplification buffer further comprises at least one sugar and / or sugar alcohol. In some embodiments, the at least one sugar and / or sugar alcohol includes sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the final concentration of the at least one sugar and / or sugar alcohol is about 0.001 M to 10 M or about 0.1% to 10% w / v (g solute / 100 mL solution).

[0053] In some embodiments, the sample amplification buffer further comprises additional reagents. In some embodiments, the additional reagents include a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof. In some embodiments, the sample amplification buffer is lyophilized.

[0054] In some embodiments, the sample amplification buffer further comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, probes, or any combination thereof. In some embodiments, the sample amplification buffer is configured to stabilize the enzymatic activity of the thermostable enzyme for use during the nucleic acid amplification. In some embodiments, the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and IsoFast. TM Bst and any mutants thereof. In some embodiments, the dNTPs include dATP, dCTP, dGTP, dTTP, or dUTP. In some embodiments, the concentration of the dNTPs in the composition is from about 40 micromoles (µM) to 5000 µM. In some embodiments, the primers are at least 4 nucleotides long. In some embodiments, the probes are at least 15 nucleotides long. In some embodiments, the sample stabilizing buffer comprises one or more reagents selected from collapse modifiers, protein stabilizers, and glass transition modifiers.

[0055] In some embodiments, the sample stabilizing buffer comprises at least one salt. In some embodiments, the sample stabilizing buffer comprises cyclodextrin. In some embodiments, the sample stabilizing buffer is configured to reconstruct a lyophilized sample. In some embodiments, the stabilizing buffer comprises at least one reducing agent. In some embodiments, the at least one reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP), or any combination thereof.

[0056] In some embodiments, the composition further comprises a sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample comprises a target nucleic acid molecule for sample processing. In some embodiments, the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

[0057] In some aspects, this disclosure provides a method for amplifying a sample, the method comprising: (a) contacting the sample with a sample processing buffer to generate a processed sample; (b) contacting the processed sample with a sample amplification buffer to provide conditions for nucleic acid amplification; and (c) performing the nucleic acid amplification on the processed sample, wherein the sample processing buffer is not removed prior to the contact in (b).

[0058] In some embodiments, the method further includes contacting the sample with a sample stabilizing buffer to stabilize the enzyme during nucleic acid amplification. In some embodiments, the method further includes contacting the sample with the sample stabilizing buffer prior to (c). In some embodiments, the sample stabilizing buffer and the sample amplification buffer are in the same mixture. In some embodiments, the sample stabilizing buffer is contacted with the sample after the sample has been contacted with the sample amplification buffer. In some embodiments, the method does not include heating the sample. In some embodiments, the sample processing buffer includes a lysis buffer and / or a recovery buffer. In some embodiments, the lysis buffer contains a detergent. In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the recovery buffer contains a solubilizer and cyclodextrin. In some embodiments, the sample processing time is a time period from the contact in (a) to the contact in (b), wherein the time period is at most 20 seconds. In some embodiments, the nucleic acid amplification includes polymerase chain reaction (PCR) or isothermal amplification. In some embodiments, the nucleic acid amplification includes thermal cycling of the sample. In some embodiments, the nucleic acid amplification generates an amplified sample. In some embodiments, the time period from contact with the amplified sample in (a) is at most 5 minutes.

[0059] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample contains one or more different target nucleic acid molecules. In some embodiments, the concentration of the one or more different target nucleic acid molecules is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher than the concentration of one or more different target nucleic acid molecules in other, equally shaped samples treated alone with the sample processing buffer. In some embodiments, the sample processing buffer further comprises cucurbituril.

[0060] In some aspects, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a sample processing buffer to generate a processed sample; (b) contacting the processed sample with a sample amplification buffer to provide conditions for nucleic acid amplification; and (c) performing the nucleic acid amplification on the processed sample in the sample amplification buffer, wherein the time period from contact in (a) to generating the processed sample before contact with the sample amplification buffer is (i) no more than the time for pipetting the sample processing buffer into the sample to mix the sample processing buffer and the sample, or (ii) at most 1 min, at most 50 seconds, at most 40 seconds, or at most 20 seconds.

[0061] In some embodiments, the sample processing buffer comprises a lysis buffer and / or a recovery buffer. In some embodiments, the lysis buffer comprises a detergent. In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the recovery buffer comprises a solubilizer and a cyclodextrin. In some embodiments, the sample processing buffer comprises a detergent, a solubilizer, and a cyclodextrin, wherein the sample processing buffer is configured to stabilize an enzyme during nucleic acid amplification, and wherein the sample processing buffer is configured to reduce and / or eliminate the activity of degrading nucleases. In some embodiments, the sample processing buffer is a composition described herein. In some embodiments, the sample amplification buffer comprises an excipient. In some embodiments, the sample processing buffer is not removed prior to (b). In some embodiments, the sample amplification buffer comprises a reaction mixture described herein, or the sample amplification buffer is a composition described herein. In some embodiments, the method does not include heating the sample.

[0062] In some embodiments, the sample is a biological sample. In some embodiments, the biological sample includes blood samples, swab samples, saliva samples, urine samples, cerebrospinal fluid samples, pleural fluid samples, rectal samples, vaginal samples, fecal samples, sputum samples, lymph samples, raw emulsions, pasteurized and / or homogenized emulsions, pasteurized and / or treated emulsions, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue samples, cell cultures, purified nucleic acid samples, environmental samples, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof. In some embodiments, the blood sample is obtained from an object. In some embodiments, the blood sample is collected in a blood collection tube. In some embodiments, the blood collection tube contains a stabilizer for stabilizing RNA. In some embodiments, the stabilizer contains tetradecyltrimethylammonium oxalate and / or tartaric acid. In some embodiments, the blood sample is contacted with a sample processing buffer without removing the stabilizer. In some embodiments, the blood sample is directly contacted with the sample processing buffer without further treatment before contact with the sample processing buffer. In some embodiments, the blood sample is not treated by centrifugation or spin column before contact with the sample processing buffer. In some embodiments, the sample is lyophilized. In some embodiments, the sample amplification buffer is lyophilized. In some embodiments, the sample processing buffer further comprises cucurbitacin.

[0063] In some aspects, this disclosure provides compositions for sample processing, the compositions comprising: a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; a stabilizer comprising tetradecyltrimethylammonium oxalate and / or tartaric acid, and wherein the compositions are configured to stabilize enzymes during nucleic acid amplification, and wherein the compositions are configured to reduce and / or eliminate the activity of degrading nucleases.

[0064] In some aspects, this disclosure provides compositions for sample processing, the compositions comprising: a sample amplification buffer comprising: a nonionic surfactant, cyclodextrin, and a sucrose / epicochlorohydrin polymer; a stabilizer comprising tetradecyltrimethylammonium oxalate and / or tartaric acid, and wherein the compositions are configured to increase the amplification rate during nucleic acid amplification.

[0065] In some embodiments, the composition does not contain ethanol. In some embodiments, the composition further contains cucurbituril. In some embodiments, the cucurbituril comprises cucurbit[n]urea, where n is an integer of 5, 6, 7, 8, or 10. In some embodiments, the cucurbituril is cucurbit[7]urea. In some embodiments, the composition further comprises a sample. In some embodiments, the sample is a biological sample.

[0066] In some embodiments, the biological sample includes blood samples, swab samples, saliva samples, urine samples, cerebrospinal fluid samples, pleural fluid samples, rectal samples, vaginal samples, fecal samples, sputum samples, lymph samples, raw emulsions, pasteurized and / or homogenized emulsions, pasteurized and / or treated emulsions, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue samples, cell cultures, purified nucleic acid samples, environmental samples, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof.

[0067] In some embodiments, the blood sample is obtained from the subject. In some embodiments, the blood sample is collected in a blood collection tube. In some embodiments, the blood collection tube contains a stabilizer for stabilizing RNA. In some embodiments, the stabilizer contains tetradecyltrimethylammonium oxalate and / or tartaric acid. In some embodiments, the blood sample is contacted with the sample processing buffer without removing the stabilizer. In some embodiments, the blood sample is directly contacted with the sample processing buffer without further treatment before contacting the processing buffer. In some embodiments, the blood sample is not treated by centrifugation or a spin column before contacting the processing buffer.

[0068] In some aspects, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a pyrolysis buffer comprising a detergent, wherein the sample comprises tetradecyltrimethylammonium oxalate and / or tartaric acid, or wherein the sample is directly derived from a sample collection tube; and / or (b) contacting the sample with a recovery buffer comprising a solubilizer and cyclodextrin, thereby processing the sample to generate a treated sample in a mixture comprising the detergent, the solubilizer, and the cyclodextrin.

[0069] In some embodiments, the method further includes contacting the sample with a sample amplification buffer.

[0070] In some aspects, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a sample amplification buffer comprising: a nonionic surfactant, cyclodextrin and a sucrose / epicochlorohydrin polymer, wherein the composition is configured to increase the amplification rate during nucleic acid amplification, and wherein the sample comprises tetradecyltrimethylammonium oxalate and / or tartaric acid, or wherein the sample is directly derived from a sample collection tube.

[0071] In some embodiments, the method further includes contacting the sample with a sample processing buffer before contacting the sample with the sample amplification buffer.

[0072] In some aspects, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a sample processing buffer comprising: a detergent, a solubilizer, and cyclodextrin; (b) contacting the sample with a sample amplification buffer comprising: a nonionic surfactant and cyclodextrin; and (c) contacting the sample with a sample stabilizing buffer configured to stabilize an enzyme in nucleic acid amplification, wherein the sample comprises tetradecyltrimethylammonium oxalate and / or tartaric acid, or wherein the sample is directly derived from a sample collection tube.

[0073] In some embodiments, the sample is not treated with an RNA extraction kit. In some embodiments, the kit comprises a rotating column. In some embodiments, the kit comprises a wash pellet. In some embodiments, the method does not include contact with a wash buffer. In some embodiments, the method does not include membrane-based extraction. In some embodiments, the method further comprises nucleic acid amplification of the sample. In some embodiments, the nucleic acid amplification comprises polymerase chain reaction (PCR) or isothermal amplification. In some embodiments, the nucleic acid amplification comprises thermal cycling of the sample. In some embodiments, the nucleic acid amplification generates an amplified sample. In some embodiments, the time period from the contact in (a) to the generation of the amplified sample is at most 5 minutes. In some embodiments, the sample processing time is the time period from the contact in (a) to the generation of the treated sample before contact with the amplification buffer, wherein the processing time is at most 1 min, at most 50 seconds, at most 30 seconds, or at most 20 seconds. In some embodiments, the method does not include heating the sample. In some embodiments, the sample is a blood sample. In some embodiments, the method further includes obtaining the sample from the object and collecting the sample into the sample collection tube. In some embodiments, the sample processing buffer further comprises cucurbituril.

[0074] Further aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious respects, all without departing from this disclosure. Therefore, the drawings and descriptions should be considered illustrative in nature and not restrictive.

[0075] Incorporation All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated herein by reference. Attached Figure Description

[0076] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of the invention, along with the accompanying drawings (also referred to herein as “Figures”): Figure 1An exemplary scheme depicting a direct sample preparation method is shown. A sample can be provided and brought into contact with a lysis buffer. Optionally, the sample in the lysis buffer can be incubated at room temperature for a period of time. Optionally, the sample can be incubated at a higher temperature (e.g., 95°C, 98°C, 100°C, etc.). A recovery buffer can then be added to the sample in the lysis buffer and mixed to obtain a treated sample. The recovery buffer can be liquid or lyophilized. The treated sample can be mixed with a reaction mixture for nucleic acid amplification. The recovery buffer can be lyophilized together with the reaction mixture.

[0077] Figure 2 The amplification results of different swab samples using the direct sample preparation method are shown. The target RNA sequence of the ribonuclease P protein subunit p30 (RPP30) was amplified by a single reaction.

[0078] Figure 3 This shows the amplification results of a human nasal swab sample prepared using the direct sample preparation method. Neisseria gonorrhoeae (Neisseria gonorrhoeae) Neisseria gonorrhoeae Cultures of Chlamydia trachomatis ( Chlamydia trachomatis The culture and RPP30 sample were subjected to a triple isothermal reaction.

[0079] Figures 4A-4B The amplification results of human nasal swab samples prepared using the direct sample preparation method are shown. Figure 4A The results of the triple isothermal reaction of Neisseria gonorrhoeae RNA, Chlamydia trachomatis RNA, and RPP30 samples were described. Figure 4B The results of the direct method for NTC samples are described.

[0080] Figure 5 The results show amplification of attenuated SARS-CoV-2 virus at approximately 2,000 copies / reaction in NP matrix, direct sample preparation, and triple isothermal reactions using DTECT chemistry (SARS-CoV-2 spike, SARS-CoV-2 NSP2, and RPP30).

[0081] Figure 6 The results show the amplification of synthetic monkeypox DNA (left) and pan-orthozoma (right) at approximately 2,000 copies / reaction in NP matrix, direct sample preparation, and double isothermal reaction (e.g., DTECT chemistry).

[0082] Figure 7 The results of PCR amplification of human buccal cell samples prepared directly from the sample are shown. The triangles represent the standard curve dilution series reactions.

[0083] Figure 8Results of amplification assays of blood samples using lysis buffers and recovery buffers as described herein are shown. Amplification of blood samples was observed after processing with lysis buffers and recovery buffers as described herein without any heating steps.

[0084] Figure 9 Results of 18S RNA amplification using differentially targeted endonuclease chemistry (DTECT) are shown. Amplification was evaluated with cucurbita[7]urea alone, cucurbita[7]urea and γ-cyclodextrin together, or γ-cyclodextrin alone. The combination of cucurbita[7]urea and γ-cyclodextrin showed a lower cycle threshold (Ct).

[0085] Figure 10 IL1RN RNA amplification using DTECT chemistry is shown. Amplification was evaluated with cucurbitacin alone, cucurbitacin and γ-cyclodextrin together, or γ-cyclodextrin alone. Cucurbitacin alone resulted in a lower cycle threshold (Ct).

[0086] Figure 11 MCTP1 RNA amplification using DTECT chemistry is shown. Amplification was evaluated with cucurbitacin alone, cucurbitacin and γ-cyclodextrin together, or γ-cyclodextrin alone. Cucurbitacin alone resulted in a lower cycle threshold (Ct).

[0087] Figure 12 Showing from Figures 9-11 A summary of the results.

[0088] Figure 13 The effect of different Tris concentrations in the recovery buffer on the cycling threshold is shown. Concentrations of 35 mM, 52.5 mM, and 17.5 mM Tris resulted in the lowest Ct values.

[0089] Figure 14 The performance of the DTECT assay using MMLV reverse transcriptase (MMLV RT) is demonstrated. MMLV plays a role in the DTECT assay using different control RNA dilutions.

[0090] Figures 15A-15O Examples of precursor steps leading to isothermal amplification cycles according to various implementation schemes described herein are shown. Figure 15A The double-stranded oligonucleotide complex that binds to the target nucleic acid strand is described. Figure 15B The endonuclease activity on the double-stranded oligonucleotide / target complex was described. Figure 15C The polymerase extending from the 3′ end of the target chain is depicted. Figure 15D The polymerase that replaces the double-stranded guide molecule was described. Figure 15E The endonuclease activity on the oligonucleotide / extension product complex was described. Figure 15F The substitution of polymerase and guide extending from the 3′ end of the cleaved oligonucleotide is described. Figure 15G The activities of endonucleases on novel synthetic moieties complementary to the target strand were described. Figure 15H The substitution of the target strand by polymerase and synthetic complement extending from the 3′ end of the cleavage site is depicted. Figure 15I The replaced complement was described as a novel target for the second complementary strand duplex oligonucleotide complex. Figure 15J The polymerase that replaces the second complementary double-stranded guide molecule was described. Figure 15K The extensions completed on the new guide molecules are depicted. Figure 15L The endonuclease activity on the second complementary oligonucleotide / extension product complex was described. Figure 15M The polymerase extending from the 3' end of the cleavage site of the second complementary strand of the oligonucleotide / extension product complex was described. Figure 15N The endonuclease activity on the complementary strand of a newly synthesized nucleic acid guided by the second complementary strand was described. Figure 15O The substitution of single-stranded synthetic fragments as starting material for chain substitution amplification reactions is described. Detailed Implementation

[0091] definition Although various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0092] Unless otherwise indicated, the practice of some of the methods disclosed herein employs techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols in Molecular Biology series (edited by FM Ausubel et al.); Methods in Enzymology series (Academic Press, Inc.), PCR 2: A Practical Approach (edited by MJ MacPherson, BD Hames, and GR Taylor (1995)), edited by Harlow and Lane (1988); Antibodies, A Laboratory Manual; and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th edition (edited by RI Freshney (2010)) (all of which are incorporated herein by reference).

[0093] Unless the context explicitly indicates otherwise, the singular forms “a / an,” “a type,” and “described” as used herein are intended to include the plural forms as well. Furthermore, with regard to the terms “including / includes,” “having / has / with,” or variations thereof used in the detailed description and / or claims, these terms are intended to indicate inclusion in a manner similar to the term “comprising.”

[0094] The terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” may refer to one or more standard deviations. Alternatively, “about” may refer to a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0095] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include nucleotide analogs. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphate (dNTP) (such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof). Such derivatives can include, for example, [αS]dATP, 7-denitro-dGTP, and 7-denitro-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. Synthetic nucleotide analogs can include locked nucleic acids (LNA), bridging nucleic acids (BNA), fluorinated nucleic acids (also known as fluorine-modified nucleic acids), and peptide nucleic acids (PNA). As used herein, the term “locked nucleic acid” (“LNA”) generally refers to a nucleic acid analog in which the ribose ring is “locked” by an additional bridge connecting the 2′-oxygen atom and the 4′-carbon atom of the nucleotide, such as a methylene bridge (see, for example, WO 99 / 14226, which is incorporated herein by reference in its entirety). As used herein, the term “bridging nucleic acid (BNA)” generally refers to a restricted or inaccessible nucleic acid molecule having a fixed bridge structure at the 2′- or 4′-position. As used herein, “fluorinated nucleic acid” generally refers to a nucleic acid having an incorporated fluorine atom (typically at the 2′- or 4′-position). As used herein, the term “peptide nucleic acid (PNA)” generally refers to a nucleotide analog in which the backbone of the analog (e.g., the sugar backbone in DNA) is a pseudopeptide. The PNA backbone may include, for example, a sequence of repeating N-(2-amino-ethyl)-glycine units. Peptide nucleic acid analogs can react as DNA would in a specified environment and can additionally bind complementary nucleic acid sequences and various proteins. Due to its non-natural backbone, PNA is insensitive to endonuclease cleavage even if endonucleases will cut equivalent DNA / RNA sequences. As used herein, the term "nucleotide" can refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled, such as using portions that include optically detectable parts (e.g., fluorophores). Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0096] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to generally refer to polymeric forms of nucleotides of any length, whether single-stranded, double-stranded, or multi-stranded, whether deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can contain one or more nucleotide analogues (e.g., those with altered backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. Some non-limiting examples of analogues include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, ethylene glycol nucleic acid, threonine, dideoxynucleotide, cordycepin, 7-denitro-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, piracetamidine, wyosine, PNA, and LNA.

[0097] As used herein, the terms “restriction endonuclease,” “restriction enzyme,” or their grammatical equivalents generally refer to enzymes originating from bacterial host defense and are understood to recognize specific sequences on imported viral DNA and cleave the DNA at the recognition sequence or at a unique sequence site. One group of restriction endonucleases has been identified as type IIS. This group can recognize asymmetric DNA sequences and cleave the DNA at a site beyond the cleavage site, at a defined distance from the recognition site. In some cases, type IIS restriction endonucleases cleave DNA 1 to 20 nucleotides from the relevant recognition site.

[0098] As used herein, the term "restriction endonuclease recognition sequence" generally refers to a location on a nucleic acid molecule (e.g., a DNA molecule) containing a specific nucleotide sequence that is recognized by various restriction enzymes. These sequences can range in length from 4–8 base pairs to 12–40 base pairs. These sites can be palindromic sequences.

[0099] As used herein, the term "sample" refers to a substance (e.g., solid or liquid) containing the target nucleic acid sequence to be amplified. The target nucleic acid may be DNA. The target nucleic acid molecule may be RNA. As used herein, "processed sample" refers to a sample that has been contacted with the lysis buffer and / or recovery buffer of this disclosure.

[0100] As used herein, the term "template" typically refers to a portion of the sample's target RNA or DNA that is amplified by DNA polymerase to produce one or more amplified nucleic acid products.

[0101] As used in this article, “amplified product,” “amplified nucleic acid product,” or “amplifier” generally refers to the final product generated from nucleic acid methods such as PCR or isothermal amplification.

[0102] As used herein, the term "polymerase" generally refers to an enzyme that uses a nucleic acid as a template strand to produce a complementary copy of a nucleic acid molecule. DNA polymerases bind to the template strand and then move down the template strand, adding nucleotides to the free hydroxyl group at the 3' end of the growing strand of the nucleic acid. DNA polymerases synthesize complementary DNA molecules from a DNA template (e.g., DNA-dependent DNA polymerase) or an RNA template (e.g., RNA-dependent DNA polymerase or reverse transcriptase), and RNA polymerases synthesize RNA molecules from a DNA template (e.g., DNA-dependent RNA polymerase involved in transcription). DNA polymerases typically initiate strand growth using a short, pre-existing RNA or DNA strand (called a primer). Some DNA polymerases replicate a single-stranded template, while others replace the strand upstream of the site where they add bases to the strand.

[0103] As used herein, the term "strand displacement," when referring to a polymerase, generally refers to the activity of removing the complementary strand that has base-paired with the template strand that the polymerase is reading. Exemplary polymerases with strand displacement activity include large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, and sequencing-grade T7 exo-polymerase.

[0104] As used herein, "primer" or "primer sequence" generally refers to a linear oligonucleotide that is complementary to and annealed to the target sequence. Because very short primers (e.g., less than 5 nucleotides) do not form thermodynamically stable double strands under most hybridization conditions, the lower limit of primer length is determined by hybridization capability. Primer length can vary from 4 to 50 nucleotides. In some embodiments, primer length is between about 10 and 20 nucleotides. In some embodiments, primer length can be more than about 100 nucleotides. In some embodiments, primers can be oligonucleotides capable of hybridizing with the target nucleic acid sequence. In some embodiments, primers can be probes. In some embodiments, primers can include guide oligonucleotides. For example, a primer (e.g., a guide oligonucleotide) can be an oligonucleotide containing a target-binding region that hybridizes with the target polynucleotide sequence and a non-target-binding region that does not hybridize with the target sequence. In some embodiments, the non-target-binding region of the primer can contain a palindromic sequence. In some embodiments, the palindromic sequence can allow for the recruitment of a restriction enzyme to treat the target sequence.

[0105] As used herein, the terms “amplify”, “amplifies”, “amplification” generally refer to any method used to replicate nucleic acids. Replication can be performed using primer-dependent polymerases. Replication can be enzyme-free amplification. In some cases, the amplification or replication of the target nucleic acid strand also includes the replication or amplification of the complementary strand of the target nucleic acid strand. The amplified products can be subjected to subsequence analysis, including but not limited to unwinding curve analysis, nucleotide sequencing, single-strand conformation polymorphism determination, allele-specific oligonucleotide hybridization, Southern blotting analysis, and restriction endonuclease digestion.

[0106] As used herein, the terms “hybridization” and “annealing” generally refer to a reaction in which one or more polynucleotides interact to form a complex that is stabilized via hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-sensitive or specific mechanism. The complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. Hybridization reactions can constitute a step in a broader process, such as the initiation of PCR or the enzymatic cleavage of polynucleotides by ribozymes. A first sequence that can be stabilized via hydrogen bonding with the bases of a second sequence is generally “hybridizable” to the second sequence. In this case, the second sequence may also be hybridizable to the first sequence.

[0107] As used herein, the terms “complement,” “complementary,” and “complementarity” generally refer to a sequence that is completely complementary to a specified sequence and is capable of hybridizing with that specified sequence. In some cases, a first sequence capable of hybridizing with a second sequence or a second sequence group may hybridize specifically or selectively with the second sequence or a second sequence group, making hybridization with the second sequence or a second sequence group possible. Hybridizable sequences may share a degree of sequence complementarity over all or part of their respective lengths, such as complementarity between 25% and 100%, including at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.

[0108] Overview This application provides compositions of sample processing buffers, sample stabilizing buffers, and amplification reaction buffers, kits containing one or more buffers described herein, and methods for using the compositions. The sample processing buffers, sample stabilizing buffers, and amplification reaction buffers can be directly mixed in suitable steps without washing steps or removing the buffers from any previous steps. The compositions, methods, and kits described herein can be used as part of a framework system to enhance the research and development process of samples (e.g., biological samples). The compositions, methods, and kits described herein, and their uses, can be designed to be flexible and adaptable, thereby reducing the time required to develop products.

[0109] The buffer compositions described herein (e.g., lysis buffers, recovery buffers, amplification and / or stabilization buffers) can be multifunctional and can function at various component concentrations.

[0110] The compositions and methods described herein can be used to treat a variety of samples and can function in the presence of any inhibitors that may be present in the sample.

[0111] The compositions described herein may be part of an amplification buffer system, a sample processing buffer system, a stabilizing buffer system, or any combination thereof. An amplification buffer (e.g., a core amplification buffer) may comprise an enzyme, primers and / or probes (e.g., guide oligonucleotides), reverse transcriptase primers, molecular beacons, dNTPs, or any combination thereof. A sample processing buffer (e.g., Sample Direct) may comprise reagents for lysis and / or recovery buffers described herein. A sample processing buffer may comprise salts and / or buffers that can be tuned to optimize amplification reactions (e.g., PCR and / or isothermal amplification). A stabilizing buffer may comprise cyclodextrin, protein stabilizers, pie structure modifiers (Tc, Tg, Tg'), salts, buffers, or any combination thereof. A pie structure modifier may comprise reagents that alter the glass transition temperature (Tg), the glass transition temperature (Tg') of the master mixed solute at maximum freeze concentration before drying, the initial crystallization temperature (Tc), or any combination thereof. One or more pie structure modifiers may increase the critical collapse temperature of the compositions described herein (e.g., sample stabilizing buffers). In some embodiments, one or more pie structure modifiers can enable more efficient (e.g., higher temperature) drying cycles for the compositions described herein (e.g., sample stabilizing buffers). Pie structure modifiers can improve the structural properties of the dried composition (e.g., the dried pie). Enhanced structural properties can make the dried composition (e.g., the dried pie) more resistant to crushing, breakage, cracking, or any combination thereof. In some cases, the glass transition temperature can vary, for example, from about 140 °C to 370 °C. The reagents of the stabilizing buffer can be optimized for freeze-drying.

[0112] Screening assays in representative sample matrices can reduce the risk of downstream sample-assay integration. Consistent drying cycles can be used for immediate research and development applications. The benefits of the compositions, methods, and kits described herein can include (i) reduced time to develop and integrate assays into commercially viable, shelf-stable formulations; (ii) screening and optimization of assays in representative sample matrices (e.g., matrix-based screening, compositions of total sample types); (iii) the ability to lyophilize built-in excipients using compatible lyophilization cycles; and (iv) eliminating the distinction between chemistry (e.g., chemical reagents) intended for immediate operation (e.g., research and development experiments) and chemistry intended for lyophilization. The combination of the compositions, methods, and kits described herein provides greater efficiency in sample handling, stabilization, and amplification.

[0113] The method of using the compositions described herein may be referred to as a “sample direct” preparation method. The method provided herein can rapidly (e.g., up to about 5 minutes, up to about 4 minutes, up to about 3 minutes, up to about 2 minutes, up to about 1 minute, up to about 45 seconds, up to about 30 seconds, up to about 20 seconds or less) process samples and improve amplification reaction performance.

[0114] Compositions for sample processing, stabilization, and amplification This disclosure provides compositions and methods for processing samples containing target nucleic acid molecules for nucleic acid amplification. The target nucleic acid molecules may be DNA and / or RNA.

[0115] In some aspects, this disclosure provides compositions for sample processing in nucleic acid amplification methods. In some embodiments, the composition comprises a detergent, a solubilizer, and a cyclodextrin. Without wishing to be bound by theory, the composition can be configured to stabilize an enzyme during nucleic acid amplification. The composition can also assist in reducing the activity of degrading nucleases during nucleic acid amplification. The composition can eliminate the activity of degrading nucleases during nucleic acid amplification. The composition can degrade nucleases or inactivate nucleases prior to nucleic acid amplification. The composition can be configured to lyse cell walls and / or nuclear membranes.

[0116] In some embodiments, the enzyme stabilized by the compositions provided herein is a polymerase, endonuclease, reverse transcriptase, ligase, helicase, recombinase, or any combination thereof.

[0117] In some embodiments, the nuclease is a ribonuclease. In some embodiments, the ribonuclease includes endonucleases or exonucleases. In some embodiments, the endonucleases include, but are not limited to, RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V, RNase E, and RNase G. In some embodiments, the exonucleases include, but are not limited to, RNase PH, RNase R, RNase D, RNase T, oligonucleotide nucleases, exonucleases I, II, and polynucleotide phosphorylases (e.g., PNP enzymes).

[0118] In some embodiments, the detergent is sodium lauryl sulfate (SDS). In some embodiments, the detergent includes sodium lauryl sulfate (SDS), sodium lauryl sulfate, lithium lauryl sulfate, or functional variants thereof. In some embodiments, the detergent is an ionic detergent. In some embodiments, the detergent is a nonionic detergent. In some embodiments, the detergent is part of a lysis buffer. The lysis buffer is capable of lysing cells but preserving intact nucleic acids (e.g., not denaturing the nucleic acid chains to the point of breaking them down into individual nucleic acids). In some embodiments, the lysis buffer is capable of handling challenging solid and liquid sample types.

[0119] In some embodiments, the detergent is present at a final concentration when mixed with the sample to be treated in the lysis buffer. The detergent may be present at a final concentration that effectively lyses cells in the mixture in the presence of the sample. The concentration of any agent described herein when mixed with the sample to be treated may be referred to as the final concentration. In some embodiments, the concentration (e.g., final concentration) of detergent in the mixture in the presence of the sample is at least about 0.05% w / v (where w / v means g solute / 100 mL solution), at least about 0.1% w / v, at least about 0.15% w / v, at least about 0.2% w / v, at least about 0.25% w / v, at least about 0.3% w / v, at least about 0.35% w / v, at least about 0.4% w / v, at least about 0.45% w / v, at least about 0.5% w / v, at least about 0.55% w / v, at least about 0.6% w / v, at least about 0.65% w / v, at least about 0.7% w / v, at least about 0.75% w / v, at least about 0.8% w / v, at least about 0.85% w / v, at least about 0.9% w / v, at least about 0.95% w / v, and at least about 1.0%. w / v, at least about 2.0% w / v, at least about 3.0% w / v, at least about 4.0% w / v, at least about 5.0% w / v, at least about 6.0% w / v, at least about 7.0% w / v, at least about 8.0% w / v, at least about 9.0% w / v, or at least about 10.0% w / v.

[0120] In some embodiments, the concentration (e.g., final concentration) of detergent in the mixture in the presence of the sample is at most about 10.0% w / v, at most about 9.0% w / v, at most about 8.0% w / v, at most about 7.0% w / v, at most about 6.0% w / v, at most about 5.0% w / v, at most about 4.0% w / v, at most about 3.0% w / v, at most about 2.0% w / v, at most about 1.0% w / v, at most about 0.95% w / v, at most about 0.9% w / v, at most about 0.85% w / v, at most about 0.8% w / v, at most about 0.75% w / v, at most about 0.7% w / v, at most about 0.65% w / v, at most about 0.6% w / v, at most about 0.55% w / v, or at most about 0.5%. w / v, up to about 0.45% w / v, up to about 0.4% w / v, up to about 0.35% w / v, up to about 0.3% w / v, up to about 0.25% w / v, up to about 0.2% w / v, up to about 0.15% w / v, up to about 0.1% w / v, or up to about 0.05% w / v.

[0121] In some embodiments, the concentration of detergent in the mixture (e.g., final concentration) in the presence of the sample is about 0.1% w / v to about 2% w / v. In some embodiments, the concentration (e.g., final concentration) of detergent in the mixture in the presence of the sample is about 0.1% w / v to about 0.2% w / v, about 0.1% w / v to about 0.3% w / v, about 0.1% w / v to about 0.4% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.6% w / v, about 0.1% w / v to about 0.7% w / v, about 0.1% w / v to about 0.8% w / v, about 0.1% w / v to about 0.9% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 1.5% w / v, about 0.1% w / v to about 2% w / v, about 0.2% w / v to about 0.3% w / v, about 0.2% w / v to about 0.4% w / v, or about 0.2%. w / v to about 0.5% w / v, about 0.2% w / v to about 0.6% w / v, about 0.2% w / v to about 0.7% w / v, about 0.2% w / v to about 0.8% w / v, about 0.2% w / v to about 0.9% w / v, about 0.2% w / v to about 1% w / v, about 0.2% w / v to about 1.5% w / v, about 0.2% w / v to about 2% w / v, about 0.3% w / v to about 0.4% w / v, about 0.3% w / v to about 0.5% w / v, about 0.3% w / v to about 0.6% w / v, about 0.3% w / v to about 0.7% w / v, about 0.3% w / v to about 0.8% w / v, about 0.3% w / v to about 0.9% w / v, about 0.3% w / v to about 1% w / v w / v, about 0.3% w / v to about 1.5% w / v, about 0.3% w / v to about 2% w / v, about 0.4% w / v to about 0.5% w / v, about 0.4% w / v to about 0.6% w / v, about 0.4% w / v to about 0.7% w / v, about 0.4% w / v to about 0.8% w / v, about 0.4% w / v to about 0.9% w / v, about 0.4% w / v to about 1% w / v, about 0.4% w / v to about 1.5% w / v, about 0.4% w / v to about 2% w / v, about 0.5% w / v to about 0.6% w / v, about 0.5% w / v to about 0.7% w / v, about 0.5% w / v to about 0.8% w / v, about 0.5% w / v to about 0.9% w / v, about 0.5% w / v w / v to about 1% w / v, about 0.5% w / v to about 1.5% w / v, about 0.5% w / v to about 2% w / v, about 0.6% w / v to about 0.7% w / v, about 0.6% w / v to about 0.8% w / v, about 0.6% w / v to about 0.9% w / v, about 0.6% w / v to about 1% w / v, about 0.6% w / v to about 1.5% w / v, about 0.6% w / v to about 2% w / v, about 0.7% w / v to about 0.8% w / v, about 0.7% w / v to about 0.9% w / v, about 0.7% w / v to about 1% w / v, about 0.7% w / v to about 1.5% w / v, about 0.7% w / v to about 2% w / v, about 0.8% w / v to about 0.9% w / v, about 0.8% w / v to about 1% w / v, about 0.8% w / v to about 1.5% w / v, about 0.8% w / v to about 2% w / v, about 0.9% w / v to about 1% w / v, about 0.9% w / v to about 1.5% w / v w / v, approximately 0.9% w / v to approximately 2% w / v, approximately 1% w / v to approximately 1.5% w / v, approximately 1% w / v to approximately 2% w / v, or approximately 1.5% w / v to approximately 2% w / v.

[0122] In some embodiments, the lysis buffer further comprises an additive, including but not limited to ethyl acetate (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), and / or tris(hydroxymethyl)aminomethane (e.g., Tris). In some embodiments, the lysis buffer comprises SDS, ethyl acetate (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), and / or tris(hydroxymethyl)aminomethane (e.g., Tris). In some embodiments, the lysis buffer further comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

[0123] In some embodiments, the lysis buffer comprises a chelating agent. In some embodiments, the lysis buffer comprises one, two, three, four or more chelating agents. In some embodiments, the chelating agent includes deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, or N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).

[0124] In some embodiments, the pyrolysis buffer comprises a reducing agent. In some embodiments, the pyrolysis buffer comprises one, two, three, four, five or more reducing agents. In some embodiments, the reducing agent includes oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

[0125] In some implementations, EGTA is present at a final concentration that effectively binds calcium, magnesium, and / or other ions in the lysis buffer. In some embodiments, the concentration (e.g., final concentration) of EGTA in the mixture in the presence of the sample is at least about 0.1 mM, at least about 0.25 mM, at least about 0.5 mM, at least about 0.75 mM, at least about 1.0 mM, at least about 1.5 mM, at least about 2.0 mM, at least about 2.5 mM, at least about 3.0 mM, at least about 3.5 mM, at least about 4.0 mM, at least about 4.5 mM, at least about 5.0 mM, at least about 5.5 mM, at least about 6.0 mM, at least about 6.5 mM, at least about 7.0 mM, at least about 7.5 mM, at least about 8.0 mM, at least about 8.5 mM, at least about 9.0 mM, at least about 9.5 mM, at least about 10.0 mM, at least about 11.0 mM, at least about 12.0 mM, at least about 13.0 mM, and at least about 14.0 mM. mM, at least about 15.0 mM, at least about 20.0 mM, at least about 25.0 mM, at least about 30.0 mM, at least about 35.0 mM, at least about 40.0 mM, at least about 45.0 mM or at least about 50.0 mM.

[0126] In some embodiments, the concentration (e.g., final concentration) of EGTA in the mixture in the presence of the sample is at most about 50.0 mM, at most about 45.0 mM, at most about 40.0 mM, at most about 35.0 mM, at most about 30.0 mM, at most about 25.0 mM, at most about 20.0 mM, at most about 15.0 mM, at most about 10.0 mM, at most about 9.5 mM, at most about 9.0 mM, at most about 8.5 mM, at most about 8.0 mM, at most about 7.5 mM, at most about 7.0 mM, at most about 6.5 mM, at most about 6.0 mM, at most about 5.5 mM, at most about 5.0 mM, at most about 4.5 mM, at most about 4.0 mM, at most about 3.5 mM, at most about 3.0 mM, at most about 2.5 mM. mM, up to about 2.0 mM, up to about 1.5 mM, up to about 1.0 mM, up to about 0.75 mM, up to about 0.5 mM, up to about 0.25 mM or up to about 0.1 mM.

[0127] In some embodiments, the concentration of EGTA in the mixture (e.g., the final concentration) in the presence of the sample is from about 0.5 mM to about 20 mM. In some embodiments, the concentration of EGTA in the mixture (e.g., the final concentration) in the presence of the sample is at most about 20 mM. In some embodiments, the concentration (e.g., final concentration) of EGTA in the mixture in the presence of the sample is about 0.5 mM to about 1 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 8 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 12 mM, about 0.5 mM to about 15 mM, about 0.5 mM to about 20 mM, about 1 mM to about 2 mM, about 1 mM to about 3 mM, about 1 mM to about 4 mM, about 1 mM to about 5 mM, about 1 mM to about 6 mM, about 1 mM to about 8 mM, about 1 mM to about 10 mM, about 1 mM to about 12 mM, about 1 mM to about 15 mM, about 1 mM to about 20 mM, about 2 mM to about 3 mM. mM, about 2 mM to about 4 mM, about 2 mM to about 5 mM, about 2 mM to about 6 mM, about 2 mM to about 8 mM, about 2 mM to about 10 mM, about 2 mM to about 12 mM, about 2 mM to about 15 mM, about 2 mM to about 20 mM, about 3 mM to about 4 mM, about 3 mM to about 5 mM, about 3 mM to about 6 mM, about 3 mM to about 8 mM, about 3 mM to about 10 mM, about 3 mM to about 12 mM, about 3 mM to about 15 mM, about 3 mM to about 20 mM, about 4 mM to about 5 mM, about 4 mM to about 6 mM, about 4 mM to about 8 mM, about 4 mM to about 10 mM, about 4 mM to about 12 mM, about 4 mM to about 15 mM, about 4 mM to about 20 mM, about 5 mM to about 6 mM, about 5 mM to about 8 mM, about 5 mM to about 10 mM, about 5 mM to about 12 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 6 mM to about 8 mM, about 6 mM to about 10 mM, about 6 mM to about 12 mM, about 6 mM to about 15 mM, about 6 mM to about 20 mM, about 8 mM to about 10 mM, about 8 mM to about 12 mM, about 8 mM to about 15 mM, about 8 mM to about 20 mM, about 10 mM to about 12 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, about 12 mM to about 15 mM, about 12 mM to about 20 mM or about 15 mM to about 20 mM.

[0128] In some implementations, EDTA is present at a final concentration that effectively binds magnesium, calcium, and / or other ions in the lysis buffer. In some embodiments, the concentration (e.g., final concentration) of EDTA in the mixture in the presence of the sample is at least about 0.1 mM, at least about 0.2, at least about 0.3 mM, at least about 0.4 mM, at least about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, at least about 1.0 mM, at least about 1.1 mM, at least about 1.2 mM, at least about 1.3 mM, at least about 1.4 mM, at least about 1.5 mM, at least about 1.6 mM, at least about 1.7 mM, at least about 1.8 mM, at least about 1.9 mM, at least about 2.0 mM, at least about 3.0 mM, at least about 4.0 mM, at least about 5.0 mM, at least about 6.0 mM, at least about 7.0 mM, at least about 8.0 mM, at least about 9.0 mM, and at least about 10.0 mM. mM, at least about 20.0 mM, at least about 30.0 mM, at least about 40.0 mM or at least about 50.0 mM.

[0129] In some embodiments, the concentration (e.g., final concentration) of EDTA in the mixture in the presence of the sample is at most about 50.0 mM, at most about 40.0 mM, at most about 30.0 mM, at most about 20.0 mM, at most about 10.0 mM, at most about 9.0 mM, at most about 8.0 mM, at most about 7.0 mM, at most about 6.0 mM, at most about 5.0 mM, at most about 4.0 mM, at most about 3.0 mM, at most about 2.0 mM, at most about 1.9 mM, at most about 1.8 mM, at most about 1.7 mM, at most about 1.6 mM, at most about 1.5 mM, at most about 1.4 mM, at most about 1.3 mM, at most about 1.2 mM, at most about 1.1 mM, and at most about 1.0 mM. mM, up to about 0.9 mM, up to about 0.8 mM, up to about 0.7 mM, up to about 0.6 mM, up to about 0.5 mM, up to about 0.4 mM, up to about 0.3 mM, up to about 0.2 mM or up to about 0.1 mM.

[0130] In some embodiments, the concentration of EDTA in the mixture (e.g., the final concentration) in the presence of the sample is from about 0.1 mM to about 25 mM. In some embodiments, the concentration of EDTA in the mixture (e.g., the final concentration) in the presence of the sample is at most about 25 mM.In some embodiments, the concentration (e.g., final concentration) of EDTA in the mixture in the presence of the sample is about 0.1 mM to about 0.25 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 0.75 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 2.5 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 25 mM, about 0.25 mM to about 0.5 mM, about 0.25 mM to about 0.75 mM, about 0.25 mM to about 1 mM, about 0.25 mM to about 1.5 mM, about 0.25 mM to about 2 mM, about 0.25 mM to about 2.5 mM, about 0.25 mM to about 3 mM. mM, about 0.25 mM to about 5 mM, about 0.25 mM to about 10 mM, about 0.25 mM to about 25 mM, about 0.5 mM to about 0.75 mM, about 0.5 mM to about 1 mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 2.5 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 25 mM, about 0.75 mM to about 1 mM, about 0.75 mM to about 1.5 mM, about 0.75 mM to about 2 mM, about 0.75 mM to about 2.5 mM, about 0.75 mM to about 3 mM, about 0.75 mM to about 5 mM, about 0.75 mM to about 10 mM, about 0.75 mM to about 25 mM, about 1 mM to about 1.5 mM, about 1 mM to about 2 mM, about 1 mM to about 2.5 mM, about 1 mM to about 3 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 25 mM, about 1.5 mM to about 2 mM, about 1.5 mM to about 2.5 mM, about 1.5 mM to about 3 mM, about 1.5 mM to about 5 mM, about 1.5 mM to about 10 mM, about 1.5 mM to about 25 mM, about 2 mM to about 2.5 mM, about 2 mM to about 3 mM, about 2 mM to about 5 mM, about 2 mM to about 10 mM, about 2 mM to about 25 mM, about 2.5 mM to about 3 mM, about 2.5 mM to about 5 mM, about 2.5 mM to about 10 mM, about 2.5 mM to about 25 mM, about 3 mM to about 5 mM, about 3 mM to about 10 mM, about 3 mM to about 25 mM, about 5 mM to about 10 mM, about 5 mM to about 25 mM or about 10 mM to about 25 mM.

[0131] In some implementations, the reducing agent includes tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the concentration (e.g., final concentration) of TCEP in the mixture in the presence of the sample is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, or at least about 100 mM.

[0132] In some embodiments, the concentration (e.g., final concentration) of TCEP in the mixture in the presence of the sample is at most about 100 mM, at most about 90 mM, at most about 80 mM, at most about 70 mM, at most about 60 mM, at most about 50 mM, at most about 45 mM, at most about 40 mM, at most about 35 mM, at most about 30 mM, at most about 25 mM, at most about 20 mM, at most about 19 mM, at most about 18 mM, at most about 17 mM, at most about 16 mM, at most about 15 mM, at most about 14 mM, at most about 13 mM, at most about 12 mM, at most about 11 mM, at most about 10 mM, at most about 9 mM, at most about 8 mM, at most about 7 mM, at most about 6 mM, at most about 5 mM, at most about 4 mM, at most about 3 ... mM, up to about 2 mM or up to about 1 mM.

[0133] In some embodiments, the concentration of TCEP in the mixture (e.g., the final concentration) in the presence of the sample is from about 0.5 mM to about 75 mM. In some embodiments, the concentration of TCEP in the mixture in the presence of the sample ( For exampleThe final concentrations are approximately 0.5 mM to 1 mM, approximately 0.5 mM to 2 mM, approximately 0.5 mM to 3 mM, approximately 0.5 mM to 5 mM, approximately 0.5 mM to 10 mM, approximately 0.5 mM to 12 mM, approximately 0.5 mM to 15 mM, approximately 0.5 mM to 20 mM, approximately 0.5 mM to 25 mM, approximately 0.5 mM to 50 mM, approximately 0.5 mM to 75 mM, approximately 1 mM to 2 mM, approximately 1 mM to 3 mM, approximately 1 mM to 5 mM, approximately 1 mM to 10 mM, approximately 1 mM to 12 mM, approximately 1 mM to 15 mM, approximately 1 mM to 20 mM, approximately 1 mM to 25 mM, approximately 1 mM to 50 mM, approximately 1 mM to 75 mM, approximately 2 mM to 3 mM, approximately 2 mM to 5 mM. mM, about 2 mM to about 10 mM, about 2 mM to about 12 mM, about 2 mM to about 15 mM, about 2 mM to about 20 mM, about 2 mM to about 25 mM, about 2 mM to about 50 mM, about 2 mM to about 75 mM, about 3 mM to about 5 mM, about 3 mM to about 10 mM, about 3 mM to about 12 mM, about 3 mM to about 15 mM, about 3 mM to about 20 mM, about 3 mM to about 25 mM, about 3 mM to about 50 mM, about 3 mM to about 75 mM, about 5 mM to about 10 mM, about 5 mM to about 12 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 25 mM, about 5 mM to about 50 mM, about 5 mM to about 75 mM, about 10 mM to about 12 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, about 10 mM to about 25 mM, about 10 mM to about 50 mM, about 10 mM to about 75 mM, about 12 mM to about 15 mM, about 12 mM to about 20 mM, about 12 mM to about 25 mM, about 12 mM to about 50 mM, about 12 mM to about 75 mM, about 15 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 50 mM, about 15 mM to about 75 mM, about 20 mM to about 25 mM, about 20 mM to about 50 mM, about 20 mM to about 75 mM, about 25 mM to about 50 mM, about 25 mM to about 75 mM or about 50 mM to about 75 mM.

[0134] In some embodiments, the reducing agent includes Tris. In some embodiments, the concentration (e.g., final concentration) of Tris in the mixture in the presence of the sample is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, or at least about 100 mM.

[0135] In some embodiments, the concentration (e.g., final concentration) of Tris in the mixture in the presence of the sample is at most about 100 mM, at most about 90 mM, at most about 80 mM, at most about 70 mM, at most about 60 mM, at most about 50 mM, at most about 45 mM, at most about 40 mM, at most about 35 mM, at most about 30 mM, at most about 25 mM, at most about 20 mM, at most about 19 mM, at most about 18 mM, at most about 17 mM, at most about 16 mM, at most about 15 mM, at most about 14 mM, at most about 13 mM, at most about 12 mM, at most about 11 mM, at most about 10 mM, at most about 9 mM, at most about 8 mM, at most about 7 mM, at most about 6 mM, at most about 5 mM, at most about 4 mM, at most about 3 ... mM, up to about 2 mM or up to about 1 mM.

[0136] In some embodiments, Tris is present in the sample at a concentration of at least about 0.5 mM to about 75 mM in the mixture (e.g., the final concentration). In some embodiments, the concentration (e.g., final concentration) of Tris in the mixture in the presence of the sample is about 0.5 mM to about 1 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 12 mM, about 0.5 mM to about 15 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 25 mM, about 0.5 mM to about 50 mM, about 0.5 mM to about 75 mM, about 1 mM to about 2 mM, about 1 mM to about 3 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 12 mM, about 1 mM to about 15 mM, about 1 mM to about 20 mM, about 1 mM to about 25 mM, about 1 mM to about 50 mM, about 1 mM to about 75 mM. mM, about 2 mM to about 3 mM, about 2 mM to about 5 mM, about 2 mM to about 10 mM, about 2 mM to about 12 mM, about 2 mM to about 15 mM, about 2 mM to about 20 mM, about 2 mM to about 25 mM, about 2 mM to about 50 mM, about 2 mM to about 75 mM, about 3 mM to about 5 mM, about 3 mM to about 10 mM, about 3 mM to about 12 mM, about 3 mM to about 15 mM, about 3 mM to about 20 mM, about 3 mM to about 25 mM, about 3 mM to about 50 mM, about 3 mM to about 75 mM, about 5 mM to about 10 mM, about 5 mM to about 12 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 25 mM, about 5 mM to about 50 mM, about 5 mM to about 75 mM, about 10 mM to about 12 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, about 10 mM to about 25 mM, about 10 mM to about 50 mM, about 10 mM to about 75 mM, about 12 mM to about 15 mM, about 12 mM to about 20 mM, about 12 mM to about 25 mM, about 12 mM to about 50 mM, about 12 mM to about 75 mM, about 15 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 50 mM, about 15 mM to about 75 mM, about 20 mM to about 25 mM, about 20 mM to about 50 mM, about 20 mM to about 75 mM, about 25 mM to about 50 mM, about 25 mM to about 75 mM or about 50 mM to about 75 mM.

[0137] In some embodiments, the lysis buffer may comprise SDS, EGTA, EDTA, TCEP, and / or Tris. The lysis buffer may comprise SDS having a final concentration of about 0.01% w / v to 0.4% w / v in the presence of the sample; EGTA having a final concentration of about 0.1 mM to 3 mM in the presence of the sample; EDTA having a final concentration of about 0.01 mM to 1 mM in the presence of the sample; TCEP having a final concentration of about 1.0 mM to 4.0 mM in the presence of the sample; and Tris having a final concentration of about 1.0 mM to 4.5 mM in the presence of the sample. For example, for each component, the lysis buffer used in the presence of the sample may comprise a final concentration of 0.2% SDS, 2 mM EGTA, 0.5 mM EDTA, 1 mM TCEP, and 1 mM Tris.

[0138] In some embodiments, the lysis buffer has a pH value sufficient for lysing the cells required for lysis. In some embodiments, the lysis buffer has a pH value of at least about 1, at least about 2, at least about 3, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7, at least about 8, or at least about 9. In some embodiments, the lysis buffer has a pH value of at most about 9, at most about 8, at most about 7, at most about 6.5, at most about 6, at most about 5.5, at most about 5, at most about 4.5, at most about 4, at most about 3, at most about 2, or at most about 1.

[0139] In some embodiments, the lysis buffer has a pH of about 1 to about 10. In some embodiments, the lysis buffer has a pH of up to about 10. In some embodiments, the lysis buffer has a pH of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 5.5, about 1 to about 6, about 1 to about 6.5, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 5.5, about 2 to about 6, about 2 to about 6.5, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 5, about 3 to about 5.5, about 3 to about 6, about 3 to about 6.5, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 5.5, about 4 to about 6, about 4 to about pH values ​​of 6.5, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 5.5, about 5 to about 6, about 5 to about 6.5, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5.5 to about 6, about 5.5 to about 6.5, about 5.5 to about 7, about 5.5 to about 8, about 5.5 to about 9, about 5.5 to about 10, about 6 to about 6.5, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10. In some embodiments, the lysis buffer has a pH of about 8. In some embodiments, the lysis buffer has a pH of about 7. In some embodiments, the lysis buffer has a pH of about 6.

[0140] In some embodiments, the pyrolysis buffer does not contain detergent. In some embodiments, the pyrolysis buffer does not contain solubilizer.

[0141] The final volume of the lysis buffer can depend on the type of sample or amplification method. In some embodiments, the final volume of the lysis buffer is at least about 50 microliters (µl), at least about 100 µl, at least about 150 µl, at least about 200 µl, at least about 250 µl, at least about 300 µl, at least about 350 µl, at least about 400 µl, at least about 450 µl, at least about 500 µl, at least about 550 µl, at least about 600 µl, at least about 650 µl, at least about 700 µl, at least about 750 µl, at least about 800 µl, at least about 850 µl, at least about 900 µl, at least about 950 µl, at least about 1000 µl, at least about 1100 µl, at least about 1200 µl, at least about 1300 µl, at least about 1400 µl, at least about 1500 µl, at least about 2000 µl, at least about 2500 µl, at least about 3000 µl. µl, at least about 4000 µl, at least about 5000 µl, at least about 7500 µl, or at least about 10,000 µl.

[0142] In some embodiments, the final volume of the lysis buffer is up to about 10,000 µl, up to about 7,500 µl, up to about 5,000 µl, up to about 4,000 µl, up to about 3,000 µl, up to about 2,500 µl, up to about 2,000 µl, up to about 1,500 µl, up to about 1,400 µl, up to about 1,300 µl, up to about 1,200 µl, up to about 1,100 µl, up to about 1,000 µl, up to about 950 µl, up to about 900 µl, up to about 850 µl, up to about 800 µl, up to about 750 µl, up to about 700 µl, up to about 650 µl, up to about 600 µl, up to about 550 µl, up to about 500 µl, up to about 450 µl. µl, up to about 400 µl, up to about 350 µl, up to about 300 µl, up to about 250 µl, up to about 200 µl, up to about 150 µl, up to about 100 µl, or up to about 50 µl.

[0143] In some embodiments, the final volume of the lysis buffer is from about 50 µl to about 2,000 µl. In some embodiments, the final volume of the lysis buffer is at most about 2,000 µl. In some embodiments, the final volume of the lysis buffer is about 50 µl to about 100 µl, about 50 µl to about 200 µl, about 50 µl to about 300 µl, about 50 µl to about 400 µl, about 50 µl to about 500 µl, about 50 µl to about 600 µl, about 50 µl to about 750 µl, about 50 µl to about 1,000 µl, about 50 µl to about 1,500 µl, about 50 µl to about 1,750 µl, about 50 µl to about 2,000 µl, about 100 µl to about 200 µl, about 100 µl to about 300 µl, about 100 µl to about 400 µl, about 100 µl to about 500 µl, about 100 µl to about 600 µl, about 100 µl to about 750 µl, about 100 µl to about 1,000 µl. µl, about 100 µl to about 1,500 µl, about 100 µl to about 1,750 µl, about 100 µl to about 2,000 µl, about 200 µl to about 300 µl, about 200 µl to about 400 µl, about 200 µl to about 500 µl, about 200 µl to about 600 µl, about 200 µl to about 750 µl, about 200 µl to about 1,000 µl, about 200 µl to about 1,500 µl, about 200 µl to about 1,750 µl, about 200 µl to about 2,000 µl, about 300 µl to about 400 µl, about 300 µl to about 500 µl, about 300 µl to about 600 µl, about 300 µl to about 750 µl, about 300 µl to about 1,000 µl, about 300 µl About 1,500 µl to 1,750 µl, about 300 µl to 2,000 µl, about 400 µl to 500 µl, about 400 µl to 600 µl, about 400 µl to 750 µl, about 400 µl to 1,000 µl, about 400 µl to 1,500 µl, about 400 µl to 1,750 µl, about 400 µl to 2,000 µl, about 500 µl to 600 µl, about 500 µl to 750 µl, about 500 µl to 1,000 µl, about 500 µl to 1,500 µl, about 500 µl to 1,750 µl, about 500 µl to 2,000 µl, about 600 µl to 750 µl, about 600 µl to 1,500 µl, about 300 µl to 1,750 µl, about 500 µl to 2,000 µl, about 600 µl to 750 µl, about 600 µl to 1,500 µl, about 300 µl to 1,750 µl, about 3 ...1,750 µl, about 300 µl to 1,500 µl, about 300 µl to 1,750 µl, about 300 µl to 1,750 µl, about 500 µl to 1,500 µ µl to about 1,000 µl, about 600 µl to about 1,500 µl, about 600 µl to about 1,750 µl, about 600 µl to about 2,000 µl, about 750 µl to about 1,000 µl, about 750 µl to about 1,500 µl, about 750 µl to about 1,750 µl, about 750 µl to about 2,000 µl, about 1,000 µl to about 1,500 µl, about 1,000 µl to about 1,750 µl, about 1,000 µl to about 2,000 µl, about 1,500 µl to about 1,750 µl, about 1,500 µl to about 2,000 µl, or about 1,750 µl to about 2,000 µl.

[0144] In some implementations, the final volume of the lysis buffer is about 2 ml to about 10 ml.

[0145] In some embodiments, the lysis buffer is lyophilized. In other embodiments, the lysis buffer is not lyophilized. In some embodiments, the final volume of the lysis buffer is approximately 2 ml to approximately 2.5 ml, approximately 2 ml to approximately 3 ml, approximately 2 ml to approximately 3.5 ml, approximately 2 ml to approximately 4 ml, approximately 2 ml to approximately 4.5 ml, approximately 2 ml to approximately 5 ml, approximately 2 ml to approximately 6 ml, approximately 2 ml to approximately 7 ml, approximately 2 ml to approximately 8 ml, approximately 2 ml to approximately 9 ml, approximately 2 ml to approximately 10 ml, approximately 2.5 ml to approximately 3 ml, approximately 2.5 ml to approximately 3.5 ml, approximately 2.5 ml to approximately 4 ml, approximately 2.5 ml to approximately 4.5 ml, approximately 2.5 ml to approximately 5 ml, approximately 2.5 ml to approximately 6 ml, approximately 2.5 ml to approximately 7 ml, approximately 2.5 ml to approximately 8 ml, approximately 2.5 ml to approximately 9 ml, approximately 2.5 ml to approximately 10 ml, approximately 3 ml to approximately 3.5 ml, approximately 3 ml to approximately 4 ml, approximately 3 ml to approximately 4.5 ml, approximately 3 ml to approximately 5 ml, approximately 3 ml to approximately 6 ml, approximately 3 ml to approximately 7 ml, approximately 3 ml to approximately 8 ml, approximately 3 ml to approximately 3 ml. Approximately 3 ml to 10 ml, approximately 3.5 ml to 4 ml, approximately 3.5 ml to 4.5 ml, approximately 3.5 ml to 5 ml, approximately 3.5 ml to 6 ml, approximately 3.5 ml to 7 ml, approximately 3.5 ml to 8 ml, approximately 3.5 ml to 9 ml, approximately 3.5 ml to 10 ml, approximately 4 ml to 4.5 ml, approximately 4 ml to 5 ml, approximately 4 ml to 6 ml, approximately 4 ml to 7 ml, approximately 4 ml to 8 ml, approximately 4 ml to 9 ml, approximately 4 ml to 10 ml, approximately 4.5 ml to 5 ml, approximately 4.5 ml to 6 ml, approximately 4.5 ml to 7 ml, approximately 4.5 ml to 8 ml, approximately 4.5 ml to 9 ml, approximately 4.5 ml to 10 ml, approximately 5 ml to 6 ml, approximately 5 ml to 7 ml, approximately 5 ml to 8 ml, approximately 5 ml to 9 ml, approximately 5 ml to 10 ml, approximately 6 ml to 7 ml, approximately 6 ml to 8 ml ml, about 6 ml to about 9 ml, about 6 ml to about 10 ml, about 7 ml to about 8 ml, about 7 ml to about 9 ml, about 7 ml to about 10 ml, about 8 ml to about 9 ml, about 8 ml to about 10 ml or about 9 ml to about 10 ml.

[0146] In some embodiments, the solubilizer is a nonionic surfactant. In some embodiments, the solubilizer comprises polysorbate. The polysorbate may be polyoxyethylene (20) sorbitol monooleate (e.g., polysorbate 80), polyoxyethylene (20) sorbitol monolaurate (e.g., polysorbate 20), polyoxyethylene (20) sorbitol monopalmitate (e.g., polysorbate 40), polyoxyethylene (20) sorbitol monostearate (e.g., polysorbate 60), or a functional variant thereof. In some embodiments, the solubilizer is Tergitol. TM Surfactants, Triton TM Surfactants or Igepal ® Surfactant. In some embodiments, the solubilizer is an alkoxylate or cocamide. In some embodiments, the solubilizer is decyl glucoside, alkyl glycoside, lauryl glucoside, sorbitan tristearate, or a niosome. In some embodiments, the recovery buffer contains one, two, three, four, or more solubilizers. The solubilizer can be mixed with the detergent of this composition. In some embodiments, the solubilizer is capable of forming micelles comprising the detergent of this application.

[0147] In some embodiments, the solubilizer is polysorbate 80. In some embodiments, the concentration of the solubilizer in the mixture in the presence of the sample ( For example The final concentration is at least about 0.05% v / v, at least about 0.1% v / v, at least about 0.5% v / v, at least about 1% v / v, at least about 5% v / v, at least about 10% v / v, at least about 15% v / v, at least about 20% v / v, at least about 22.5% v / v, at least about 25% v / v, at least about 27.5% v / v, at least about 30% v / v, at least about 32.5% v / v, at least about 35% v / v, at least about 37.5% v / v, at least about 40% v / v, at least about 42.5% v / v, at least about 45% v / v, at least about 47.5% v / v, at least about 50% v / v, at least about 52.5% v / v, at least about 55% v / v, at least about 57.5% v / v, at least about 60% v / v, at least about 70%. v / v or at least about 75% v / v.

[0148] In some implementations, the concentration of the solubilizer in the mixture in the presence of the sample ( For exampleThe final concentrations are as follows: approximately 75% v / v, approximately 70% v / v, approximately 65% ​​v / v, approximately 60% v / v, approximately 57.5% v / v, approximately 55% v / v, approximately 52.5% v / v, approximately 50% v / v, approximately 47.5% v / v, approximately 45% v / v, approximately 42.5% v / v, approximately 40% v / v, approximately 37.5% v / v, approximately 35% v / v, approximately 32.5% v / v, approximately 30% v / v, approximately 27.5% v / v, approximately 25% v / v, approximately 22.5% v / v, approximately 20% v / v, approximately 15% v / v, and approximately 10%. v / v, up to about 5% v / v, up to about 1% v / v, up to about 0.5% v / v, up to about 0.1% v / v or up to about 0.05% v / v.

[0149] In some implementations, the concentration of the solubilizer in the mixture in the presence of the sample ( For example The final concentration is approximately 0.1% v / v to approximately 80% v / v. In some embodiments, the concentration of the solubilizer in the mixture in the presence of the sample ( For exampleThe final concentrations are approximately 0.1% v / v to approximately 5% v / v, approximately 0.1% v / v to approximately 10% v / v, approximately 0.1% v / v to approximately 15% v / v, approximately 0.1% v / v to approximately 20% v / v, approximately 0.1% v / v to approximately 25% v / v, approximately 0.1% v / v to approximately 30% v / v, approximately 0.1% v / v to approximately 40% v / v, approximately 0.1% v / v to approximately 50% v / v, approximately 0.1% v / v to approximately 60% v / v, approximately 0.1% v / v to approximately 70% v / v, and approximately 0.1% v / v to about 80% v / v, about 5% v / v to about 10% v / v, about 5% v / v to about 15% v / v, about 5% v / v to about 20% v / v, about 5% v / v to about 25% v / v, about 5% v / v to about 30% v / v, about 5% v / v to about 40% v / v, about 5% v / v to about 50% v / v, about 5% v / v to about 60% v / v, about 5% v / v to about 70% v / v, about 5% v / v to about 80% v / v, about 10% v / v to about 15% v / v, about 10% v / v to about 20% v / v, about 10% v / v to about 25% v / v, about 10% v / v to about 30% v / v, about 10% v / v to about 40% v / v, about 10% v / v to about 50% v / v v / v, about 10% v / v to about 60% v / v, about 10% v / v to about 70% v / v, about 10% v / v to about 80% v / v, about 15% v / v to about 20% v / v, about 15% v / v to about 25% v / v, about 15% v / v to about 30% v / v, about 15% v / v to about 40% v / v, about 15% v / v to about 50% v / v, about 15% v / v to about 60% v / v, about 15% v / v to about 70% v / v, about 15% v / v to about 80% v / v, about 20% v / v to about 25% v / v, about 20% v / v to about 30% v / v, about 20% v / v to about 40% v / v, about 20% v / v to about 50% v / v, about 20% v / v to about 60% v / v v / v, approximately 20% v / v to approximately 70% v / v, approximately 20% v / v to approximately 80% v / v, approximately 25% v / v to approximately 30% v / v, approximately 25% v / v to approximately 40% v / v, approximately 25% v / v to approximately 50% v / v, approximately 25% v / v to approximately 60% v / v, approximately 25% v / v to approximately 70% v / v, approximately 25% v / v to approximately 80% v / v, approximately 30% v / v to approximately 40% v / v, approximately 30% v / v to approximately 50% v / v, approximately 40% v / v to approximately 60% v / v, approximately 40% v / v to approximately 70% v / v v / v, approximately 40% v / v to approximately 80% v / v, approximately 50% v / v to approximately 60% v / v, approximately 50% v / v to approximately 70% v / v, approximately 50% v / v to approximately 80% v / v, approximately 60% v / v to approximately 70% v / v, approximately 60% v / v to approximately 80% v / v, or approximately 70% v / v to approximately 80% v / v.

[0150] In some embodiments, the composition comprises cyclodextrin. The cyclodextrin is configured to form a complex with the detergent of this application. Not wishing to be bound by theory, the complex formed between the cyclodextrin and the detergent assists in stabilizing the enzyme in the composition. The cyclodextrin increases the efficiency of complex formation. As a complexing agent, cyclodextrin can increase the water solubility of poorly soluble drugs and increase their bioavailability and stability in solution. In some embodiments, cyclodextrins include (2-hydroxypropyl)β-cyclodextrin, (2-hydroxypropyl)γ-cyclodextrin, (2-hydroxypropyl)α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof. Cyclodextrins may include anionic cyclodextrins. Anionic cyclodextrins may include carboxymethyl-α-cyclodextrin, carboxymethyl-β-cyclodextrin, succinyl-α-cyclodextrin, succinyl-β-cyclodextrin, succinyl-γ-cyclodextrin, (2-carboxyl)-α-cyclodextrin, (2-carboxyl)-β-cyclodextrin, α-cyclodextrin phosphate, β-cyclodextrin phosphate, γ-cyclodextrin phosphate, sulfonated β-cyclodextrin, α-cyclodextrin sulfate, β-cyclodextrin sulfate, γ-cyclodextrin sulfate, carboxymethyl-γ-cyclodextrin, (2-carboxyl)-γ-cyclodextrin, sulfonated-α-cyclodextrin, succinyl-(2-hydroxypropyl)-β-cyclodextrin, succinyl-(2-hydroxypropyl)-γ-cyclodextrin, sulfonated-γ-cyclodextrin, methyl-β-cyclodextrin, or any combination thereof. In some embodiments, the cyclodextrin in the recovered buffer may comprise two or more different cyclodextrin species described herein. For example, the cyclodextrin in the recovered buffer may comprise (2-hydroxypropyl)β-cyclodextrin and (2-hydroxypropyl)γ-cyclodextrin. As another example, the cyclodextrin in the recovered buffer may comprise (2-hydroxypropyl)α-cyclodextrin and methyl-β-cyclodextrin. In some cases, the cyclodextrin in the recovered buffer may comprise (2-hydroxypropyl)β-cyclodextrin and methyl-β-cyclodextrin. In some cases, changing the molar substitution rate of a specific modified cyclodextrin species (e.g., (2-hydroxypropyl)β-cyclodextrin, methyl-β-cyclodextrin, etc.) can improve reaction performance, such as shortening the resultant time value, Ct value, or Cq value.

[0151] In some embodiments, the recovered buffer does not contain components of the pyrolysis buffer. For example, the recovered buffer may not contain detergents or reducing agents. In some cases, the recovered buffer may not contain one or more agents selected from the following: etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), and tetrahydropyran (THP).

[0152] In some embodiments, cyclodextrin is present in the presence of a sample to effectively isolate the final concentration of detergent within the composition of the present invention. In some embodiments, the concentration of cyclodextrin in the mixture in the presence of a sample ( For example The final concentration is at least about 0.05 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 5.0 mM, at least about 10.0 mM, at least about 15.0 mM, at least about 20.0 mM, at least about 25.0 mM, at least about 30.0 mM, at least about 35.0 mM, at least about 40.0 mM, at least about 50.0 mM, at least about 55.0 mM, at least about 60.0 mM, at least about 65.0 mM, at least about 70.0 mM, at least about 75.0 mM, at least about 80.0 mM, at least about 85.0 mM, at least about 90.0 mM, at least about 95.0 mM, at least about 100.0 mM, at least about 125.0 mM, at least about 150.0 mM, and at least about 175.0 mM. mM, at least about 200.0 mM, at least about 250.0 mM or at least about 300.0 mM.

[0153] In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample ( For exampleThe final concentrations were approximately 300.0 mM, 250.0 mM, 200.0 mM, 175.0 mM, 150.0 mM, 125.0 mM, 100.0 mM, 95.0 mM, 90.0 mM, 85.0 mM, 80.0 mM, 75.0 mM, 70.0 mM, 65.0 mM, 60.0 mM, 55.0 mM, 50.0 mM, 45.0 mM, 40.0 mM, 35.0 mM, 30.0 mM, 25.0 mM, and 20.0 mM, respectively. mM, up to about 15.0 mM, up to about 10.0 mM, up to about 5.0 mM, up to about 1.0 mM, up to about 0.5 mM, up to about 0.1 mM or up to about 0.05 mM.

[0154] In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample ( For example The final concentration is approximately 10 mM to approximately 300 mM. In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample ( For example The final concentration is at least about 10 mM. In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample is ( For example The final concentration is at most about 300 mM. In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample ( For exampleThe final concentrations are approximately 10 mM to 20 mM, approximately 10 mM to 25 mM, approximately 10 mM to 30 mM, approximately 10 mM to 32.5 mM, approximately 10 mM to 35 mM, approximately 10 mM to 37.5 mM, approximately 10 mM to 40 mM, approximately 10 mM to 50 mM, approximately 10 mM to 100 mM, approximately 10 mM to 200 mM, approximately 10 mM to 300 mM, approximately 20 mM to 25 mM, approximately 20 mM to 30 mM, approximately 20 mM to 32.5 mM, approximately 20 mM to 35 mM, approximately 20 mM to 37.5 mM, approximately 20 mM to 40 mM, approximately 20 mM to 50 mM, approximately 20 mM to 100 mM, approximately 20 mM to 200 mM, approximately 20 mM to 200 mM, approximately 20 mM to 40 mM, approximately 20 mM to 50 mM, approximately 20 mM to 100 mM, approximately 20 mM to 200 mM, approximately 20 mM to 200 mM, approximately 20 mM to 3 ... Approximately 300 mM to 300 mM, approximately 25 mM to 30 mM, approximately 25 mM to 32.5 mM, approximately 25 mM to 35 mM, approximately 25 mM to 37.5 mM, approximately 25 mM to 40 mM, approximately 25 mM to 50 mM, approximately 25 mM to 100 mM, approximately 25 mM to 200 mM, approximately 25 mM to 300 mM, approximately 30 mM to 32.5 mM, approximately 30 mM to 35 mM, approximately 30 mM to 37.5 mM, approximately 30 mM to 40 mM, approximately 30 mM to 50 mM, approximately 30 mM to 100 mM, approximately 30 mM to 200 mM, approximately 30 mM to 300 mM, approximately 32.5 mM to 35 mM, approximately 32.5 mM to 37.5 mM, approximately 32.5 mM mM to about 40 mM, about 32.5 mM to about 50 mM, about 32.5 mM to about 100 mM, about 32.5 mM to about 200 mM, about 32.5 mM to about 300 mM, about 35 mM to about 37.5 mM, about 35 mM to about 40 mM, about 35 mM to about 50 mM, about 35 mM to about 100 mM, about 35 mM to about 200 mM, about 35 mM to about 300 mM, about 37.5 mM to about 40 mM, about 37.5 mM to about 50 mM, about 37.5 mM to about 100 mM, about 37.5 mM to about 200 mM, about 37.5 mM to about 300 mM, about 40 mM to about 50 mM, about 40 mM to about 100 mM, about 40 mM to about 200 mM, about 40 mM to about 300 mM mM, about 50 mM to about 100 mM, about 50 mM to about 200 mM, about 50 mM to about 300 mM, about 100 mM to about 200 mM, about 100 mM to about 300 mM or about 200 mM to about 300 mM.

[0155] In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample ( For example The final concentration is from about 0.1 mM to about 100 mM. In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample is ( For example The final concentration is at most about 100 mM. In some embodiments, the concentration of cyclodextrin in the mixture in the presence of the sample ( For exampleThe final concentrations are approximately 0.1 mM to 1 mM, approximately 0.1 mM to 5 mM, approximately 0.1 mM to 10 mM, approximately 0.1 mM to 20 mM, approximately 0.1 mM to 30 mM, approximately 0.1 mM to 35 mM, approximately 0.1 mM to 40 mM, approximately 0.1 mM to 50 mM, approximately 0.1 mM to 60 mM, approximately 0.1 mM to 75 mM, approximately 0.1 mM to 100 mM, approximately 1 mM to 5 mM, approximately 1 mM to 10 mM, approximately 1 mM to 20 mM, approximately 1 mM to 30 mM, approximately 1 mM to 35 mM, approximately 1 mM to 40 mM, approximately 1 mM to 50 mM, approximately 1 mM to 60 mM, approximately 1 mM to 75 mM, approximately 1 mM to 100 mM, approximately 5 mM to 10 mM. mM, about 5 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 35 mM, about 5 mM to about 40 mM, about 5 mM to about 50 mM, about 5 mM to about 60 mM, about 5 mM to about 75 mM, about 5 mM to about 100 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 35 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 10 mM to about 60 mM, about 10 mM to about 75 mM, about 10 mM to about 100 mM, about 20 mM to about 30 mM, about 20 mM to about 35 mM, about 20 mM to about 40 mM, about 20 mM to about 50 mM, about 20 mM to about 60 mM, about 20 mM to about 75 mM, about 20 mM to about 100 mM, about 30 mM to about 35 mM, about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 75 mM, about 30 mM to about 100 mM, about 35 mM to about 40 mM, about 35 mM to about 50 mM, about 35 mM to about 60 mM, about 35 mM to about 75 mM, about 35 mM to about 100 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 75 mM, about 40 mM to about 100 mM, about 50 mM to about 60 mM, about 50 mM to about 75 mM, about 50 mM to about 100 mM, about 60 mM to about 75 mM, about 60 mM to about 100 mM or about 75 mM to about 100 mM.

[0156] In some embodiments, the recovery buffer may comprise cyclodextrin having a final concentration of about 6 mM to 11 mM in the presence of the sample and polysorbate 80 having a final concentration of about 0.1% v / v to 3.0% v / v in the presence of the sample. For example, the recovery buffer may comprise 2 mM cyclodextrin and 1.5% v / v polysorbate 80 for each component in the presence of the sample.

[0157] In some embodiments, cyclodextrins exhibit a higher binding affinity for detergents compared to the binding affinity of solubilizers. In some embodiments, the binding affinity of cyclodextrins for detergents can be an association constant. In some embodiments, the binding affinity of cyclodextrins for detergents has at least about 2.5 x 10⁻⁶ for the detergent. 3 M -1 At least approximately 3x10 3 M -1 At least approximately 3.5 x 10 3 M -1 At least approximately 4x10 3 M -1 At least approximately 5x10 3 M -1 At least approximately 1x10 4 M -1 At least approximately 2x10 4 M -1 At least approximately 3x10 4 M -1 At least approximately 4x10 4 M -1 At least approximately 5x10 4 M -1 At least approximately 1x10 5 M -1 At least approximately 5x10 5 M -1 Or at least about 1x10 6 M -1 association constant (K) a In some implementations, the binding affinity of cyclodextrin to detergent is at most about 1 x 10⁻⁶. 6 M -1 At most approximately 5x10 5 M -1 At most approximately 1x10 5 M -1 At most approximately 5x10 4 M -1 At most approximately 4x10 4 M -1 At most approximately 3x10 4 M -1At most approximately 2x10 4 M -1 At most approximately 1x10 4 M -1 At most approximately 5x10 3 M -1 At most approximately 4x10 3 M -1 At most approximately 3x10 3 M -1 Or at most about 2.5x10 3 M -1 association constant (K) a ).

[0158] In some implementations, the solubilizer and cyclodextrin are configured to shorten the cycle threshold or result time in nucleic acid amplification compared to the cycle threshold or result time value in nucleic acid amplification of other aspects of the same sample treated alone with SDS, polysorbate 80, or cyclodextrin. The term "cycle threshold" refers to the number of cycles required to amplify the target nucleic acid molecule to a detectable level (e.g., when the signal exceeds a background threshold level). A lower cycle threshold can indicate a larger quantity of the target nucleic acid in the sample. In some cases, when isothermal amplification is used, the result time value may also be used, and it refers to the time required to amplify the target nucleic acid molecule to a detectable level. In some implementations, the solubilizers and / or cyclodextrins described herein are configured to reduce the cycling threshold to at most about 60, at most about 50, at most about 40, at most about 30, at most about 25, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1. In some implementations, the solubilizers and / or cyclodextrins described herein are configured to reduce the result time value to at most about 15 minutes, at most about 14 minutes, at most about 13 minutes, at most about 12 minutes, at most about 11 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, at most about 5 minutes or less.

[0159] In some embodiments, the solubilizer and / or cyclodextrin are configured to reduce the coefficient of variation in nucleic acid amplification compared to the coefficient of variation in nucleic acid amplification of other aspects of the same sample treated alone with SDS, polysorbate 80, or cyclodextrin. The term "coefficient of variation" refers to a measure of the precision of the amplification method. In some embodiments, the solubilizer and / or cyclodextrin are configured to reduce the coefficient of variation value to at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4.5%, at most about 4%, at most about 3.5%, at most about 3%, at most about 2.5%, at most about 2%, at most about 1.5%, or at most about 1%.

[0160] In some embodiments, the solubilizer and / or cyclodextrin are configured to lower the detection limit of nucleic acid amplification compared to the detection limit of other aspects of the same sample treated with SDS, polysorbate 80, or cyclodextrin alone. "Detection limit" refers to the lowest amount of a component in a sample that can be reliably detected in an amplification method. In some embodiments, the solubilizer and / or cyclodextrin are configured to reduce the detection limit to about 1 target molecule, about 1.5 target molecules, about 2 target molecules, about 2.5 target molecules, about 3 target molecules, about 3.5 target molecules, about 4 target molecules, about 4.5 target molecules, about 5 target molecules, about 6 target molecules, about 7 target molecules, about 8 target molecules, about 9 target molecules, or about 10 target molecules.

[0161] In some embodiments, the solubilizer and cyclodextrin are part of the recovery buffer. In some embodiments, the recovery buffer contains a salt. In some embodiments, the recovery buffer does not contain a salt. In some embodiments, the salt includes a sodium salt. In some embodiments, the recovery buffer contains a pH buffer. In some embodiments, the recovery buffer does not contain a pH buffer. In some embodiments, the pH of the recovery buffer is at least about 3, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7, at least about 7.5, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12. In some embodiments, the pH of the recovery buffer is at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7.5, at most about 7, at most about 6.5, at most about 6, at most about 5.5, at most about 5, at most about 4.5, at most about 4, or at most about 3.

[0162] In some implementations, the recovery buffer is lyophilized. The recovery buffer can be liquid. The recovery buffer can be lyophilized together with the reaction buffer / reaction mixture used for nucleic acid amplification.

[0163] In some embodiments, the pyrolysis buffer and the recovery buffer are in the same mixture. In some embodiments, the pyrolysis buffer and the recovery buffer are mixed manually. In some embodiments, the pyrolysis buffer and the recovery buffer are mixed by vortexing. In some embodiments, the pyrolysis buffer and the recovery buffer are mixed by automated machinery, consumables, or a microfluidic system. In some embodiments, the pyrolysis buffer and the recovery buffer are mixed until they are homogeneous.

[0164] In some aspects, this disclosure provides compositions for sample treatment containing a buffer, the compositions comprising: (i) a detergent, (ii) a solubilizer, and (iii) a cyclodextrin. In some embodiments, the buffer stabilizes an enzyme during nucleic acid amplification. In some embodiments, the buffer is configured to inactivate a degrading enzyme. In some embodiments, the enzyme is a ribonuclease.

[0165] In some embodiments, the compositions of this disclosure further comprise an agent capable of reducing disulfide bonds. In some embodiments, the agent capable of reducing the disulfide bonds includes dithiothreitol (DTT), hydroxylamine, hydroxylamine-HCl, 2-mercaptoethanol (BME), or TCEP. In some embodiments, the agent capable of reducing the disulfide bonds includes monothiols, dithiols, or phosphine compounds.

[0166] In some embodiments, the composition further comprises a sample (e.g., blood sample, swab sample, saliva sample, urine sample, cerebrospinal fluid sample, pleural fluid sample, rectal sample, vaginal sample, fecal sample, sputum sample, and / or lymph sample), raw emulsion, pasteurized and / or homogenized emulsion, pasteurized and / or treated emulsion, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue sample, cell culture, purified nucleic acid sample, environmental sample, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof for nucleic acid amplification. In some embodiments, the swab sample includes vaginal swabs, oral swabs, and / or rectal swabs. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject has a disease, symptom, or infection. In some embodiments, the sample comprises a biological sample. In some embodiments, the sample comprises a purified sample. In some embodiments, the biological sample comprises a target nucleic acid molecule for sample processing.

[0167] In some embodiments, the composition further comprises a reaction mixture for nucleic acid amplification. In some embodiments, the reaction mixture is lyophilized. In some embodiments, the reaction mixture is not lyophilized. In some embodiments, the reaction mixture comprises (i) a thermostable enzyme, (ii) a deoxynucleoside triphosphate (dNTP), (iii) primers and / or (iv) a probe. In some embodiments, the thermostable enzyme comprises *Bacillus stearothermophilus* polymerase, a large fragment of *Bacillus stearothermophilus* polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, *Thermophyton floccosum* (… Thermus aquaticus () For example Taq-polA), Thermopyrocephalus ( Thermotoga maritima () For example The composition may be Tma-polA, Pfu-polB, a Pab-polB, OmniTaq 2 LA DNA polymerase, or any mutant thereof. A large fragment of the thermophilic Bacillus stearothermophilus polymerase is a part of the thermophilic Bacillus stearothermophilus DNA polymerase, containing 5'→3' polymerase activity but lacking the 5'→3' exonuclease domain. In some embodiments, the composition is configured to stabilize the enzyme activity of the thermostable enzyme for use during nucleic acid amplification.

[0168] In some embodiments, the dNTPs of the reaction mixture include dATP, dCTP, dGTP, dTTP, and / or dUTP. In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is at least about 25 micromoles (µM), at least about 50 µM, at least about 75 µM, at least about 100 µM, at least about 150 µM, at least about 200 µM, at least about 250 µM, at least about 300 µM, at least about 350 µM, at least about 400 µM, at least about 450 µM, at least about 500 µM, at least about 750 µM, at least about 1000 µM, at least about 1500 µM, at least about 2000 µM, at least about 2500 µM, at least about 3000 µM, at least about 3500 µM, at least about 4000 µM, at least about 4500 µM, at least about 5000 µM, at least about 6000 µM, at least about 7000 µM, at least about 8000 µM, at least about 9000 µM, or at least about 10000 µM. µM.

[0169] In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is up to about 10,000 µM, up to about 9,000 µM, up to about 8,000 µM, up to about 7,000 µM, up to about 6,000 µM, up to about 5,000 µM, up to about 4,500 µM, up to about 4,000 µM, up to about 3,500 µM, up to about 3,000 µM, up to about 2,500 µM, up to about 2,000 µM, up to about 1,500 µM, up to about 1,000 µM, up to about 750 µM, up to about 500 µM, up to about 450 µM, up to about 400 µM, up to about 350 µM, up to about 300 µM, up to about 250 µM, up to about 200 µM, up to about 150 µM, up to about 100 µM. µM, up to about 75 µM, up to about 50 µM or up to about 25 µM.

[0170] In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is from about 50 µM to about 7,500 µM. In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is approximately 50 µM to approximately 100 µM, approximately 50 µM to approximately 250 µM, approximately 50 µM to approximately 500 µM, approximately 50 µM to approximately 750 µM, approximately 50 µM to approximately 1,000 µM, approximately 50 µM to approximately 1,250 µM, approximately 50 µM to approximately 1,500 µM, approximately 50 µM to approximately 2,000 µM, approximately 50 µM to approximately 4,000 µM, approximately 50 µM to approximately 5,000 µM, approximately 50 µM to approximately 7,500 µM, approximately 100 µM to approximately 250 µM, approximately 100 µM to approximately 500 µM, approximately 100 µM to approximately 750 µM, approximately 100 µM to approximately 1,000 µM, approximately 100 µM to approximately 1,250 µM, approximately 100 µM to approximately 1,500 µM. µM, about 100 µM to about 2,000 µM, about 100 µM to about 4,000 µM, about 100 µM to about 5,000 µM, about 100 µM to about 7,500 µM, about 250 µM to about 500 µM, about 250 µM to about 750 µM, about 250 µM to about 1,000 µM, about 250 µM to about 1,250 µM, about 250 µM to about 1,500 µM, about 250 µM to about 2,000 µM, about 250 µM to about 4,000 µM, about 250 µM to about 5,000 µM, about 250 µM to about 7,500 µM, about 500 µM to about 750 µM, about 500 µM to about 1,000 µM, about 500 µM to about 1,250 µM, about 500 µM approximately 1,500 µM to 2,000 µM, approximately 500 µM to 4,000 µM, approximately 500 µM to 5,000 µM, approximately 500 µM to 7,500 µM, approximately 750 µM to 1,000 µM, approximately 750 µM to 1,250 µM, approximately 750 µM to 1,500 µM, approximately 750 µM to 2,000 µM, approximately 750 µM to 4,000 µM, approximately 750 µM to 5,000 µM, approximately 750 µM to 7,500 µM, approximately 1,000 µM to 1,250 µM, approximately 1,000 µM to 1,500 µM, approximately 1,000 µM to 2,000 µM, approximately 1,000 µM to 4,000 µM µM, about 1,000 µM to about 5,000 µM, about 1,000 µM to about 7,500 µM, about 1,250 µM to about 1,500 µM, about 1,250 µM to about 2,000 µM, about 1,250 µM to about 4,000 µM, about 1,250 µM to about 5,000 µM, about 1,250 µM to about 7,500 µM, about 1,500 µM to about 2,000 µM, about 1,500 µM to about 4,000 µM, about 1,500 µM to about 5,000 µM, about 1,500 µM to about 7,500 µM, about 2,000 µM to about 4,000 µM, about 2,000 µM to about 5,000 µM, about 2,000 µM to about 7,500 µM, about 4,000 µM to about 5,000 µM, about 4,000 µM to about 7,500 µM, or about 5,000 µM to about 7,500 µM.

[0171] In some embodiments, the primer or probe may be a segment of nucleotides that hybridizes to the target nucleic acid sequence. In some embodiments, the primer length is at least about 3 nucleotides, at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 nucleotides. In some implementations, the primer length is up to about 200 nucleotides, up to about 150 nucleotides, up to about 100 nucleotides, up to about 90 nucleotides, up to about 80 nucleotides, up to about 70 nucleotides, up to about 60 nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, up to about 35 nucleotides, up to about 30 nucleotides, up to about 25 nucleotides, up to about 20 nucleotides, up to about 15 nucleotides, up to about 10 nucleotides, up to about 5 nucleotides, or up to about 3 nucleotides.

[0172] In some embodiments, the primer length is from about 3 nucleotides to about 100 nucleotides. In some embodiments, the primer length is at most about 100 nucleotides. In some embodiments, the primer length is from about 3 nucleotides to about 5 nucleotides, from about 3 nucleotides to about 10 nucleotides, from about 3 nucleotides to about 20 nucleotides, from about 3 nucleotides to about 30 nucleotides, from about 3 nucleotides to about 40 nucleotides, from about 3 nucleotides to about 50 nucleotides, from about 3 nucleotides to about 60 nucleotides, from about 3 nucleotides to about 70 nucleotides, from about 3 nucleotides to about 80 nucleotides, from about 3 nucleotides to about 90 nucleotides, from about 3 nucleotides to about 100 nucleotides, from about 5 nucleotides to about 10 nucleotides, from about 5 nucleotides to about 20 nucleotides, from about 5 nucleotides to about 30 nucleotides, from about 5 nucleotides to about 40 nucleotides. Nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 60 nucleotides, about 5 nucleotides to about 70 nucleotides, about 5 nucleotides to about 80 nucleotides, about 5 nucleotides to about 90 nucleotides, about 5 nucleotides to about 100 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 60 nucleotides, about 10 nucleotides to about 70 nucleotides, about 10 nucleotides to about 80 nucleotides, about 10 nucleotides to about 90 nucleotides, about 10 nucleotides to about 100 nucleotides Nucleotides, approximately 20 nucleotides to approximately 30 nucleotides, approximately 20 nucleotides to approximately 40 nucleotides, approximately 20 nucleotides to approximately 50 nucleotides, approximately 20 nucleotides to approximately 60 nucleotides, approximately 20 nucleotides to approximately 70 nucleotides, approximately 20 nucleotides to approximately 80 nucleotides, approximately 20 nucleotides to approximately 90 nucleotides, approximately 20 nucleotides to approximately 100 nucleotides, approximately 30 nucleotides to approximately 40 nucleotides, approximately 30 nucleotides to approximately 50 nucleotides, approximately 30 nucleotides to approximately 60 nucleotides, approximately 30 nucleotides to approximately 70 nucleotides, approximately 30 nucleotides to approximately 80 nucleotides, approximately 30 nucleotides to approximately 90 nucleotides, approximately 30 nucleotides Approximately 100 nucleotides, approximately 40 nucleotides to approximately 50 nucleotides, approximately 40 nucleotides to approximately 60 nucleotides, approximately 40 nucleotides to approximately 70 nucleotides, approximately 40 nucleotides to approximately 80 nucleotides, approximately 40 nucleotides to approximately 90 nucleotides, approximately 40 nucleotides to approximately 100 nucleotides, approximately 50 nucleotides to approximately 60 nucleotides, approximately 50 nucleotides to approximately 70 nucleotides, approximately 50 nucleotides to approximately 80 nucleotides, approximately 50 nucleotides to approximately 90 nucleotides, approximately 50 nucleotides to approximately 100 nucleotides, approximately 60 nucleotides to approximately 70 nucleotides, approximately 60 nucleotides to approximately 80 nucleotides, approximately 60 nucleotides to approximately 90 nucleotides.Approximately 60 nucleotides to approximately 100 nucleotides, approximately 70 nucleotides to approximately 80 nucleotides, approximately 70 nucleotides to approximately 90 nucleotides, approximately 70 nucleotides to approximately 100 nucleotides, approximately 80 nucleotides to approximately 90 nucleotides, approximately 80 nucleotides to approximately 100 nucleotides, or approximately 90 nucleotides to approximately 100 nucleotides.

[0173] In some embodiments, the reaction mixture includes a probe that visualizes the amplified nucleic acid product. In some embodiments, the probe includes a strand displacement probe, an embedded fluorophore, a pH-sensitive dye, and / or a pyrophosphate detection product.

[0174] The reaction mixture described herein may contain excipients. Excipients may include sugars (e.g., monosaccharides, disaccharides, polysaccharides, or any combination thereof). Excipients may include surfactants (e.g., nonoxynol-9). In some embodiments, the excipients include a polymer comprising sucrose crosslinked with epichlorohydrin. The polymer may be polysucrose 400. In some embodiments, the excipients include Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, nonoxynol-9, trehalose, dextran, polysucrose 400, cyclodextrin, or any combination thereof. Excipients may include dithiothreitol (DTT).

[0175] The cyclodextrin excipient can be hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, anionic cyclodextrin, or any combination thereof.

[0176] In some embodiments, the excipients may include Tris, sodium chloride and / or potassium chloride, EDTA, nonyl alcohol ether-9, one or more sugars (e.g., dextran and / or trehalose), polysucrose 400 and / or cyclodextrin in a final concentration.

[0177] In some embodiments, the concentration (e.g., final concentration) of Tris in the excipient is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM. mM, at least about 750 mM, at least about 1000 mM, at least about 1500 mM, at least about 2000 mM or greater than about 2000 mM.

[0178] In some embodiments, the concentration of Tris in the excipient (e.g., the final concentration) is up to about 2000 mM, up to about 1500 mM, up to about 1000 mM, up to about 750 mM, up to about 500 mM, up to about 250 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 19 mM, up to about 18 mM, up to about 17 mM, up to about 16 mM, up to about 15 mM, up to about 14 mM, up to about 13 mM, up to about 12 mM, up to about 11 mM. mM, up to about 10 mM, up to about 9 mM, up to about 8 mM, up to about 7 mM, up to about 6 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM or up to about 1 mM.

[0179] In some embodiments, the concentration of Tris in the excipient (e.g., the final concentration) can be between about 0.0001 M and about 5 M. In some embodiments, the concentration of Tris in the excipient (e.g., the final concentration) can be at most about 5 M. In some embodiments, the concentration of Tris in the excipient (e.g., the final concentration) can be from about 0.0001 M to about 0.0005 M, about 0.0001 M to about 0.001 M, about 0.0001 M to about 0.005 M, about 0.0001 M to about 0.0075 M, about 0.0001 M to about 0.01 M, about 0.0001 M to about 0.025 M, about 0.0001 M to about 0.05 M, about 0.0001 M to about 0.1 M, about 0.0001 M to about 0.5 M, about 0.0001 M to about 1 M, about 0.0001 M to about 5 M, about 0.0005 M to about 0.001 M, about 0.0005 M to about 0.005 M, about 0.0005 M to about 0.0075 M, or about 0.0005 M to about 0.01 M. M, about 0.0005 M to about 0.025 M, about 0.0005 M to about 0.05 M, about 0.0005 M to about 0.1 M, about 0.0005 M to about 0.5 M, about 0.0005 M to about 1 M, about 0.0005 M to about 5 M, about 0.001 M to about 0.005 M, about 0.001 M to about 0.0075 M, about 0.001 M to about 0.01 M, about 0.001 M to about 0.025 M, about 0.001 M to about 0.05 M, about 0.001 M to about 0.1 M, about 0.001 M to about 0.5 M, about 0.001 M to about 1 M, about 0.001 M to about 5 M, about 0.005 M to about 0.0075 M, about 0.005 M to about 0.01 M, about 0.005 M to about 0.025 M, about 0.005 M to about 0.05 M, about 0.005M to about 0.1 M, about 0.005 M to about 0.5 M, about 0.005 M to about 1 M, about 0.005 M to about 5 M, about 0.0075 M to about 0.01 M, about 0.0075 M to about 0.025 M, about 0.0075 M to about 0.05 M, about 0.0075 M to about 0.1 M, about 0.0075 M to about 0.5 M, about 0.0075 M to about 1 M, about 0.0075 M to about 5 M, about 0.01 M to about 0.025 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 5 M, about 0.025 M to approximately 0.0.5 M, approximately 0.025 M to approximately 0.1 M, approximately 0.025 M to approximately 0.5 M, approximately 0.025 M to approximately 1 M, approximately 0.025 M to approximately 5 M, approximately 0.05 M to approximately 0.1 M, approximately 0.05 M to approximately 0.5 M, approximately 0.05 M to approximately 1 M, approximately 0.05 M to approximately 5 M, approximately 0.1 M to approximately 0.5 M, approximately 0.1 M to approximately 1 M, approximately 0.1 M to approximately 5 M, approximately 0.5 M to approximately 1 M, approximately 0.5 M to approximately 5 M, or between approximately 1 M and approximately 5 M.

[0180] In some embodiments, the concentration (e.g., final concentration) of sodium chloride and / or potassium chloride in the excipient is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 250 mM. mM, at least about 500 mM, at least about 750 mM, at least about 1000 mM, at least about 1500 mM, at least about 2000 mM or greater than about 2000 mM.

[0181] In some embodiments, the concentration (e.g., final concentration) of sodium chloride and / or potassium chloride in the excipient is at most about 2000 mM, at most about 1500 mM, at most about 1000 mM, at most about 750 mM, at most about 500 mM, at most about 250 mM, at most about 100 mM, at most about 90 mM, at most about 80 mM, at most about 70 mM, at most about 60 mM, at most about 50 mM, at most about 45 mM, at most about 40 mM, at most about 35 mM, at most about 30 mM, at most about 25 mM, at most about 20 mM, at most about 19 mM, at most about 18 mM, at most about 17 mM, at most about 16 mM, at most about 15 mM, at most about 14 mM, at most about 13 mM, at most about 12 mM. mM, up to about 11 mM, up to about 10 mM, up to about 9 mM, up to about 8 mM, up to about 7 mM, up to about 6 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM or up to about 1 mM.

[0182] In some embodiments, the concentration (e.g., final concentration) of sodium chloride and / or potassium chloride in the excipient can be between about 0.0001 M and about 5 M. In some embodiments, the concentration (e.g., final concentration) of sodium chloride and / or potassium chloride in the excipient can be at most about 5 M. In some embodiments, the concentration (e.g., final concentration) of sodium chloride and / or potassium chloride in the excipient can be from about 0.0001 M to about 0.0005 M, from about 0.0001 M to about 0.001 M, from about 0.0001 M to about 0.005 M, from about 0.0001 M to about 0.0075 M, from about 0.0001 M to about 0.01 M, from about 0.0001 M to about 0.025 M, from about 0.0001 M to about 0.05 M, from about 0.0001 M to about 0.1 M, from about 0.0001 M to about 0.5 M, from about 0.0001 M to about 1 M, from about 0.0001 M to about 5 M, from about 0.0005 M to about 0.001 M, from about 0.0005 M to about 0.005 M, from about 0.0005 M to about 0.0075 M, from about 0.0005 M to about 0.0005 ... M to about 0.01 M, about 0.0005 M to about 0.025 M, about 0.0005 M to about 0.05 M, about 0.0005 M to about 0.1 M, about 0.0005 M to about 0.5 M, about 0.0005 M to about 1 M, about 0.0005 M to about 5 M, about 0.001 M to about 0.005 M, about 0.001 M to about 0.0075 M, about 0.001 M to about 0.01 M, about 0.001 M to about 0.025 M, about 0.001 M to about 0.05 M, about 0.001 M to about 0.1 M, about 0.001 M to about 0.5 M, about 0.001 M to about 1 M, about 0.001 M to about 5 M, about 0.005 M to about 0.0075 M, about 0.005 M to about 0.01 M, about 0.005 M to about 0.025 M, about 0.005 M to about 0.05 M, about 0.005 M to about 0.1 M, about 0.005 M to about 0.5 M, about 0.005 M to about 1 M, about 0.005 M to about 5 M, about 0.0075 M to about 0.01 M, about 0.0075 M to about 0.025 M, about 0.0075 M to about 0.05 M, about 0.0075 M to about 0.1 M, about 0.0075 M to about 0.5 M, about 0.0075 M to about 1 M, about 0.0075 M to about 5 M, about 0.01 M to about 0.025 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 5 M, approximately 0.Between 0.025 M and 0.05 M, between 0.025 M and 0.1 M, between 0.025 M and 0.5 M, between 0.025 M and 1 M, between 0.025 M and 5 M, between 0.05 M and 0.1 M, between 0.05 M and 0.5 M, between 0.05 M and 1 M, between 0.05 M and 5 M, between 0.1 M and 0.5 M, between 0.1 M and 1 M, between 0.1 M and 5 M, between 0.5 M and 1 M, or between 1 M and 5 M.

[0183] In some embodiments, the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient is at least about 0.01 mM, at least about 0.05 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM. mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM, at least about 750 mM, at least about 1000 mM, at least about 1500 mM, at least about 2000 mM or greater than about 2000 mM.

[0184] In some embodiments, the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient is up to about 2000 mM, up to about 1500 mM, up to about 1000 mM, up to about 750 mM, up to about 500 mM, up to about 250 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 19 mM, up to about 18 mM, up to about 17 mM, up to about 16 mM, up to about 15 mM, up to about 14 mM, up to about 13 mM, up to about 12 mM. mM, up to about 11 mM, up to about 10 mM, up to about 9 mM, up to about 8 mM, up to about 7 mM, up to about 6 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM, up to about 1 mM, up to about 0.5 mM, up to about 0.1 mM, up to about 0.05 mM, up to about 0.01 mM or less than about 0.01 mM.

[0185] In some embodiments, the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient can be between about 0.0001 M and about 5 M. In some embodiments, the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient can be at most about 5 M. In some embodiments, the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient can be from about 0.0001 M to about 0.0005 M, from about 0.0001 M to about 0.001 M, from about 0.0001 M to about 0.005 M, from about 0.0001 M to about 0.0075 M, from about 0.0001 M to about 0.01 M, from about 0.0001 M to about 0.025 M, from about 0.0001 M to about 0.05 M, from about 0.0001 M to about 0.1 M, from about 0.0001 M to about 0.5 M, from about 0.0001 M to about 1 M, from about 0.0001 M to about 5 M, from about 0.0005 M to about 0.001 M, from about 0.0005 M to about 0.005 M, from about 0.0005 M to about 0.0075 M, from about 0.0005 M to about 0.0005 M. M to about 0.01 M, about 0.0005 M to about 0.025 M, about 0.0005 M to about 0.05 M, about 0.0005 M to about 0.1 M, about 0.0005 M to about 0.5 M, about 0.0005 M to about 1 M, about 0.0005 M to about 5 M, about 0.001 M to about 0.005 M, about 0.001 M to about 0.0075 M, about 0.001 M to about 0.01 M, about 0.001 M to about 0.025 M, about 0.001 M to about 0.05 M, about 0.001 M to about 0.1 M, about 0.001 M to about 0.5 M, about 0.001 M to about 1 M, about 0.001 M to about 5 M, about 0.005 M to about 0.0075 M, about 0.005 M to about 0.01 M, about 0.005 M to about 0.025 M, about 0.005 M to about 0.05 M, about 0.005 M to about 0.1 M, about 0.005 M to about 0.5 M, about 0.005 M to about 1 M, about 0.005 M to about 5 M, about 0.0075 M to about 0.01 M, about 0.0075 M to about 0.025 M, about 0.0075 M to about 0.05 M, about 0.0075 M to about 0.1 M, about 0.0075 M to about 0.5 M, about 0.0075 M to about 1 M, about 0.0075 M to about 5 M, about 0.01 M to about 0.025 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.0.1 M to approximately 5 M, approximately 0.025 M to approximately 0.05 M, approximately 0.025 M to approximately 0.1 M, approximately 0.025 M to approximately 0.5 M, approximately 0.025 M to approximately 1 M, approximately 0.025 M to approximately 5 M, approximately 0.05 M to approximately 0.1 M, approximately 0.05 M to approximately 0.5 M, approximately 0.05 M to approximately 1 M, approximately 0.05 M to approximately 5 M, approximately 0.1 M to approximately 0.5 M, approximately 0.1 M to approximately 1 M, approximately 0.1 M to approximately 5 M, approximately 0.5 M to approximately 1 M, approximately 0.5 M to approximately 5 M, or between approximately 1 M and approximately 5 M.

[0186] In some embodiments, the concentration (e.g., final concentration) of EDTA in the excipient is at least about 0.01 mM, at least about 0.05 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM. mM, at least about 90 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM, at least about 750 mM, at least about 1000 mM, at least about 1500 mM, at least about 2000 mM or greater than about 2000 mM.

[0187] In some embodiments, the concentration (e.g., final concentration) of EDTA in the excipient is up to about 2000 mM, up to about 1500 mM, up to about 1000 mM, up to about 750 mM, up to about 500 mM, up to about 250 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 19 mM, up to about 18 mM, up to about 17 mM, up to about 16 mM, up to about 15 mM, up to about 14 mM, up to about 13 mM, up to about 12 mM, up to about 11 mM. mM, up to about 10 mM, up to about 9 mM, up to about 8 mM, up to about 7 mM, up to about 6 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM, up to about 1 mM, up to about 0.5 mM, up to about 0.1 mM, up to about 0.05 mM, up to about 0.01 mM or less than about 0.01 mM.

[0188] In some embodiments, the concentration of EDTA in the excipient (e.g., the final concentration) can be between about 0.01 mM and about 5 mM. In some embodiments, the concentration of EDTA in the excipient (e.g., the final concentration) can be at most about 5 mM. In some embodiments, the concentration of EDTA in the excipient (e.g., the final concentration) can be from about 0.01 mM to about 0.05 mM, from about 0.01 mM to about 0.1 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.75 mM, from about 0.01 mM to about 1 mM, from about 0.01 mM to about 1.25 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 1.75 mM, from about 0.01 mM to about 2 mM, from about 0.01 mM to about 3 mM, from about 0.01 mM to about 5 mM, from about 0.05 mM to about 0.1 mM, from about 0.05 mM to about 0.5 mM, from about 0.05 mM to about 0.75 mM, from about 0.05 mM to about 1 mM, or from about 0.05 mM to about 1.25 mM. mM, about 0.05 mM to about 1.5 mM, about 0.05 mM to about 1.75 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 5 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 0.75 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 1.25 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 1.75 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 5 mM, about 0.5 mM to about 0.75 mM, about 0.5 mM to about 1 mM, about 0.5 mM to about 1.25 mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 1.75 mM mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 5 mM, about 0.75 mM to about 1 mM, about 0.75 mM to about 1.25 mM, about 0.75 mM to about 1.5 mM, about 0.75 mM to about 1.75 mM, about 0.75 mM to about 2 mM, about 0.75 mM to about 3 mM, about 0.75 mM to about 5 mM, about 1 mM to about 1.25 mM, about 1 mM to about 1.5 mM, about 1 mM to about 1.75 mM, about 1 mM to about 2 mM, about 1 mM to about 3 mM, about 1 mM to about 5 mM, about 1.25 mM to about 1.5 mM, about 1.25 mM to about 1.75 mM, about 1.25 mM to about 2 mM, about 1.25 mM to about 3 mM, about 1.Between 25 mM and 5 mM, about 1.5 mM and 1.75 mM, about 1.5 mM and 2 mM, about 1.5 mM and 3 mM, about 1.5 mM and 5 mM, about 1.75 mM and 2 mM, about 1.75 mM and 3 mM, about 1.75 mM and 5 mM, about 2 mM and 3 mM, about 2 mM and 5 mM, or about 3 mM and 5 mM.

[0189] In some embodiments, the concentration (e.g., final concentration) of nonoxynol-9 in the excipient is at least about 0.001% v / v, at least about 0.005% v / v, at least about 0.01% v / v, at least about 0.05% v / v, at least about 0.1% v / v, at least about 0.5% v / v, at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, or greater than about 5% v / v. In some embodiments, the concentration of nonoxynol-9 in the excipient ( For example The final concentration is at most about 5% v / v, at most about 4% v / v, at most about 3% v / v, at most about 2% v / v, at most about 1% v / v, at most about 0.5% v / v, at most about 0.1% v / v, at most about 0.05% v / v, at most about 0.01% v / v, at most about 0.005% v / v, at most about 0.001% v / v, or less than about 0.001% v / v.

[0190] In some embodiments, the concentration (e.g., final concentration) of nonoxynol-9 in the excipient can be between about 0.01% v / v and about 5% v / v. In some embodiments, the concentration (e.g., final concentration) of nonoxynol-9 in the excipient can be at most about 5% v / v. In some embodiments, the concentration (e.g., final concentration) of nonoxynol-9 in the excipient can be from about 0.01% v / v to about 0.05% v / v, from about 0.01% v / v to about 0.1% v / v, from about 0.01% v / v to about 0.5% v / v, from about 0.01% v / v to about 0.75% v / v, from about 0.01% v / v to about 1% v / v, from about 0.01% v / v to about 1.25% v / v, from about 0.01% v / v to about 1.5% v / v, from about 0.01% v / v to about 1.75% v / v, from about 0.01% v / v to about 2% v / v, from about 0.01% v / v to about 3% v / v, from about 0.01% v / v to about 5% v / v, or from about 0.05% v / v. v / v to about 0.1% v / v, about 0.05% v / v to about 0.5% v / v, about 0.05% v / v to about 0.75% v / v, about 0.05% v / v to about 1% v / v, about 0.05% v / v to about 1.25% v / v, about 0.05% v / v to about 1.5% v / v, about 0.05% v / v to about 1.75% v / v, about 0.05% v / v to about 2% v / v, about 0.05% v / v to about 3% v / v, about 0.05% v / v to about 5% v / v, about 0.1% v / v to about 0.5% v / v, about 0.1% v / v to about 0.75% v / v, about 0.1% v / v to about 1% v / v %v / v, about 0.1% v / v to about 1.25% v / v, about 0.1% v / v to about 1.5% v / v, about 0.1% v / v to about 1.75% v / v, about 0.1% v / v to about 2% v / v, about 0.1% v / v to about 3% v / v, about 0.1% v / v to about 5% v / v, about 0.5% v / v to about 0.75% v / v, about 0.5% v / v to about 1% v / v, about 0.5% v / v to about 1.25% v / v, about 0.5% v / v to about 1.5% v / v, about 0.5% v / v to about 1.75% v / v, about 0.5% v / v to about 2% v / v, about 0.5% v / v to about 3% v / v, about 0.5% v / v to about 5% v / v, about 0.75% v / v to about 1% v / v, about 0.75% v / v to about 1.25% v / v, about 0.75% v / v to about 1.5% v / v, about 0.75% v / v to about 1.75% v / v, about 0.75% v / v to about 2% v / v, about 0.75% v / v to about 3% v / v, about 0.75% v / v to about 5% v / v, about 1% v / v to about 1.25% v / v, about 1% v / v to about 1.5% v / v, about 1% v / v to about 1.75% v / v, about 1% v / v to about 2% v / v, about 1% v / v to about 3% v / v, about 1% v / v to about 5% v / v, about 1.25% v / v to about 1.5% v / v, about 1.25% v / v to about 1.75% v / v. Between % v / v, approximately 1.25% v / v to approximately 2% v / v, approximately 1.25% v / v to approximately 3% v / v, approximately 1.25% v / v to approximately 5% v / v, approximately 1.5% v / v to approximately 1.75% v / v, approximately 1.5% v / v to approximately 2% v / v, approximately 1.5% v / v to approximately 3% v / v, approximately 1.5% v / v to approximately 5% v / v, approximately 1.75% v / v to approximately 2% v / v, approximately 1.75% v / v to approximately 3% v / v, approximately 1.75% v / v to approximately 5% v / v, approximately 2% v / v to approximately 3% v / v, approximately 2% v / v to approximately 5% v / v, or approximately 3% v / v to approximately 5% v / v.

[0191] In some implementations, the concentration of trehalose in the excipient ( For example The final concentration is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM, at least about 750 mM, at least about 1000 mM. mM, at least about 2000 mM, at least about 3000 mM, at least about 4000 mM, at least about 5000 mM or greater than about 5000 mM.

[0192] In some embodiments, the concentration (e.g., final concentration) of trehalose in the excipient is up to about 5000 mM, up to about 4000 mM, up to about 3000 mM, up to about 2000 mM, up to about 1000 mM, up to about 750 mM, up to about 500 mM, up to about 250 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 19 mM, up to about 18 mM, up to about 17 mM, up to about 16 mM, up to about 15 mM, up to about 14 mM. The concentrations of trehalose in the excipients can be from about 0.001 M to about 5 M. In some embodiments, the concentration of trehalose in the excipients (e.g., the final concentration) can be between about 0.001 M and about 5 M. In some embodiments, the concentration (e.g., final concentration) of trehalose in the excipient can be from about 0.001 M to about 0.005 M, from about 0.001 M to about 0.0075 M, from about 0.001 M to about 0.01 M, from about 0.001 M to about 0.05 M, from about 0.001 M to about 0.075 M, from about 0.001 M to about 0.1 M, from about 0.001 M to about 0.25 M, from about 0.001 M to about 0.5 M, from about 0.001 M to about 0.75 M, from about 0.001 M to about 1 M, from about 0.001 M to about 5 M, from about 0.005 M to about 0.0075 M, from about 0.005 M to about 0.01 M, from about 0.005 M to about 0.05 M, from about 0.005 M to about 0.075 M, from about 0.005 M to about 0.05 M, from about 0.005 M to about 0.075 M, from about 0.005 M to about 0.005 M. M to about 0.1M, about 0.005 M to about 0.25 M, about 0.005 M to about 0.5 M, about 0.005 M to about 0.75 M, about 0.005 M to about 1M, about 0.005 M to about 5 M, about 0.0075 M to about 0.01 M, about 0.0075 M to about 0.05 M, about 0.0075 M to about 0.075 M, about 0.0075 M to about 0.1 M, about 0.0075 M to about 0.25 M, about 0.0075 M to about 0.5 M, about 0.0075 M to about 0.75 M, about 0.0075 M to about 1 M, about 0.0075 M to about 5 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.075 M, about 0.0.1 M to about 0.1 M, about 0.01 M to about 0.25 M, about 0.01 M to about 0.5 M, about 0.01 M to about 0.75 M, about 0.01 M to about 1 M, about 0.01 M to about 5 M, about 0.05 M to about 0.075 M, about 0.05 M to about 0.1 M, about 0.05 M to about 0.25 M, about 0.05 M to about 0.5 M, about 0.05 M to about 0.75 M, about 0.05 M to about 1 M, about 0.05 M to about 5 M, about 0.075 M to about 0.1 M, about 0.075 M to about 0.25 M, about 0.075 M to about 0.5 M, about 0.075 M to about 0.75 M, about 0.075 M to about 1 M, about 0.075 M to about 5 M, about 0.1 M to about 0.25 M, about 0.1 M to approximately 0.5 M, approximately 0.1 M to approximately 0.75 M, approximately 0.1 M to approximately 1 M, approximately 0.1 M to approximately 5 M, approximately 0.25 M to approximately 0.5 M, approximately 0.25 M to approximately 0.75 M, approximately 0.25 M to approximately 1 M, approximately 0.25 M to approximately 5 M, approximately 0.5 M to approximately 0.75 M, approximately 0.5 M to approximately 1 M, approximately 0.5 M to approximately 5 M, approximately 0.75 M to approximately 1 M, approximately 0.75 M to approximately 5 M, or between approximately 1 M and approximately 5 M.

[0193] In some embodiments, the concentration (e.g., final concentration) of dextran in the excipient is at least about 0.001% w / v, at least about 0.005% w / v, at least about 0.01% w / v, at least about 0.05% w / v, at least about 0.1% w / v, at least about 0.5% w / v, at least about 1% w / v, at least about 2% w / v, at least about 3% w / v, at least about 4% w / v, at least about 5% w / v, at least about 7.5% w / v, at least about 10% w / v, at least about 15% w / v, or greater than about 15% w / v. In some embodiments, the concentration of dextran in the excipient ( For example The final concentration is up to about 15% w / v, up to about 10% w / v, up to about 7.5% w / v, up to about 5% w / v, up to about 4% w / v, up to about 3% w / v, up to about 2% w / v, up to about 1% w / v, up to about 0.5% w / v, up to about 0.1% w / v, up to about 0.05% w / v, up to about 0.01% w / v, up to about 0.005% w / v, up to about 0.001% w / v, or less than about 0.001% w / v.

[0194] In some embodiments, the concentration of dextran in the excipient (e.g., the final concentration) can be between about 0.1% w / v and about 8% w / v. In some embodiments, the concentration of dextran in the excipient (e.g., the final concentration) can be at most about 8% w / v. In some embodiments, the concentration of dextran in the excipient (e.g., the final concentration) can be from about 0.1% w / v to about 0.5% w / v, from about 0.1% w / v to about 1% w / v, from about 0.1% w / v to about 1.5% w / v, from about 0.1% w / v to about 2% w / v, from about 0.1% w / v to about 2.5% w / v, from about 0.1% w / v to about 3% w / v, from about 0.1% w / v to about 3.5% w / v, from about 0.1% w / v to about 4% w / v, from about 0.1% w / v to about 5% w / v, from about 0.1% w / v to about 6% w / v, from about 0.1% w / v to about 8% w / v, from about 0.5% w / v to about 1% w / v, or from about 0.5% w / v to about 1.5%. w / v, about 0.5% w / v to about 2% w / v, about 0.5% w / v to about 2.5% w / v, about 0.5% w / v to about 3% w / v, about 0.5% w / v to about 3.5% w / v, about 0.5% w / v to about 4% w / v, about 0.5% w / v to about 5% w / v, about 0.5% w / v to about 6% w / v, about 0.5% w / v to about 8% w / v, about 1% w / v to about 1.5% w / v, about 1% w / v to about 2% w / v, about 1% w / v to about 2.5% w / v, about 1% w / v to about 3% w / v, about 1% w / v to about 3.5% w / v, about 1% w / v to about 4% w / v, about 1% w / v to about 5% w / v % w / v, about 1% w / v to about 6% w / v, about 1% w / v to about 8% w / v, about 1.5% w / v to about 2% w / v, about 1.5% w / v to about 2.5% w / v, about 1.5% w / v to about 3% w / v, about 1.5% w / v to about 3.5% w / v, about 1.5% w / v to about 4% w / v, about 1.5% w / v to about 5% w / v, about 1.5% w / v to about 6% w / v, about 1.5% w / v to about 8% w / v, about 2% w / v to about 2.5% w / v, about 2% w / v to about 3% w / v, about 2% w / v to about 3% w / v.5% w / v, about 2% w / v to about 4% w / v, about 2% w / v to about 5% w / v, about 2% w / v to about 6% w / v, about 2% w / v to about 8% w / v, about 2.5% w / v to about 3% w / v, about 2.5% w / v to about 3.5% w / v, about 2.5% w / v to about 4% w / v, about 2.5% w / v to about 5% w / v, about 2.5% w / v to about 6% w / v, about 2.5% w / v to about 8% w / v, about 3% w / v to about 3.5% w / v, about 3% w / v to about 4% w / v, about 3% w / v to about 5% w / v, about 3% w / v to about 6% w / v, about 3% w / v to about 8% w / v w / v, approximately 3.5% w / v to approximately 4% w / v, approximately 3.5% w / v to approximately 5% w / v, approximately 3.5% w / v to approximately 6% w / v, approximately 3.5% w / v to approximately 8% w / v, approximately 4% w / v to approximately 5% w / v, approximately 4% w / v to approximately 6% w / v, approximately 4% w / v to approximately 8% w / v, approximately 5% w / v to approximately 6% w / v, approximately 5% w / v to approximately 8% w / v, or between approximately 6% w / v and approximately 8% w / v.

[0195] In some embodiments, the concentration (e.g., final concentration) of polysucrose 400 in the excipient is at least about 0.001% w / v, at least about 0.005% w / v, at least about 0.01% w / v, at least about 0.05% w / v, at least about 0.1% w / v, at least about 0.5% w / v, at least about 1% w / v, at least about 2% w / v, at least about 3% w / v, at least about 4% w / v, at least about 5% w / v, at least about 7.5% w / v, at least about 10% w / v, at least about 15% w / v, or greater than about 15% w / v (g solute / 100 mL solution). In some embodiments, the concentration (e.g., final concentration) of polysucrose 400 in the excipient is up to about 15% w / v, up to about 10% w / v, up to about 7.5% w / v, up to about 5% w / v, up to about 4% w / v, up to about 3% w / v, up to about 2% w / v, up to about 1% w / v, up to about 0.5% w / v, up to about 0.1% w / v, up to about 0.05% w / v, up to about 0.01% w / v, up to about 0.005% w / v, up to about 0.001% w / v, or less than about 0.001% w / v (g solute / 100 mL solution).

[0196] In some embodiments, the concentration (e.g., final concentration) of polysucrose 400 in the excipient can be between about 0.001% w / v and about 5% w / v. In some embodiments, the concentration (e.g., final concentration) of polysucrose 400 in the excipient can be at most about 5% w / v. In some embodiments, the concentration (e.g., final concentration) of sucrose 400 in the excipient can be from about 0.001% w / v to about 0.01% w / v, from about 0.001% w / v to about 0.1% w / v, from about 0.001% w / v to about 0.2% w / v, from about 0.001% w / v to about 0.3% w / v, from about 0.001% w / v to about 0.4% w / v, from about 0.001% w / v to about 0.5% w / v, from about 0.001% w / v to about 0.75% w / v, from about 0.001% w / v to about 1% w / v, from about 0.001% w / v to about 2% w / v, from about 0.001% w / v to about 3% w / v, or from about 0.001% w / v to about 5%. w / v, about 0.01% w / v to about 0.1% w / v, about 0.01% w / v to about 0.2% w / v, about 0.01% w / v to about 0.3% w / v, about 0.01% w / v to about 0.4% w / v, about 0.01% w / v to about 0.5% w / v, about 0.01% w / v to about 0.75% w / v, about 0.01% w / v to about 1% w / v, about 0.01% w / v to about 2% w / v, about 0.01% w / v to about 3% w / v, about 0.01% w / v to about 5% w / v, about 0.1% w / v to about 0.2% w / v, about 0.1% w / v to about 0.3% w / v, about 0.1% w / v to about 0.4% w / v, about 0.1% w / v to about 0.5% w / v, about 0.1% w / v to about 0.75% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 2% w / v, about 0.1% w / v to about 3% w / v, about 0.1% w / v to about 5% w / v, about 0.2% w / v to about 0.3% w / v, about 0.2% w / v to about 0.4% w / v, about 0.2% w / v to about 0.5% w / v, about 0.2% w / v to about 0.75% w / v, about 0.2% w / v to about 1% w / v, about 0.2% w / v to about 2% w / v, about 0.2% w / v to about 3% w / v w / v, about 0.2% w / v to about 5% w / v, about 0.3% w / v to about 0.4% w / v, about 0.3% w / v to about 0.5% w / v, about 0.3% w / v to about 0.75% w / v, about 0.3% w / v to about 1% w / v, about 0.3% w / v to about 2% w / v, about 0.3% w / v to about 3% w / v, about 0.3% w / v to about 5% w / v, about 0.4% w / v to about 0.5% w / v, about 0.4% w / v to about 0.75% w / v, about 0.4% w / v to about 1% w / v, about 0.4% w / v to about 2% w / v, about 0.4% w / v to about 3% w / v, about 0.4% w / v to about 5% w / v, about 0.5% w / v to about 0.75% w / v, about 0.5% w / v to about 1% w / v w / v, approximately 0.5% w / v to approximately 2% w / v, approximately 0.5% w / v to approximately 3% w / v, approximately 0.5% w / v to approximately 5% w / v, approximately 0.75% w / v to approximately 1% w / v, approximately 0.75% w / v to approximately 2% w / v, approximately 0.75% w / v to approximately 3% w / v, approximately 0.75% w / v to approximately 5% w / v, approximately 1% w / v to approximately 2% w / v, approximately 1% w / v to approximately 3% w / v, approximately 1% w / v to approximately 5% w / v, approximately 2% w / v to approximately 3% w / v, approximately 2% w / v to approximately 5% w / v, or between approximately 3% w / v and approximately 5% w / v.

[0197] In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the excipient is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 250 mM, at least about 500 mM. mM, at least about 750 mM, at least about 1000 mM, at least about 2000 mM, at least about 3000 mM, at least about 4000 mM, at least about 5000 mM or greater than about 5000 mM.

[0198] In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the excipient is up to about 5000 mM, up to about 4000 mM, up to about 3000 mM, up to about 2000 mM, up to about 1000 mM, up to about 750 mM, up to about 500 mM, up to about 250 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM, up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 19 mM, up to about 18 mM, up to about 17 mM, up to about 16 mM, up to about 15 mM, up to about 14 mM. mM, up to about 13 mM, up to about 12 mM, up to about 11 mM, up to about 10 mM, up to about 9 mM, up to about 8 mM, up to about 7 mM, up to about 6 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM or up to about 1 mM.

[0199] In some embodiments, the concentration of cyclodextrin in the excipient (e.g., the final concentration) can be between about 0.001 M and about 5 M. In some embodiments, the concentration of cyclodextrin in the excipient (e.g., the final concentration) can be at most about 5 M.In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the excipient can be from about 0.001 M to about 0.005 M, about 0.001 M to about 0.01 M, about 0.001 M to about 0.02 M, about 0.001 M to about 0.03 M, about 0.001 M to about 0.04 M, about 0.001 M to about 0.05 M, about 0.001 M to about 0.1 M, about 0.001 M to about 0.5 M, about 0.001 M to about 1 M, about 0.001 M to about 3 M, about 0.001 M to about 5 M, about 0.005 M to about 0.01 M, about 0.005 M to about 0.02 M, about 0.005 M to about 0.03 M, about 0.005 M to about 0.04 M, about 0.005 M to about 0.05 M, or about 0.005 M to about 0.1 M. M, about 0.005 M to about 0.5 M, about 0.005 M to about 1 M, about 0.005 M to about 3 M, about 0.005 M to about 5 M, about 0.01 M to about 0.02 M, about 0.01 M to about 0.03 M, about 0.01 M to about 0.04 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 3 M, about 0.01 M to about 5 M, about 0.02 M to about 0.03 M, about 0.02 M to about 0.04 M, about 0.02 M to about 0.05 M, about 0.02 M to about 0.1 M, about 0.02 M to about 0.5 M, about 0.02 M to about 1 M, about 0.02 M to about 3 M, about 0.02 M to about 5 M, about 0.03 M to about 0.04 M, about 0.03 M to about 0.05 M, about 0.03 M to about 0.1 M, about 0.03 M to about 0.5 M, about 0.03 M to about 1 M, about 0.03 M to about 3 M, about 0.03 M to about 5 M, about 0.04 M to about 0.05 M, about 0.04 M to about 0.1 M, about 0.04 M to about 0.5 M, about 0.04 M to about 1 M, about 0.04 M to about 3 M, about 0.04 M to about 5 M, about 0.05 M to about 0.1 M, about 0.05 M to about 0.5 M, about 0.05 M to about 1 M, about 0.05 M to about 3 M, about 0.05 M to about 5 M, about 0.1 M to about 0.5 M, about 0.1 M to about 1 M, about 0.1 M to about 3 M, about 0.1 M to about 5 M, about 0.5 Between M and 1 M, between 0.5 M and 3 M, between 0.5 M and 5 M, between 1 M and 3 M, between 1 M and 5 M, or between 3 M and 5 M.

[0200] The excipient may contain at least one additional reagent (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more additional reagents). In some embodiments, the additional reagent may include a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof. The additional reagent of the excipient may be configured to stabilize the enzyme. The additional reagent may reduce the Cq value of nucleic acid amplification. In some embodiments, nucleic acid amplification according to the composition containing the excipient described herein may have a lower Cq value compared to nucleic acid amplification without the composition (containing the excipient).

[0201] The compositions described herein may further comprise a sample stabilizing buffer. The sample stabilizing buffer may comprise one or more reagents. These reagents may be collapse modifiers, protein stabilizers, glass transition modifiers, or any combination thereof. In some embodiments, the sample stabilizing buffer may comprise at least one salt (e.g., 1, 2, 3, 4, 5, or more salts). The sample stabilizing buffer may comprise cyclodextrin, wherein the cyclodextrin may be the cyclodextrin described herein and / or a concentration of cyclodextrin. One or more reagents of the sample stabilizing buffer may be optimized for lyophilization. The sample stabilizing buffer may be configured to reconstruct lyophilized samples. Application of the sample stabilizing buffer can reconstruct lyophilized samples and provide improved nucleic acid amplification of the samples. In some embodiments, the sample stabilizing buffer comprises one or more reducing agents. These reducing agents may be oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP), or any combination thereof.

[0202] Without being bound by theory, the composition provided in this paper can stabilize nucleic acids during nucleic acid amplification, thereby improving the accuracy and / or efficiency of amplification.

[0203] In some embodiments, the compositions described herein (e.g., recovery buffers) may contain one or more cucurbiturils. Cucurbiturils are macrocyclic molecules composed of glycourea monomers linked by methylene bridges. Cucurbiturils can form host-guest complexes in the compositions described herein. Not wishing to be bound by theory, cucurbiturils may be advantageous in sample preparation because they host one or more inhibitory substances in a sample (e.g., a PAX sample), allowing the sample to be run directly without purification. As used herein, a PAX sample refers to a sample collected from a PAX gene. ®Samples are collected in tubes (or PAX tubes as used here). ® The samples in the tubes can be used directly for further sample processing or for amplification reactions using the amplification buffer described herein. PAXgene ® Blood RNA tubes contain one or more solutions formulated to stabilize intracellular RNA. In some cases, PAXgene... ® Blood RNA tubes may contain tetradecyltrimethylammonium oxalate and / or tartaric acid. For use with PAXgene ® Blood RNA tubes, samples can undergo centrifugation. The compositions and / or methods described herein, containing one or more buffers, can eliminate the need for centrifugation of samples for sample processing, sample stabilization, and / or sample amplification. In some embodiments, with PAXgene... ® Compared to the blood RNA system, the compositions and / or methods described herein, which include one or more buffers as described herein, can improve the stability of genetic material (e.g., RNA). Using the compositions and / or methods described herein can enhance the stability of genetic material (e.g., RNA) to improve the quality of amplified genetic products, reduce the time required to generate amplified products, or any combination thereof.

[0204] Cucurbita may contain one or more glycoure units (e.g., glycoure monomers). In some embodiments, the cucurbita of the compositions described herein (e.g., recovery buffers) may contain at least about 1 glycoure unit, at least about 2 glycoure units, at least about 3 glycoure units, at least about 4 glycoure units, at least about 5 glycoure units, at least about 6 glycoure units, at least about 7 glycoure units, at least about 8 glycoure units, at least about 9 glycoure units, at least about 10 glycoure units, or more than about 10 glycoure units. Cucurbita may be abbreviated as cucurbit[n]ure, where n is an integer indicating the number of glycoure units. In some embodiments, the compositions described herein (e.g., recovery buffers) may contain cucurbit[1]ure, cucurbit[2]ure, cucurbit[3]ure, cucurbit[4]ure, cucurbit[5]ure, cucurbit[6]ure, cucurbit[7]ure, cucurbit[8]ure, cucurbit[9]ure, or cucurbit

[10] ure.

[0205] In some respects, this document provides compositions for sample amplification. Compositions for sample amplification may contain nonionic surfactants, cyclodextrins, sucrose / epicochlorohydrin polymers, or any combination thereof. The compositions may be configured to increase the amplification rate. Amplification may be nucleic acid amplification (e.g., PCR or isothermal nucleic acid amplification).

[0206] For example, a composition for sample amplification may comprise: a nonionic surfactant, cyclodextrin, and a sucrose / epicochlorohydrin polymer, wherein the composition is configured to increase the amplification rate during nucleic acid amplification.

[0207] The composition for sample amplification can be configured to stabilize one or more enzymes (e.g., thermostable enzymes). The enzyme can be stabilized during amplification (e.g., nucleic acid amplification). The enzyme can be a polymerase, endonuclease, or reverse transcriptase, or any combination thereof. In some embodiments, the reverse transcriptase can be avian myeloid leukemia virus (AMV) reverse transcriptase or murine leukemia virus (MMLV) reverse transcriptase. The nonionic surfactant of the composition for sample amplification can be nonoxynol-9.

[0208] Compositions for sample amplification may contain cyclodextrin as described herein. Compositions for sample amplification may contain a concentration of cyclodextrin. In some embodiments, cyclodextrin is present at a final concentration in the presence of the sample. In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the composition in the presence of the sample is at least about 0.05 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 5.0 mM, at least about 10.0 mM, at least about 15.0 mM, at least about 20.0 mM, at least about 25.0 mM, at least about 30.0 mM, at least about 35.0 mM, at least about 40.0 mM, at least about 50.0 mM, at least about 55.0 mM, at least about 60.0 mM, at least about 65.0 mM, at least about 70.0 mM, at least about 75.0 mM, at least about 80.0 mM, at least about 85.0 mM, at least about 90.0 mM, at least about 95.0 mM, at least about 100.0 mM, at least about 500 mM, at least about 1000 mM. mM, at least about 2500 mM, at least about 5000 mM, at least about 7500 mM, at least about 10000 mM, at least about 20000 mM or greater than about 20000 mM. In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the composition in the presence of the sample is at most about 20,000 mM, at most about 10,000 mM, at most about 7,500 mM, at most about 5,000 mM, at most about 2,500 mM, at most about 1,000 mM, at most about 500 mM, at most about 100.0 mM, at most about 95.0 mM, at most about 90.0 mM, at most about 85.0 mM, at most about 80.0 mM, at most about 75.0 mM, at most about 70.0 mM, at most about 65.0 mM, at most about 60.0 mM, at most about 55.0 mM, at most about 50.0 mM, at most about 45.0 mM, at most about 40.0 mM, at most about 35.0 mM, at most about 30.0 mM. mM, up to about 25.0 mM, up to about 20.0 mM, up to about 15.0 mM, up to about 10.0 mM, up to about 5.0 mM, up to about 1.0 mM, up to about 0.5 mM, up to about 0.1 mM or up to about 0.05 mM.

[0209] In some embodiments, the concentration of cyclodextrin in the composition in the presence of a sample ( For exampleThe final concentration is at least about 0.001% v / v, at least about 0.005% v / v, at least about 0.01% v / v, at least about 0.05% v / v, at least about 0.1% v / v, at least about 0.5% v / v, at least about 1.0% v / v, at least about 1.5% v / v, at least about 2.0% v / v, at least about 3.0% v / v, at least about 4.0% v / v, at least about 5.0% v / v, or greater than about 5.0% v / v. In some embodiments, the concentration of cyclodextrin in the composition in the presence of the sample ( For example The final concentration is up to about 5.0% v / v, up to about 4.0% v / v, up to about 3.0% v / v, up to about 2.0% v / v, up to about 1.5% v / v, up to about 1.0% v / v, up to about 0.5% v / v, up to about 0.1% v / v, up to about 0.05% v / v, up to about 0.01% v / v, up to about 0.005% v / v, up to about 0.001% v / v, or less than about 0.001% v / v.

[0210] The sucrose / epicochlorohydrin polymer in the composition may be polysucrose 400. In some embodiments, the concentration of polysucrose 400 in the composition ( For example The final concentration is at least about 0.001% w / v, at least about 0.005% w / v, at least about 0.01% w / v, at least about 0.05% w / v, at least about 0.1% w / v, at least about 0.5% w / v, at least about 1% w / v, at least about 2% w / v, at least about 3% w / v, at least about 4% w / v, at least about 5% w / v, at least about 7.5% w / v, at least about 10% w / v, at least about 15% w / v, or greater than about 15% w / v (g solute / 100 mL solution). In some embodiments, the concentration of sucrose 400 in the composition ( For example The final concentration is up to about 15% w / v, up to about 10% w / v, up to about 7.5% w / v, up to about 5% w / v, up to about 4% w / v, up to about 3% w / v, up to about 2% w / v, up to about 1% w / v, up to about 0.5% w / v, up to about 0.1% w / v, up to about 0.05% w / v, up to about 0.01% w / v, up to about 0.005% w / v, up to about 0.001% w / v, or less than about 0.001% w / v (g solute / 100 mL solution).

[0211] The composition used for sample amplification may contain one or more salts (e.g., 1, 2, 3, 4, 5 or more salts). The salts may be sodium chloride, potassium chloride, potassium phosphate, or any combination thereof. The final concentration of at least one salt in the composition in the presence of the sample may be at least about 0.0001 M, at least about 0.001 M, at least about 0.005 M, at least about 0.01 M, at least about 0.05 M, at least about 0.1 M, at least about 0.5 M, at least about 1.0 M, at least about 2.0 M, at least about 3.0 M, at least about 4.0 M, at least about 5.0 M, at least about 6.0 M, at least about 7.0 M, at least about 8.0 M, at least about 9.0 M, at least about 10 M, at least about 15 M, or greater than about 15 M. The final concentration of at least one salt in the composition in the presence of the sample may be at most about 15 M, at most about 10 M, at most about 9.0 M, at most about 8.0 M, at most about 7.0 M, or greater than about 7.0 M. M, up to about 6.0 M, up to about 5.0 M, up to about 4.0 M, up to about 3.0 M, up to about 2.0 M, up to about 1.0 M, up to about 0.5 M, up to about 0.1 M, up to about 0.05 M, up to about 0.01 M, up to about 0.005 M, up to about 0.001 M, up to about 0.0001 M or less than about 0.0001 M.

[0212] In some embodiments, the composition may comprise etanercept (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformin, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), tetrahydropyran (THP), or any combination thereof. The composition may comprise a concentration of EDTA and / or Tris as described herein. Compositions for sample amplification may contain one or more agents capable of reducing disulfide bonds (e.g., dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethanol (βME), or any combination thereof).

[0213] The composition may comprise one or more sugars and / or sugar alcohols. The one or more sugars and / or sugar alcohols may include sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof. In some embodiments, the composition described herein may comprise a final concentration of sugars and / or sugar alcohols of at least about 0.0001 M, at least about 0.001 M, at least about 0.005 M, at least about 0.01 M, at least about 0.05 M, at least about 0.1 M, at least about 0.5 M, at least about 1.0 M, at least about 2.0 M, at least about 3.0 M, at least about 4.0 M, at least about 5.0 M, at least about 6.0 M, at least about 7.0 M, at least about 8.0 M, at least about 9.0 M, at least about 10 M, at least about 15 M, or greater than about 15 M. In some embodiments, the compositions described herein may comprise a final concentration of sugars and / or sugar alcohols of up to about 15 M, up to about 10 M, up to about 9.0 M, up to about 8.0 M, up to about 7.0 M, up to about 6.0 M, up to about 5.0 M, up to about 4.0 M, up to about 3.0 M, up to about 2.0 M, up to about 1.0 M, up to about 0.5 M, up to about 0.1 M, up to about 0.05 M, up to about 0.01 M, up to about 0.005 M, up to about 0.001 M, up to about 0.0001 M, or less than about 0.0001 M.

[0214] In some embodiments, the compositions described herein may comprise a final concentration of sugars and / or sugar alcohols of at least about 0.001% w / v, at least about 0.005% w / v, at least about 0.01% w / v, at least about 0.05% w / v, at least about 0.1% w / v, at least about 0.5% w / v, at least about 1% w / v, at least about 2% w / v, at least about 3% w / v, at least about 4% w / v, at least about 5% w / v, at least about 7.5% w / v, at least about 10% w / v, at least about 15% w / v, or greater than about 15% w / v (g solute / 100 mL solution). In some embodiments, the compositions described herein may contain a final concentration of sugars and / or sugar alcohols of up to about 15% w / v, up to about 10% w / v, up to about 7.5% w / v, up to about 5% w / v, up to about 4% w / v, up to about 3% w / v, up to about 2% w / v, up to about 1% w / v, up to about 0.5% w / v, up to about 0.1% w / v, up to about 0.05% w / v, up to about 0.01% w / v, up to about 0.005% w / v, up to about 0.001% w / v, or less than about 0.001% w / v (g solute / 100 mL solution).

[0215] Compositions for sample amplification may contain at least one additional reagent (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more additional reagents). In some embodiments, the additional reagent may include a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof. The compositions described herein may be lyophilized. Compositions for sample amplification may be configured to stabilize the enzymatic activity of a thermostable enzyme for use during nucleic acid amplification. The thermostable enzyme may be a large fragment of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, IsoFast, etc. TM Bst, any of their mutants, or any combination thereof.

[0216] In some respects, this document provides compositions comprising sample processing buffers. Sample processing buffers may comprise one or more of detergents, solubilizers, or cyclodextrins. Compositions may comprise sample amplification buffers. Sample amplification buffers may comprise one or more of nonionic surfactants, cyclodextrins, or sucrose / epicochlorohydrin polymers. Compositions may comprise sample stabilizing buffers. Sample stabilizing buffers may be configured to stabilize one or more enzymes. Enzymes may be used in nucleic acid amplification.

[0217] For example, the composition may include: a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose / epicochlorohydrin polymer; and a sample stabilizing buffer configured to stabilize enzymes during nucleic acid amplification.

[0218] The solubilizer and cyclodextrin in the composition can be configured to shorten the cycle threshold (Ct) and / or result time. In some cases, quantitative cycle Cq values ​​are also used and can be used interchangeably with Ct values. In some embodiments, the solubilizer and cyclodextrin in the composition can be configured to shorten the cycle threshold (Ct) and / or result time in nucleic acid amplification compared to the cycle threshold (Ct) and / or result time in nucleic acid amplification of samples with the same other aspects treated by SDS, polysorbate 80, or cyclodextrin alone. In some implementations, the solubilizers and / or cyclodextrins described herein are configured to reduce the cycling threshold to at most about 60, at most about 50, at most about 40, at most about 30, at most about 25, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1. In some implementations, the solubilizers and / or cyclodextrins described herein are configured to reduce the result time value to at most about 15 minutes, at most about 14 minutes, at most about 13 minutes, at most about 12 minutes, at most about 11 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, at most about 5 minutes or less.

[0219] The detergent may be part of a lysis buffer, wherein the lysis buffer may contain a chelating agent. The chelating agent may be deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), or any combination thereof. The composition may contain a reducing agent. The reducing agent may be oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), tetrahydropyran (THP), or any combination thereof. The lysis buffer may contain any compound (e.g., component) as described herein. The composition may contain the final concentrations of EGTA, EDTA, TCEP, and / or Tris in the lysis buffer in the presence of the sample. The final concentrations of EGTA, EDTA, TCEP, and / or Tris can be as described in this document.

[0220] The concentration of Tris in the recovery buffer described herein may be greater than the concentration of Tris after mixing with the sample. For example, the concentration of Tris in the recovery buffer described herein may be at least about 100 mM, at least about 200 mM, at least about 300 mM, at least about 400 mM, at least about 500 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, at least about 1000 mM, at least about 1500 mM, at least about 2000 mM, or greater than about 2000 mM. The concentration of Tris in the recovery buffer described herein can be up to about 2000 mM, up to about 1500 mM, up to about 1000 mM, up to about 900 mM, up to about 800 mM, up to about 700 mM, up to about 600 mM, up to about 500 mM, up to about 400 mM, up to about 300 mM, up to about 200 mM, up to about 100 mM, or less than about 100 mM.

[0221] The detergent may be present at a concentration sufficient to lyse cells (e.g., the final concentration). For example, the detergent in a sample processing buffer can lyse cells when mixed with a sample described herein. The cyclodextrin may be present at a concentration sufficient to isolate the detergent in the composition (e.g., the final concentration). For example, the cyclodextrin in a sample processing buffer can isolate the detergent (e.g., a portion of the detergent) in the composition when mixed with a sample described herein. The final concentrations of the detergent, solubilizer, and / or cyclodextrin may include the final concentrations described herein.

[0222] As another example, the composition for sample processing may include a sample amplification buffer comprising: a nonionic surfactant, cyclodextrin and a sucrose / epicochlorohydrin polymer; a stabilizer comprising tetradecyltrimethylammonium oxalate and / or tartaric acid, and wherein the composition is configured to increase the amplification rate during nucleic acid amplification.

[0223] As another example, the composition for sample processing may include a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; a stabilizer comprising tetradecyltrimethylammonium oxalate and / or tartaric acid; and wherein the composition is configured to stabilize an enzyme during nucleic acid amplification, and wherein the composition is configured to reduce and / or eliminate the activity of the nuclease.

[0224] The composition may contain one or more cucurbiturils. The cucurbituril may contain one or more glyurene units (e.g., glyurene monomers). In some embodiments, the cucurbituril of the composition described herein may contain at least about 1 glyurene unit, at least about 2 glyurene units, at least about 3 glyurene units, at least about 4 glyurene units, at least about 5 glyurene units, at least about 6 glyurene units, at least about 7 glyurene units, at least about 8 glyurene units, at least about 9 glyurene units, at least about 10 glyurene units, or more than about 10 glyurene units. Cucurbituril may be denoted as cucurbit[n]urene, where n is an integer indicating the number of glyurene units. In some embodiments, the composition described herein may contain cucurbit[1]urene, cucurbit[2]urene, cucurbit[3]urene, cucurbit[4]urene, cucurbit[5]urene, cucurbit[6]urene, cucurbit[7]urene, cucurbit[8]urene, cucurbit[9]urene, or cucurbit

[10] urene.

[0225] The composition may further comprise a sample. The sample may be a biological sample. The sample may be a blood sample, swab sample, saliva sample, urine sample, cerebrospinal fluid sample, pleural fluid sample, rectal sample, vaginal sample, fecal sample, sputum sample and / or lymph sample, raw emulsion, pasteurized and / or homogenized emulsion, pasteurized and / or treated emulsion, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue sample, cell culture, purified nucleic acid sample, environmental sample, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof.

[0226] Blood samples can be obtained from the subject. Blood samples can be obtained at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, or more than about 24 hours before sample processing. Blood samples can be collected in blood collection tubes. In some embodiments, the blood collection tubes may contain a stabilizer. The stabilizer can stabilize genetic material (e.g., DNA and / or RNA). In some embodiments, the stabilizer may contain tetradecyltrimethylammonium oxalate, tartaric acid, or any combination thereof. In some embodiments, the blood sample may be contacted with the sample processing buffer described herein. Blood samples may be in direct contact with the sample processing buffer. Blood samples may not be processed before contact with the sample processing buffer (e.g., contacted with another sample processing composition and / or method). Blood samples may be processed before contact with the sample processing buffer (e.g., contacted with another sample processing composition and / or method). In some embodiments, blood samples may not be processed by centrifugation, spin column, or any combination thereof before contact with the sample processing buffer described herein. In other embodiments, blood samples may be processed by centrifugation, spin column, or any combination thereof before contact with the sample processing buffer described herein.

[0227] Methods for sample processing, stabilization, and amplification In some aspects, this disclosure provides methods for processing samples. In some aspects, the method includes mixing the sample with a lysis buffer described herein. In some embodiments, the method includes mixing the sample with a recovery buffer described herein. In some embodiments, the method includes mixing the sample in a mixture of the lysis buffer and the recovery buffer described herein.

[0228] In one aspect, this disclosure provides a method for processing a sample, comprising (a) contacting the sample with a lysis buffer containing a detergent, and (b) contacting the sample with a recovery buffer containing a solubilizer and cyclodextrin, thereby processing the sample to generate a treated sample in a mixture containing detergent, solubilizer, and cyclodextrin. In some cases, the method includes contacting the lysis buffer in a first step and subsequently contacting the sample mixed with the lysis buffer with a recovery buffer to provide a treated sample. The treated sample may then be contacted with a reaction buffer for amplification. The lysis buffer, recovery buffer, and amplification buffer may be contacted with the sample in different steps. Separating the lysis buffer and recovery buffer can provide a flexible system in which the treated sample can be compatible with any existing amplification reaction, such as PCR or isothermal amplification.

[0229] In some embodiments, the sample is obtained from the subject prior to the sample processing method of this disclosure. In some embodiments, the subject has or is suspected of having a disease, condition, symptom, or infection. In some embodiments, the subject is a non-infected individual. In some embodiments, the subject is a healthy subject.

[0230] In some embodiments, the sample includes blood samples, swab samples, saliva samples, urine samples, cerebrospinal fluid samples, pleural fluid samples, rectal samples, vaginal samples, fecal samples, sputum samples, and / or lymph samples for nucleic acid amplification. In some embodiments, the swab sample includes vaginal swabs, oral swabs, and / or rectal swabs. In some embodiments, the sample includes raw emulsion, pasteurized and / or homogenized emulsion, pasteurized and / or treated emulsion, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue samples, cell cultures, purified nucleic acid samples, environmental samples, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject has a disease, symptom, or infection. In some embodiments, the sample includes a biological sample. In some embodiments, the sample includes a purified sample. In some embodiments, the sample is a combination of two, three, four, five, or more types of samples. In some implementation schemes, the sample contains one, two, three, four, five, six, seven, eight, nine, ten or more target nucleic acid molecules.

[0231] The sample may come into contact with the lysis buffer. The sample may be a liquid or a solid sample. In some cases, the sample is a liquid sample and may be mixed with the lysis buffer at a volume-to-volume ratio of 1:10, 1:20, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:500, or 1:1000, such that the final concentration of the lysis buffer is 1x. In some cases, the sample may be obtained on a swab, and in this case, the swab may come into contact with 1x of the lysis buffer. The amount of sample to be mixed with the lysis buffer may depend on the type of sample and / or the amplification method performed on the sample. In some embodiments, the sample is a certain amount of liquid mixed with the lysis buffer for downstream applications (e.g., nucleic acid amplification).

[0232] In some embodiments, the amount of sample to be mixed with the lysis buffer includes at least about 0.1 µl, at least about 0.2 µl, at least about 0.3 µl, at least about 0.4 µl, at least about 0.5 µl, at least about 0.6 µl, at least about 0.7 µl, at least about 0.8 µl, at least about 0.9 µl, at least about 1.0 µl, at least about 2.0 µl, at least about 3.0 µl, at least about 4.0 µl, at least about 5.0 µl, at least about 6.0 µl, at least about 7.0 µl, at least about 8.0 µl, at least about 9.0 µl, at least about 10.0 µl, at least about 11.0 µl, at least about 12.0 µl, at least about 13.0 µl, at least about 14.0 µl, at least about 15.0 µl, at least about 16.0 µl, at least about 17.0 µl, at least about 18.0 µl, and at least about 19.0 µl. µl, at least about 20.0 µl, at least about 25.0 µl, at least about 30.0 µl, at least about 35.0 µl, at least about 40.0 µl, at least about 45.0 µl, at least about 50.0 µl, at least about 75.0 µl, or at least about 100.0 µl.

[0233] In some embodiments, the sample amount includes up to about 100.0 µl, up to about 75.0 µl, up to about 50.0 µl, up to about 45.0 µl, up to about 40.0 µl, up to about 35.0 µl, up to about 30.0 µl, up to about 25.0 µl, up to about 20.0 µl, up to about 19.0 µl, up to about 18.0 µl, up to about 17.0 µl, up to about 16.0 µl, up to about 15.0 µl, up to about 14.0 µl, up to about 13.0 µl, up to about 12.0 µl, up to about 11.0 µl, up to about 10.0 µl, up to about 9.0 µl, up to about 8.0 µl, up to about 7.0 µl, up to about 6.0 µl, and up to about 5.0 µl. µl, up to about 4.0 µl, up to about 3.0 µl, up to about 2.0 µl, up to about 1.0 µl, up to about 0.9 µl, up to about 0.8 µl, up to about 0.7 µl, up to about 0.6 µl, up to about 0.5 µl, up to about 0.4 µl, up to about 0.3 µl, up to about 0.2 µl, or up to about 0.1 µl.

[0234] In some embodiments, the amount of sample includes about 0.1 µl to about 100 µl.In some embodiments, the sample amount includes about 0.1 µl to about 0.5 µl, about 0.1 µl to about 0.75 µl, about 0.1 µl to about 1 µl, about 0.1 µl to about 5 µl, about 0.1 µl to about 7.5 µl, about 0.1 µl to about 10 µl, about 0.1 µl to about 20 µl, about 0.1 µl to about 25 µl, about 0.1 µl to about 50 µl, about 0.1 µl to about 75 µl, about 0.1 µl to about 100 µl, about 0.5 µl to about 0.75 µl, about 0.5 µl to about 1 µl, about 0.5 µl to about 5 µl, about 0.5 µl to about 7.5 µl, about 0.5 µl to about 10 µl, about 0.5 µl to about 20 µl, about 0.5 µl to about 25 µl, about 0.5 µl to about 50 µl, about 0.5 µl... About 75 µl, about 0.5 µl to about 100 µl, about 0.75 µl to about 1 µl, about 0.75 µl to about 5 µl, about 0.75 µl to about 7.5 µl, about 0.75 µl to about 10 µl, about 0.75 µl to about 20 µl, about 0.75 µl to about 25 µl, about 0.75 µl to about 50 µl, about 0.75 µl to about 75 µl, about 0.75 µl to about 100 µl, about 1 µl to about 5 µl, about 1 µl to about 7.5 µl, about 1 µl to about 10 µl, about 5 µl to about 75 µl, about 1 µl to about 100 µl, about 5 µl to about 7.5 µl, about 5 µl to about 10 µl, about 5 µl to about 7 ... About 5 µl to about 20 µl, about 5 µl to about 25 µl, about 5 µl to about 50 µl, about 5 µl to about 75 µl, about 5 µl to about 100 µl, about 7.5 µl to about 10 µl, about 7.5 µl to about 20 µl, about 7.5 µl to about 25 µl, about 7.5 µl to about 50 µl, about 7.5 µl to about 75 µl, about 7.5 µl to about 100 µl, about 10 µl to about 20 µl, about 10 µl to about 25 µl, about 10 µl to about 50 µl, about 10 µl to about 75 µl, about 10 µl to about 100 µl, about 20 µl to about 25 µl, about 20 µl to about 50 µl, about 20 µl to about 75 µl, about 20 µl to about 100 µl, about 25 µl to about 50 µl, about 25 µl to about 75 µl µl, about 25 µl to about 100 µl, about 50 µl to about 75 µl, about 50 µl to about 100 µl, or about 75 µl to about 100 µl.

[0235] In some implementations, the sample contains a certain copy number of the target nucleic acid molecule to be processed. In some implementations, the copy number of the target nucleic acid molecule can be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, or at least about 4. 00, at least about 450, at least about 500, at least about 750, at least about 1000, at least about 1500, at least about 2000, at least about 2500, at least about 5000, at least about 7500, at least about 10,000, at least about 25,000, at least about 50,000, at least about 75,000, at least about 100,000, at least about 250,000 or at least about 500,000 copies. In some implementations, the copy number of the target nucleic acid molecule can be up to about 500,000, up to about 250,000, up to about 100,000, up to about 75,000, up to about 50,000, up to about 25,000, up to about 10,000, up to about 7,500, up to about 5,000, up to about 2,500, up to about 1,000, up to about 750, up to about 500,000. 0, up to about 450, up to about 400, up to about 350, up to about 300, up to about 250, up to about 200, up to about 150, up to about 100, up to about 75, up to about 50, up to about 40, up to about 30, up to about 20, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2 or up to about 1.

[0236] In some embodiments, the copy number of the target nucleic acid molecule can be from about 1 to about 500,000. In some embodiments, the copy number of the target nucleic acid molecule can be from about 1 to about 5, from about 1 to about 10, from about 1 to about 50, from about 1 to about 100, from about 1 to about 1,000, from about 1 to about 2,500, from about 1 to about 5,000, from about 1 to about 10,000, from about 1 to about 50,000, from about 1 to about 100,000, from about 1 to about 500,000, from about 5 to about 10, from about 5 to about 50,000, from about 5 to about 100,000, from about 5 to about 1,000, from about 5 to about 2,500, from about 5 to about 5,000, from about 5 to about 10,000, from about 5 to about 50,000, from about 5 to about 100,000. 00, about 5 to about 500,000, about 10 to about 50, about 10 to about 100, about 10 to about 1,000, about 10 to about 2,500, about 10 to about 5,000, about 10 to about 10,000, about 10 to about 50,000, about 10 to about 100,000, about 10 to about 500,000, about 50 to about 100, about 50 to about 1,000, about 50 to about 2,500, about 50 to about 5,000, about 50 to about 10,000, about 50 to about 50,000, about 50 to about 100,000, about 50 to about 500,000, about 1 00 to about 1,000, about 100 to about 2,500, about 100 to about 5,000, about 100 to about 10,000, about 100 to about 50,000, about 100 to about 100,000, about 100 to about 500,000, about 1,000 to about 2,500, about 1,000 to about 5,000, about 1,000 to about 10,000, about 1,000 to about 50,000, about 1,000 to about 100,000, about 1,000 to about 500,000, about 2,500 to about 5,000, about 2,500 to about 10,000, about 2 ,500 to about 50,000, about 2,500 to about 100,000, about 2,500 to about 500,000, about 5,000 to about 10,000, about 5,000 to about 50,000, about 5,000 to about 100,000, about 5,000 to about 500,000, about 10,000 to about 50,000, about 10,000 to about 100,000, about 10,000 to about 500,000, about 50,000 to about 100,000, about 50,000 to about 500,000, or about 100,000 to about 500,000.

[0237] In some cases, the sample preparation methods and compositions of this disclosure provide a higher concentration of target nucleic acid molecules from the treated sample compared to the concentration of target nucleic acid molecules in samples treated by different methods. In some embodiments, the concentration of one or more different target nucleic acid molecules is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, compared to the concentration of one or more different target nucleic acid molecules in other aspects of the same sample treated by SDS, polysorbate 80, and / or cyclodextrin alone.

[0238] In some embodiments, the detergent comprises sodium lauryl sulfate (SDS). In some embodiments, the detergent comprises sodium lauryl sulfate, lithium lauryl sulfate, or functional variants thereof. In some embodiments, the detergent is an ionic detergent. In some embodiments, the detergent is a nonionic detergent.

[0239] In some embodiments, the solubilizer is a nonionic surfactant. In some embodiments, the solubilizer comprises polysorbate. The polysorbate may be polyoxyethylene (20) sorbitol monooleate (e.g., polysorbate 80), polyoxyethylene (20) sorbitol monolaurate (e.g., polysorbate 20), polyoxyethylene (20) sorbitol monopalmitate (e.g., polysorbate 40), polyoxyethylene (20) sorbitol monostearate (e.g., polysorbate 60), or a functional variant thereof. In some embodiments, the solubilizer is Tergitol. TM Surfactants, Triton TM Surfactants or Igepal ® Surfactants. In some embodiments, the solubilizer is an alkoxylate or cocamide. In some embodiments, the solubilizer is decyl glucoside, alkyl glycoside, lauryl glucoside, sorbitol tristearate, or neodymium.

[0240] In some embodiments, the sample is simultaneously contacted with the pyrolysis buffer and the recovery buffer. In some embodiments, the sample is simultaneously contacted with the pyrolysis buffer and the recovery buffer in the same mixture. In some embodiments, the sample is immersed in the pyrolysis buffer. In some embodiments, the sample and the pyrolysis buffer are mixed by vortexing and / or manually. In some embodiments, the sample is not mixed with the pyrolysis buffer.

[0241] In some embodiments, the sample is incubated for a duration sufficient to lyse the cells in the sample. In some embodiments, the sample is incubated in a lysis buffer for 1 to 60 minutes. In some embodiments, the sample is incubated in a lysis buffer for at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some implementations, the sample is incubated in a lysis buffer for up to about 120 minutes, up to about 90 minutes, up to about 60 minutes, up to about 50 minutes, up to about 40 minutes, up to about 30 minutes, up to about 25 minutes, up to about 20 minutes, up to about 15 minutes, up to about 12 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6.5 minutes, up to about 6 minutes, up to about 5.5 minutes, up to about 5 minutes, up to about 4.5 minutes, up to about 4 minutes, up to about 3.5 minutes, up to about 3 minutes, up to about 2.5 minutes, up to about 2 minutes, up to about 1.5 minutes, up to about 1 minute, up to about 30 seconds, or up to about 15 seconds.

[0242] In some embodiments, the sample is incubated in a lysis buffer for about 1 minute to about 15 minutes. In some embodiments, the sample is incubated in a lysis buffer for about 1 minute to about 2 minutes, about 1 minute to about 2.5 minutes, about 1 minute to about 3 minutes, about 1 minute to about 3.5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 6 minutes, about 1 minute to about 7 minutes, about 1 minute to about 7.5 minutes, about 1 minute to about 10 minutes, about 1 minute to about 15 minutes, about 2 minutes to about 2.5 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 3.5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 5 minutes. Approximately 2 minutes to approximately 6 minutes, approximately 2 minutes to approximately 7 minutes, approximately 2 minutes to approximately 7.5 minutes, approximately 2 minutes to approximately 10 minutes, approximately 2 minutes to approximately 15 minutes, approximately 2.5 minutes to approximately 3 minutes, approximately 2.5 minutes to approximately 3.5 minutes, approximately 2.5 minutes to approximately 4 minutes, approximately 2.5 minutes to approximately 5 minutes, approximately 2.5 minutes to approximately 6 minutes, approximately 2.5 minutes to approximately 7 minutes, approximately 2.5 minutes to approximately 7.5 minutes, approximately 2.5 minutes to approximately 10 minutes, approximately 2.5 minutes to approximately 15 minutes, approximately 3 minutes to approximately 3.5 minutes, approximately 3 minutes to approximately 4 minutes, approximately 3 Approximately 3 to 5 minutes, approximately 3 to 6 minutes, approximately 3 to 7 minutes, approximately 3 to 7.5 minutes, approximately 3 to 10 minutes, approximately 3 to 15 minutes, approximately 3.5 minutes to 4 minutes, approximately 3.5 minutes to 5 minutes, approximately 3.5 minutes to 6 minutes, approximately 3.5 minutes to 7 minutes, approximately 3.5 minutes to 7.5 minutes, approximately 3.5 minutes to 10 minutes, approximately 3.5 minutes to 15 minutes, approximately 4 minutes to 5 minutes, approximately 4 minutes to 6 minutes, approximately 4 minutes to 7 minutes, approximately 4 minutes to 7.5 minutes. Approximately 4 minutes to approximately 10 minutes, approximately 4 minutes to approximately 15 minutes, approximately 5 minutes to approximately 6 minutes, approximately 5 minutes to approximately 7 minutes, approximately 5 minutes to approximately 7.5 minutes, approximately 5 minutes to approximately 10 minutes, approximately 5 minutes to approximately 15 minutes, approximately 6 minutes to approximately 7 minutes, approximately 6 minutes to approximately 7.5 minutes, approximately 6 minutes to approximately 10 minutes, approximately 6 minutes to approximately 15 minutes, approximately 7 minutes to approximately 7.5 minutes, approximately 7 minutes to approximately 10 minutes, approximately 7 minutes to approximately 15 minutes, approximately 7.5 minutes to approximately 10 minutes, approximately 7.5 minutes to approximately 15 minutes, or approximately 10 minutes to approximately 15 minutes.

[0243] In some embodiments, the sample is incubated for approximately 1 minute to approximately 120 minutes. In some embodiments, the sample is incubated for approximately 1 minute to approximately 5 minutes, approximately 1 minute to approximately 10 minutes, approximately 1 minute to approximately 15 minutes, approximately 1 minute to approximately 20 minutes, approximately 1 minute to approximately 25 minutes, approximately 1 minute to approximately 30 minutes, approximately 1 minute to approximately 45 minutes, approximately 1 minute to approximately 60 minutes, approximately 1 minute to approximately 75 minutes, approximately 1 minute to approximately 90 minutes, approximately 1 minute to approximately 120 minutes, approximately 5 minutes to approximately 10 minutes, approximately 5 minutes to approximately 15 minutes, approximately 5 minutes to approximately 20 minutes, approximately 5 minutes to approximately 25 minutes, approximately 5 minutes to approximately 30 minutes, or approximately 5 minutes to approximately 45 minutes. Approximately 5 minutes to 60 minutes, approximately 5 minutes to 75 minutes, approximately 5 minutes to 90 minutes, approximately 5 minutes to 120 minutes, approximately 10 minutes to 15 minutes, approximately 10 minutes to 20 minutes, approximately 10 minutes to 25 minutes, approximately 10 minutes to 30 minutes, approximately 10 minutes to 45 minutes, approximately 10 minutes to 60 minutes, approximately 10 minutes to 75 minutes, approximately 10 minutes to 90 minutes, approximately 10 minutes to 120 minutes, approximately 15 minutes to 20 minutes, approximately 15 minutes to 25 minutes, approximately 15 minutes to 30 minutes, approximately 15 minutes to... 45 minutes, approximately 15 minutes to approximately 60 minutes, approximately 15 minutes to approximately 75 minutes, approximately 15 minutes to approximately 90 minutes, approximately 15 minutes to approximately 120 minutes, approximately 20 minutes to approximately 25 minutes, approximately 20 minutes to approximately 30 minutes, approximately 20 minutes to approximately 45 minutes, approximately 20 minutes to approximately 60 minutes, approximately 20 minutes to approximately 75 minutes, approximately 20 minutes to approximately 90 minutes, approximately 20 minutes to approximately 120 minutes, approximately 25 minutes to approximately 30 minutes, approximately 25 minutes to approximately 45 minutes, approximately 25 minutes to approximately 60 minutes, approximately 25 minutes to approximately 75 minutes, approximately 25 minutes to approximately 90 minutes Approximately 25 minutes to approximately 120 minutes, approximately 30 minutes to approximately 45 minutes, approximately 30 minutes to approximately 60 minutes, approximately 30 minutes to approximately 75 minutes, approximately 30 minutes to approximately 90 minutes, approximately 30 minutes to approximately 120 minutes, approximately 45 minutes to approximately 60 minutes, approximately 45 minutes to approximately 75 minutes, approximately 45 minutes to approximately 90 minutes, approximately 45 minutes to approximately 120 minutes, approximately 60 minutes to approximately 75 minutes, approximately 60 minutes to approximately 90 minutes, approximately 60 minutes to approximately 120 minutes, approximately 75 minutes to approximately 90 minutes, approximately 75 minutes to approximately 120 minutes, or approximately 90 minutes to approximately 120 minutes.

[0244] In some embodiments, the sample is incubated for approximately 3 hours to approximately 48 hours. In some embodiments, the sample is incubated for approximately 3 hours to approximately 4 hours, approximately 3 hours to approximately 5 hours, approximately 3 hours to approximately 10 hours, approximately 3 hours to approximately 12 hours, approximately 3 hours to approximately 15 hours, approximately 3 hours to approximately 18 hours, approximately 3 hours to approximately 24 hours, approximately 3 hours to approximately 30 hours, approximately 3 hours to approximately 36 hours, approximately 3 hours to approximately 40 hours, approximately 3 hours to approximately 48 hours, approximately 4 hours to approximately 5 hours, approximately 4 hours to approximately 10 hours, approximately 4 hours to approximately 12 hours, approximately 4 hours to approximately 15 hours, approximately 4 hours to approximately 18 hours, approximately 4 hours to approximately 2... 4 hours, approximately 4 hours to approximately 30 hours, approximately 4 hours to approximately 36 hours, approximately 4 hours to approximately 40 hours, approximately 4 hours to approximately 48 hours, approximately 5 hours to approximately 10 hours, approximately 5 hours to approximately 12 hours, approximately 5 hours to approximately 15 hours, approximately 5 hours to approximately 18 hours, approximately 5 hours to approximately 24 hours, approximately 5 hours to approximately 30 hours, approximately 5 hours to approximately 36 hours, approximately 5 hours to approximately 40 hours, approximately 5 hours to approximately 48 hours, approximately 10 hours to approximately 12 hours, approximately 10 hours to approximately 15 hours, approximately 10 hours to approximately 18 hours, approximately 10 hours to approximately 24 hours Hours, approximately 10 to 30 hours, approximately 10 to 36 hours, approximately 10 to 40 hours, approximately 10 to 48 hours, approximately 12 to 15 hours, approximately 12 to 18 hours, approximately 12 to 24 hours, approximately 12 to 30 hours, approximately 12 to 36 hours, approximately 12 to 40 hours, approximately 12 to 48 hours, approximately 15 to 18 hours, approximately 15 to 24 hours, approximately 15 to 30 hours, approximately 15 to 36 hours, approximately 15 to 40 hours Approximately 15 hours to approximately 48 hours, approximately 18 hours to approximately 24 hours, approximately 18 hours to approximately 30 hours, approximately 18 hours to approximately 36 hours, approximately 18 hours to approximately 40 hours, approximately 18 hours to approximately 48 hours, approximately 24 hours to approximately 30 hours, approximately 24 hours to approximately 36 hours, approximately 24 hours to approximately 40 hours, approximately 24 hours to approximately 48 hours, approximately 30 hours to approximately 36 hours, approximately 30 hours to approximately 40 hours, approximately 30 hours to approximately 48 hours, approximately 36 hours to approximately 40 hours, approximately 36 hours to approximately 48 hours, or approximately 40 hours to approximately 48 hours.

[0245] In some embodiments, the sample is incubated in a lysis buffer at room temperature. In some embodiments, the sample remains in the lysis buffer at room temperature for approximately 1 minute to approximately 15 minutes. In some embodiments, the sample is incubated in the lysis buffer for approximately 1 minute to approximately 2 minutes, approximately 1 minute to approximately 2.5 minutes, approximately 1 minute to approximately 3 minutes, approximately 1 minute to approximately 3.5 minutes, approximately 1 minute to approximately 4 minutes, approximately 1 minute to approximately 5 minutes, approximately 1 minute to approximately 6 minutes, approximately 1 minute to approximately 7 minutes, approximately 1 minute to approximately 7.5 minutes, approximately 1 minute to approximately 10 minutes, approximately 1 minute to approximately 15 minutes, approximately 2 minutes to approximately 2.5 minutes, approximately 2 minutes to approximately 3 minutes, approximately 2 minutes to approximately 3.5 minutes, approximately 2 minutes to approximately 4 minutes, approximately 2 minutes to approximately 5 minutes, etc. Approximately 2 minutes to approximately 6 minutes, approximately 2 minutes to approximately 7 minutes, approximately 2 minutes to approximately 7.5 minutes, approximately 2 minutes to approximately 10 minutes, approximately 2 minutes to approximately 15 minutes, approximately 2.5 minutes to approximately 3 minutes, approximately 2.5 minutes to approximately 3.5 minutes, approximately 2.5 minutes to approximately 4 minutes, approximately 2.5 minutes to approximately 5 minutes, approximately 2.5 minutes to approximately 6 minutes, approximately 2.5 minutes to approximately 7 minutes, approximately 2.5 minutes to approximately 7.5 minutes, approximately 2.5 minutes to approximately 10 minutes, approximately 2.5 minutes to approximately 15 minutes, approximately 3 minutes to approximately 3.5 minutes, approximately 3 minutes to approximately 4 minutes, approximately 3 Approximately 3 to 5 minutes, approximately 3 to 6 minutes, approximately 3 to 7 minutes, approximately 3 to 7.5 minutes, approximately 3 to 10 minutes, approximately 3 to 15 minutes, approximately 3.5 minutes to 4 minutes, approximately 3.5 minutes to 5 minutes, approximately 3.5 minutes to 6 minutes, approximately 3.5 minutes to 7 minutes, approximately 3.5 minutes to 7.5 minutes, approximately 3.5 minutes to 10 minutes, approximately 3.5 minutes to 15 minutes, approximately 4 minutes to 5 minutes, approximately 4 minutes to 6 minutes, approximately 4 minutes to 7 minutes, approximately 4 minutes to 7.5 minutes. Approximately 4 minutes to approximately 10 minutes, approximately 4 minutes to approximately 15 minutes, approximately 5 minutes to approximately 6 minutes, approximately 5 minutes to approximately 7 minutes, approximately 5 minutes to approximately 7.5 minutes, approximately 5 minutes to approximately 10 minutes, approximately 5 minutes to approximately 15 minutes, approximately 6 minutes to approximately 7 minutes, approximately 6 minutes to approximately 7.5 minutes, approximately 6 minutes to approximately 10 minutes, approximately 6 minutes to approximately 15 minutes, approximately 7 minutes to approximately 7.5 minutes, approximately 7 minutes to approximately 10 minutes, approximately 7 minutes to approximately 15 minutes, approximately 7.5 minutes to approximately 10 minutes, approximately 7.5 minutes to approximately 15 minutes, or approximately 10 minutes to approximately 15 minutes.

[0246] In some embodiments, the lysis buffer can be stabilized for a period of time before the sample is added. In some embodiments, the lysis buffer can be stabilized for at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes, at least about 12 hours, at least about 1 day, at least about 3 days, at least about 5 days, at least about 10 days, at least about 15 days, at least about 1 month, or at least about 3 months. In some implementations, the samples are incubated for up to approximately 3 months, up to approximately 1 month, up to approximately 15 days, up to approximately 10 days, up to approximately 3 days, up to approximately 1 day, up to approximately 12 hours, up to approximately 120 minutes, up to approximately 90 minutes, up to approximately 60 minutes, up to approximately 50 minutes, up to approximately 40 minutes, up to approximately 30 minutes, up to approximately 25 minutes, up to approximately 20 minutes, up to approximately 15 minutes, up to approximately 12 minutes, up to approximately 10 minutes, up to approximately 9 minutes, up to approximately 8 minutes, up to approximately 7 minutes, up to approximately 6.5 minutes, up to approximately 6 minutes, up to approximately 5.5 minutes, up to approximately 5 minutes, up to approximately 4.5 minutes, up to approximately 4 minutes, up to approximately 3.5 minutes, up to approximately 3 minutes, up to approximately 2.5 minutes, up to approximately 2 minutes, up to approximately 1.5 minutes, up to approximately 1 minute, up to approximately 30 seconds, or up to approximately 15 seconds.

[0247] In some embodiments, the lysis buffer can be stable for about 1 hour to about 48 hours. In some embodiments, the lysis buffer can be stable for about 1 hour to about 2 hours, about 1 hour to about 3 hours, about 1 hour to about 4 hours, about 1 hour to about 5 hours, about 1 hour to about 7 hours, about 1 hour to about 10 hours, about 1 hour to about 12 hours, about 1 hour to about 18 hours, about 1 hour to about 24 hours, about 1 hour to about 36 hours, about 1 hour to about 48 hours, about 2 hours to about 3 hours, about 2 hours to about 4 hours, about 2 hours to about 5 hours, about 2 hours to about 7 hours, about 2 hours to about 10 hours, about... 2 hours to about 12 hours, about 2 hours to about 18 hours, about 2 hours to about 24 hours, about 2 hours to about 36 hours, about 2 hours to about 48 hours, about 3 hours to about 4 hours, about 3 hours to about 5 hours, about 3 hours to about 7 hours, about 3 hours to about 10 hours, about 3 hours to about 12 hours, about 3 hours to about 18 hours, about 3 hours to about 24 hours, about 3 hours to about 36 hours, about 3 hours to about 48 hours, about 4 hours to about 5 hours, about 4 hours to about 7 hours, about 4 hours to about 10 hours, about 4 hours to Approximately 12 hours, approximately 4 hours to approximately 18 hours, approximately 4 hours to approximately 24 hours, approximately 4 hours to approximately 36 hours, approximately 4 hours to approximately 48 hours, approximately 5 hours to approximately 7 hours, approximately 5 hours to approximately 10 hours, approximately 5 hours to approximately 12 hours, approximately 5 hours to approximately 18 hours, approximately 5 hours to approximately 24 hours, approximately 5 hours to approximately 36 hours, approximately 5 hours to approximately 48 hours, approximately 7 hours to approximately 10 hours, approximately 7 hours to approximately 12 hours, approximately 7 hours to approximately 18 hours, approximately 7 hours to approximately 24 hours, approximately 7 hours to approximately 36 hours, approximately 7 hours to About 48 hours, about 10 hours to about 12 hours, about 10 hours to about 18 hours, about 10 hours to about 24 hours, about 10 hours to about 36 hours, about 10 hours to about 48 hours, about 12 hours to about 18 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 18 hours to about 24 hours, about 18 hours to about 36 hours, about 18 hours to about 48 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, or about 36 hours to about 48 hours.

[0248] In some embodiments, the lysis buffer can be used as a storage buffer for transport. In some embodiments, the sample can be stored in the lysis buffer for at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some implementations, the sample may be stored in the lysis buffer for up to about 120 minutes, up to about 90 minutes, up to about 60 minutes, up to about 50 minutes, up to about 40 minutes, up to about 30 minutes, up to about 25 minutes, up to about 20 minutes, up to about 15 minutes, up to about 12 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6.5 minutes, up to about 6 minutes, up to about 5.5 minutes, up to about 5 minutes, up to about 4.5 minutes, up to about 4 minutes, up to about 3.5 minutes, up to about 3 minutes, up to about 2.5 minutes, up to about 2 minutes, up to about 1.5 minutes, up to about 1 minute, up to about 30 seconds, or up to about 15 seconds.

[0249] In some implementations, the sample may be refrigerated before contact with the lysis buffer.

[0250] The sample may be heated after incubation in a lysis buffer. The sample may be heated before incubation in a lysis buffer. In some embodiments, the sample is heated at a constant temperature for a period of time. In some embodiments, the constant temperature is at least about 30°C, at least about 40°C, at least about 50°C, at least about 60°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, at least about 91°C, at least about 92°C, at least about 93°C, at least about 94°C, at least about 95°C, at least about 96°C, at least about 97°C, at least about 98°C, at least about 99°C, at least about 100°C, at least about 105°C, at least about 110°C, at least about 115°C, at least about 120°C, at least about 125°C, at least about 130°C, at least about 140°C, or at least about 150°C. In some implementations, the constant temperature is up to about 150°C, up to about 140°C, up to about 130°C, up to about 125°C, up to about 120°C, up to about 115°C, up to about 100°C, up to about 105°C, up to about 100°C, up to about 99°C, up to about 98°C, up to about 97°C, up to about 96°C, up to about 95°C, up to about 94°C, up to about 93°C, up to about 92°C, up to about 91°C, up to about 90°C, up to about 85°C, up to about 80°C, up to about 75°C, up to about 70°C, up to about 60°C, up to about 50°C, up to about 40°C, or up to about 30°C.

[0251] In some embodiments, the constant temperature is from about 50°C to about 120°C. In some embodiments, the constant temperature is at most about 120°C. In some embodiments, the constant temperature is from about 50°C to about 60°C, from about 50°C to about 70°C, from about 50°C to about 80°C, from about 50°C to about 85°C, from about 50°C to about 90°C, from about 50°C to about 95°C, from about 50°C to about 100°C, from about 50°C to about 105°C, from about 50°C to about 110°C, from about 50°C to about 115°C, from about 50°C to about 120°C, from about 60°C to about 70°C, from about 60°C to about 80°C, from about 60°C to about 85°C, from about 60°C to about 90°C, from about 60°C to about 95°C, from about 60°C to about 90°C, from about 60°C to about 95°C, from about 60°C to about 60°C. From approximately 100°C to 100°C, from approximately 60°C to 105°C, from approximately 60°C to 110°C, from approximately 60°C to 115°C, from approximately 60°C to 120°C, from approximately 70°C to 80°C, from approximately 70°C to 85°C, from approximately 70°C to 90°C, from approximately 70°C to 95°C, from approximately 70°C to 100°C, from approximately 70°C to 105°C, from approximately 70°C to 110°C, from approximately 70°C to 115°C, from approximately 70°C to 120°C, from approximately 80°C to 85°C, from approximately 80°C to 90°C, from approximately 80°C to 95°C, from approximately 80°C to Approximately 100°C, approximately 80°C to approximately 105°C, approximately 80°C to approximately 110°C, approximately 80°C to approximately 115°C, approximately 80°C to approximately 120°C, approximately 85°C to approximately 90°C, approximately 85°C to approximately 95°C, approximately 85°C to approximately 100°C, approximately 85°C to approximately 105°C, approximately 85°C to approximately 110°C, approximately 85°C to approximately 115°C, approximately 85°C to approximately 120°C, approximately 90°C to approximately 95°C, approximately 90°C to approximately 100°C, approximately 90°C to approximately 105°C, approximately 90°C to approximately 110°C, approximately 90°C to approximately 115°C, approximately 90°C to approximately 115°C, approximately 90°C to approximately 100°C, approximately 90°C to approximately 105°C, approximately 90°C to approximately 110 ... From ℃ to about 120℃, from about 95℃ to about 100℃, from about 95℃ to about 105℃, from about 95℃ to about 110℃, from about 95℃ to about 115℃, from about 95℃ to about 120℃, from about 100℃ to about 105℃, from about 100℃ to about 110℃, from about 100℃ to about 115℃, from about 100℃ to about 120℃, from about 105℃ to about 110℃, from about 105℃ to about 115℃, from about 105℃ to about 120℃, from about 110℃ to about 115℃, from about 110℃ to about 120℃, or from about 115℃ to about 120℃.

[0252] In some embodiments, the sample is heated at a constant temperature for a period of time. In some embodiments, the sample is heated at a constant temperature for at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, or at least about 15 minutes. In some implementations, the samples are incubated for up to about 15 minutes, up to about 12 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6.5 minutes, up to about 6 minutes, up to about 5.5 minutes, up to about 5 minutes, up to about 4.5 minutes, up to about 4 minutes, up to about 3.5 minutes, up to about 3 minutes, up to about 2.5 minutes, up to about 2 minutes, up to about 1.5 minutes, up to about 1 minute, up to about 30 seconds, or up to about 15 seconds.

[0253] In some embodiments, the sample is heated at a constant temperature for about 1 minute to about 15 minutes. In some embodiments, the sample is heated at a constant temperature for about 1 minute to about 2 minutes, about 1 minute to about 2.5 minutes, about 1 minute to about 3 minutes, about 1 minute to about 3.5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 6 minutes, about 1 minute to about 7 minutes, about 1 minute to about 7.5 minutes, about 1 minute to about 10 minutes, about 1 minute to about 15 minutes, about 2 minutes to about 2.5 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 3.5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 5 minutes. Approximately 2 minutes to approximately 6 minutes, approximately 2 minutes to approximately 7 minutes, approximately 2 minutes to approximately 7.5 minutes, approximately 2 minutes to approximately 10 minutes, approximately 2 minutes to approximately 15 minutes, approximately 2.5 minutes to approximately 3 minutes, approximately 2.5 minutes to approximately 3.5 minutes, approximately 2.5 minutes to approximately 4 minutes, approximately 2.5 minutes to approximately 5 minutes, approximately 2.5 minutes to approximately 6 minutes, approximately 2.5 minutes to approximately 7 minutes, approximately 2.5 minutes to approximately 7.5 minutes, approximately 2.5 minutes to approximately 10 minutes, approximately 2.5 minutes to approximately 15 minutes, approximately 3 minutes to approximately 3.5 minutes, approximately 3 minutes to approximately 4 minutes, approximately 3 Approximately 3 to 5 minutes, approximately 3 to 6 minutes, approximately 3 to 7 minutes, approximately 3 to 7.5 minutes, approximately 3 to 10 minutes, approximately 3 to 15 minutes, approximately 3.5 minutes to 4 minutes, approximately 3.5 minutes to 5 minutes, approximately 3.5 minutes to 6 minutes, approximately 3.5 minutes to 7 minutes, approximately 3.5 minutes to 7.5 minutes, approximately 3.5 minutes to 10 minutes, approximately 3.5 minutes to 15 minutes, approximately 4 minutes to 5 minutes, approximately 4 minutes to 6 minutes, approximately 4 minutes to 7 minutes, approximately 4 minutes to 7.5 minutes. Approximately 4 minutes to approximately 10 minutes, approximately 4 minutes to approximately 15 minutes, approximately 5 minutes to approximately 6 minutes, approximately 5 minutes to approximately 7 minutes, approximately 5 minutes to approximately 7.5 minutes, approximately 5 minutes to approximately 10 minutes, approximately 5 minutes to approximately 15 minutes, approximately 6 minutes to approximately 7 minutes, approximately 6 minutes to approximately 7.5 minutes, approximately 6 minutes to approximately 10 minutes, approximately 6 minutes to approximately 15 minutes, approximately 7 minutes to approximately 7.5 minutes, approximately 7 minutes to approximately 10 minutes, approximately 7 minutes to approximately 15 minutes, approximately 7.5 minutes to approximately 10 minutes, approximately 7.5 minutes to approximately 15 minutes, or approximately 10 minutes to approximately 15 minutes.

[0254] In some embodiments, the sample is heated at a cyclic temperature for a period of time. In some embodiments, the cyclic temperature includes a temperature range. In some embodiments, the cyclic temperature is from about 30°C to about 120°C. In some embodiments, the cyclic temperature is from about 30°C to about 40°C, from about 30°C to about 50°C, from about 30°C to about 60°C, from about 30°C to about 70°C, from about 30°C to about 80°C, from about 30°C to about 85°C, from about 30°C to about 90°C, from about 30°C to about 95°C, from about 30°C to about 100°C, from about 30°C to about 110°C, from about 30°C to about 120°C, from about 40°C to about 50°C, from about 40°C to about 60°C, from about 40°C to about 70°C, from about 40°C to about 80°C, or from about 40°C to about 85°C. Approximately 40°C to approximately 90°C, approximately 40°C to approximately 95°C, approximately 40°C to approximately 100°C, approximately 40°C to approximately 110°C, approximately 40°C to approximately 120°C, approximately 50°C to approximately 60°C, approximately 50°C to approximately 70°C, approximately 50°C to approximately 80°C, approximately 50°C to approximately 85°C, approximately 50°C to approximately 90°C, approximately 50°C to approximately 95°C, approximately 50°C to approximately 100°C, approximately 50°C to approximately 110°C, approximately 50°C to approximately 120°C, approximately 60°C to approximately 70°C, approximately 60°C to approximately 80°C, approximately 60°C to approximately 85°C. Approximately 60°C to approximately 90°C, approximately 60°C to approximately 95°C, approximately 60°C to approximately 100°C, approximately 60°C to approximately 110°C, approximately 60°C to approximately 120°C, approximately 70°C to approximately 80°C, approximately 70°C to approximately 85°C, approximately 70°C to approximately 90°C, approximately 70°C to approximately 95°C, approximately 70°C to approximately 100°C, approximately 70°C to approximately 110°C, approximately 70°C to approximately 120°C, approximately 80°C to approximately 85°C, approximately 80°C to approximately 90°C, approximately 80°C to approximately 95°C, approximately 80°C to approximately 100°C, approximately 80°C to approximately 110°C Approximately 80°C to approximately 120°C, approximately 85°C to approximately 90°C, approximately 85°C to approximately 95°C, approximately 85°C to approximately 100°C, approximately 85°C to approximately 110°C, approximately 85°C to approximately 120°C, approximately 90°C to approximately 95°C, approximately 90°C to approximately 100°C, approximately 90°C to approximately 110°C, approximately 90°C to approximately 120°C, approximately 95°C to approximately 100°C, approximately 95°C to approximately 110°C, approximately 95°C to approximately 120°C, approximately 100°C to approximately 110°C, approximately 100°C to approximately 120°C, or approximately 110°C to approximately 120°C.

[0255] In some embodiments, the cycling temperature is at most about 115°C. In some embodiments, the cycling temperature is about 80°C to about 83°C, about 80°C to about 85°C, about 80°C to about 87°C, about 80°C to about 90°C, about 80°C to about 93°C, about 80°C to about 95°C, about 80°C to about 97°C, about 80°C to about 100°C, about 80°C to about 105°C, about 80°C to about 110°C, about 80°C to about 115°C, about 83°C to about 85°C, about 83°C to about 87°C, about 83°C to about 90°C, about 83°C to about 93°C, about 83°C to about 95°C, about... 83°C to about 97°C, about 83°C to about 100°C, about 83°C to about 105°C, about 83°C to about 110°C, about 83°C to about 115°C, about 85°C to about 87°C, about 85°C to about 90°C, about 85°C to about 93°C, about 85°C to about 95°C, about 85°C to about 97°C, about 85°C to about 100°C, about 85°C to about 105°C, about 85°C to about 110°C, about 85°C to about 115°C, about 87°C to about 90°C, about 87°C to about 93°C, about 87°C to about 95°C, about 87°C to about 95°C, about 87°C to about 97°C, about 87°C to about 93°C, about 87°C to about 95°C, about 87°C to about 97 ... ℃ to about 97℃, about 87℃ to about 100℃, about 87℃ to about 105℃, about 87℃ to about 110℃, about 87℃ to about 115℃, about 90℃ to about 93℃, about 90℃ to about 95℃, about 90℃ to about 97℃, about 90℃ to about 100℃, about 90℃ to about 105℃, about 90℃ to about 110℃, about 90℃ to about 115℃, about 93℃ to about 95℃, about 93℃ to about 97℃, about 93℃ to about 100℃, about 93℃ to about 105℃, about 93℃ to about 110℃, about 93°C to about 115°C, about 95°C to about 97°C, about 95°C to about 100°C, about 95°C to about 105°C, about 95°C to about 110°C, about 95°C to about 115°C, about 97°C to about 100°C, about 97°C to about 105°C, about 97°C to about 110°C, about 97°C to about 115°C, about 100°C to about 105°C, about 100°C to about 110°C, about 100°C to about 115°C, about 105°C to about 110°C, or about 110°C to about 115°C.

[0256] In some embodiments, the sample may be sonicated. Sonication can agitate particles in the sample. Not wishing to be bound by theory, sonication can assist in cell lysis during sample processing. In some embodiments, the sample is sonicated for at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, or at least about 10 minutes.

[0257] In some embodiments, ultrasonic treatment of the sample occurs before heating the sample. In some embodiments, ultrasonic treatment of the sample occurs after heating the sample. In some embodiments, ultrasonic treatment of the sample occurs simultaneously with heating the sample. In some embodiments, ultrasonic treatment occurs without heating the sample.

[0258] After incubation and / or heating at room temperature, the sample may be contacted with a recovery buffer. The recovery buffer can increase the stability and / or robustness of the enzyme. In some cases, the sample is contacted with a recovery buffer containing a solubilizer and cyclodextrin.

[0259] In some embodiments, the solubilizer includes polysorbate. The polysorbate may be polyoxyethylene (20) dehydrated sorbitol monooleate (e.g., polysorbate 80), polyoxyethylene (20) dehydrated sorbitol monolaurate (… For example Polysorbate 20), polyoxyethylene (20) dehydrated sorbitol monopalmitate ( For example Polysorbate 40), polyoxyethylene (20) dehydrated sorbitan monostearate (e.g., polysorbate 60), or functional variants thereof. In some embodiments, the solubilizer is Tergitol. TM Surfactants, Triton TM Surfactants or Igepal ® Surfactant. In some embodiments, the solubilizer is an alkoxylate or cocamide. In some embodiments, the solubilizer is decyl glucoside, alkyl glycoside, lauryl glucoside, sorbitol tristearate, or a neodymium. The solubilizer can be mixed with the detergent of this composition. In some embodiments, the solubilizer is capable of forming micelles comprising the detergent of this application.

[0260] In some implementations, the solubilizer is polysorbate 80. In some embodiments, the concentration (e.g., final concentration) of the solubilizer in the mixture in the presence of the sample is at least about 0.05% v / v, at least about 0.1% v / v, at least about 0.5% v / v, at least about 1% v / v, at least about 5% v / v, at least about 10% v / v, at least about 15% v / v, at least about 20% v / v, at least about 22.5% v / v, at least about 25% v / v, at least about 27.5% v / v, at least about 30% v / v, at least about 32.5% v / v, at least about 35% v / v, at least about 37.5% v / v, at least about 40% v / v, at least about 42.5% v / v, at least about 45% v / v, at least about 47.5% v / v, at least about 50% v / v, at least about 52.5% v / v, at least about 55% v / v, and at least about 57.5%. v / v, at least about 60% v / v, at least about 70% v / v, or at least about 75% v / v.

[0261] In some embodiments, the concentration (e.g., final concentration) of the solubilizer in the mixture in the presence of the sample is at most about 75% v / v, at most about 70% v / v, at most about 65% v / v, at most about 60% v / v, at most about 57.5% v / v, at most about 55% v / v, at most about 52.5% v / v, at most about 50% v / v, at most about 47.5% v / v, at most about 45% v / v, at most about 42.5% v / v, at most about 40% v / v, at most about 37.5% v / v, at most about 35% v / v, at most about 32.5% v / v, at most about 30% v / v, at most about 27.5% v / v, at most about 25% v / v, at most about 22.5% v / v, or at most about 20%. v / v, up to about 15% v / v, up to about 10% v / v, up to about 5% v / v, up to about 1% v / v, up to about 0.5% v / v, up to about 0.1% v / v or up to about 0.05% v / v.

[0262] In some embodiments, cyclodextrins include (2-hydroxypropyl)β-cyclodextrin, (2-hydroxypropyl)γ-cyclodextrin, (2-hydroxypropyl)α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof.

[0263] In some embodiments, the cyclodextrin is present at a final concentration when mixed with the sample, which effectively isolates the detergent within the composition of the invention. In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the mixture in the presence of the sample is at least about, and at most about or about 0.05 mM, 0.1 mM, 0.5 mM, 1.0 mM, 5.0 mM, 10.0 mM, 15.0 mM, 20.0 mM, 25.0 mM, 30.0 mM, 35.0 mM, 40.0 mM, 50.0 mM, 55.0 mM, 60.0 mM, 65.0 mM, 70.0 mM, 75.0 mM, 80.0 mM, 85.0 mM, 90.0 mM, 95.0 mM, 100.0 mM, 125.0 mM, 150.0 mM, 175.0 mM, 200.0 mM, 250.0 mM, 300.0 mM, or any two of these values.

[0264] In some embodiments, the final volume of the recovered buffer is at least about, at most about or about 100 µl, 200 µl, 300 µl, 400 µl, 450 µl, 500 µl, 550 µl, 600 µl, 650 µl, 700 µl, 750 µl, 800 µl, 900 µl, 1000 µl, or a range between any two of these values.

[0265] In some embodiments, the sample is mixed with the recovery buffer. In some embodiments, the sample and recovery buffer are mixed by vortexing and / or manually. In some embodiments, the mixing of the sample and recovery buffer is performed using automated instruments, consumables, microfluidic systems, or millifluidic systems. In some embodiments, the sample and recovery buffer are mixed until they are homogeneous.

[0266] In some embodiments, the lysis buffer can be frozen to stabilize the solution. In some embodiments, the lysis buffer can be frozen at a temperature between about -50°C and 0°C. In some embodiments, the lysis buffer can be frozen at about -50°C. In some embodiments, the lysis buffer can be frozen at about -40°C. In some embodiments, the lysis buffer can be frozen at about -30°C. In some embodiments, the lysis buffer can be frozen at about -25°C. In some embodiments, the lysis buffer can be frozen at about -20°C. In some embodiments, the lysis buffer can be frozen at about -15°C. In some embodiments, the lysis buffer can be frozen at about -10°C. In some embodiments, the lysis buffer can be frozen at about -5°C. In some embodiments, the lysis buffer can be frozen at about 0°C.

[0267] After freezing the lysis buffer, the buffer can be thawed and mixed with the sample. In some embodiments, the efficiency of the thawed lysis buffer is tested and compared with the efficiency of the unfrozen lysis buffer. In some embodiments, the efficiency of the thawed lysis buffer is similar to that of the unfrozen lysis buffer.

[0268] In some embodiments, the recovered buffer can be frozen to stabilize the solution. In some embodiments, the recovered buffer can be frozen at a temperature from about -120°C to 0°C. In some embodiments, the recovered buffer can be frozen at a temperature from about -80°C. In some embodiments, the recovered buffer can be frozen at a temperature from about -120°C to about 0°C. In some embodiments, the recovered buffer can be frozen at a temperature of at least about -120°C. In some embodiments, the recovered buffer can be frozen at a temperature of at most about 0°C. In some embodiments, the buffer can be recovered at temperatures ranging from approximately -120°C to approximately -100°C, approximately -120°C to approximately -90°C, approximately -120°C to approximately -80°C, approximately -120°C to approximately -70°C, approximately -120°C to approximately -60°C, approximately -120°C to approximately -50°C, approximately -120°C to approximately -40°C, approximately -120°C to approximately -30°C, approximately -120°C to approximately -20°C, approximately -120°C to approximately -10°C, approximately -120°C to approximately 0°C, approximately -100°C to approximately -90°C, approximately -100°C to approximately -80°C, approximately -100°C to approximately -70°C, approximately -100°C to approximately -100°C. ℃ to -60℃, about -100℃ to -50℃, about -100℃ to -40℃, about -100℃ to -30℃, about -100℃ to -20℃, about -100℃ to -10℃, about -100℃ to 0℃, about -90℃ to -80℃, about -90℃ to -70℃, about -90℃ to -60℃, about -90℃ to -50℃, about -90℃ to -40℃, about -90℃ to -30℃, about -90℃ to -20℃, about -90℃ to -10℃, about -90℃ to 0℃, about -80℃ to -70℃, about - 80°C to -60°C, approximately -80°C to -50°C, approximately -80°C to -40°C, approximately -80°C to -30°C, approximately -80°C to -20°C, approximately -80°C to -10°C, approximately -80°C to 0°C, approximately -70°C to -60°C, approximately -70°C to -50°C, approximately -70°C to -40°C, approximately -70°C to -30°C, approximately -70°C to -20°C, approximately -70°C to -10°C, approximately -70°C to 0°C, approximately -60°C to -50°C, approximately -60°C to -40°C, approximately -60°C to -30°C, approximately -60°C to Frozen at approximately -20°C, approximately -60°C to approximately -10°C, approximately -60°C to approximately 0°C, approximately -50°C to approximately -40°C, approximately -50°C to approximately -30°C, approximately -50°C to approximately -20°C, approximately -50°C to approximately -10°C, approximately -50°C to approximately 0°C, approximately -40°C to approximately -30°C, approximately -40°C to approximately -20°C, approximately -40°C to approximately -10°C, approximately -40°C to approximately 0°C, approximately -30°C to approximately -20°C, approximately -30°C to approximately -10°C, approximately -30°C to approximately 0°C, approximately -20°C to approximately -10°C, approximately -20°C to approximately 0°C, or approximately -10°C to approximately 0°C.

[0269] After freezing and recovering the buffer, the buffer can be thawed and mixed with the sample. In some embodiments, the efficiency of the thawed recovered buffer is tested and compared with the efficiency of the unfrozen recovered buffer. In some embodiments, the efficiency of the thawed recovered buffer is similar to that of the unfrozen recovered buffer.

[0270] In some implementations, the recycled buffer may be lyophilized.

[0271] After the sample has been contacted with the recycled buffer, the sample can be a treated sample. Not wishing to be bound by theory, the sample treatment methods of this disclosure can reduce the total preparation time from untreated to treated samples. In some embodiments, the sample treatment of this disclosure reduces the sample treatment time by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, or at least about 80% compared to sample treatment times using different sample treatment methods (e.g., sample treatment methods using SDS, polysorbate 80, or cyclodextrin, respectively). Different sample treatment methods may include alternative reagents, including but not limited to isopropanol and / or ethanol. In some embodiments, the sample processing of this disclosure reduces the sample processing time by up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, or up to about 3% compared to the sample processing time of different sample processing methods.

[0272] In some embodiments, the sample processing of this disclosure reduces sample processing time by about 2% to about 75%. In some embodiments, the sample processing of this disclosure reduces sample processing time by up to about 75%. In some embodiments, the sample processing of this disclosure reduces sample processing time by approximately 2% to approximately 3%, approximately 2% to approximately 5%, approximately 2% to approximately 7%, approximately 2% to approximately 10%, approximately 2% to approximately 15%, approximately 2% to approximately 20%, approximately 2% to approximately 30%, approximately 2% to approximately 40%, approximately 2% to approximately 50%, approximately 2% to approximately 60%, approximately 2% to approximately 75%, approximately 3% to approximately 5%, approximately 3% to approximately 7%, approximately 3% to approximately 10%, approximately 3% to approximately 15%, approximately 3% to approximately 20%, approximately 3% to approximately 30%, approximately 3% to approximately 40%, approximately 3% to approximately 50%, approximately 3% to approximately 60%, approximately 3% to approximately 75%, approximately 5% to approximately 7%, approximately 5% to approximately 10%, approximately 7% to approximately 15%, approximately 7% to approximately 20%, approximately 7% to approximately 75%, approximately 7% to approximately 10%, approximately 7% to approximately 15%, approximately 7% to approximately 20%, approximately 7% to approximately 7%. Approximately 30%, approximately 7% to approximately 40%, approximately 7% to approximately 50%, approximately 7% to approximately 60%, approximately 7% to approximately 75%, approximately 10% to approximately 15%, approximately 10% to approximately 20%, approximately 10% to approximately 30%, approximately 10% to approximately 40%, approximately 10% to approximately 50%, approximately 10% to approximately 60%, approximately 10% to approximately 75%, approximately 15% to approximately 20%, approximately 15% to approximately 30%, approximately 15% to approximately 40%, approximately 15% to approximately 50%, approximately 15% to approximately 60%. Approximately 15% to approximately 75%, approximately 20% to approximately 30%, approximately 20% to approximately 40%, approximately 20% to approximately 50%, approximately 20% to approximately 60%, approximately 20% to approximately 75%, approximately 30% to approximately 40%, approximately 30% to approximately 50%, approximately 30% to approximately 60%, approximately 30% to approximately 75%, approximately 40% to approximately 50%, approximately 40% to approximately 60%, approximately 40% to approximately 75%, approximately 50% to approximately 60%, approximately 50% to approximately 75%, or approximately 60% to approximately 75%.

[0273] In one aspect, this disclosure provides a method for processing a sample, the method comprising: (a) contacting the sample with a pyrolysis buffer containing a detergent; (b) incubating the sample at a first temperature or temperature range for a first time period; (c) heating the sample at a second temperature or temperature range for a second time period; and (d) contacting the sample with a recovery buffer containing a solubilizer and cyclodextrin, thereby processing the sample to generate a treated sample in a mixture containing detergent, solubilizer and cyclodextrin.

[0274] In some embodiments, heating the sample in (c) further includes heating the sample to a second temperature, cooling the sample, and then heating the sample to the second temperature after cooling. In some embodiments, the sample may be cooled to room temperature. In some embodiments, the sample may be cooled to a temperature below room temperature.

[0275] In some embodiments, the sample in (b) may be incubated at a temperature of at least about -10°C, at least about -5°C, at least about 0°C, at least about 5°C, at least about 10°C, at least about 12°C, at least about 14°C, at least about 16°C, at least about 18°C, at least about 20°C, at least about 22°C, at least about 24°C, at least about 26°C, at least about 28°C, at least about 30°C, at least about 35°C, at least about 40°C, at least about 45°C, or at least about 50°C.

[0276] In some embodiments, the sample in (b) may be incubated at temperatures of up to about 50°C, up to about 45°C, up to about 40°C, up to about 35°C, up to about 30°C, up to about 28°C, up to about 26°C, up to about 24°C, up to about 22°C, up to about 20°C, up to about 18°C, up to about 16°C, up to about 14°C, up to about 12°C, up to about 10°C, up to about 5°C, up to about 0°C, up to about -5°C, or up to about -10°C.

[0277] In some embodiments, the sample in (b) can be incubated at a temperature of about -10°C to about 50°C. In some embodiments, the sample in (b) can be incubated at temperatures ranging from about -10°C to about 0°C, from about -10°C to about 10°C, from about -10°C to about 12°C, from about -10°C to about 15°C, from about -10°C to about 17°C, from about -10°C to about 20°C, from about -10°C to about 22°C, from about -10°C to about 25°C, from about -10°C to about 27°C, from about -10°C to about 30°C, from about -10°C to about 50°C, from about 0°C to about 10°C, from about 0°C to about 12°C, from about 0°C to about 15°C, from about 0°C to about 50°C. 17°C, about 0°C to about 20°C, about 0°C to about 22°C, about 0°C to about 25°C, about 0°C to about 27°C, about 0°C to about 30°C, about 0°C to about 50°C, about 10°C to about 12°C, about 10°C to about 15°C, about 10°C to about 17°C, about 10°C to about 20°C, about 10°C to about 22°C, about 10°C to about 25°C, about 10°C to about 27°C, about 10°C to about 30°C, about 10°C to about 50°C, about 12°C to about 15°C, about 12°C to about 17°C, about 12°C Approximately 20°C, approximately 12°C to approximately 22°C, approximately 12°C to approximately 25°C, approximately 12°C to approximately 27°C, approximately 12°C to approximately 30°C, approximately 12°C to approximately 50°C, approximately 15°C to approximately 17°C, approximately 15°C to approximately 20°C, approximately 15°C to approximately 22°C, approximately 15°C to approximately 25°C, approximately 15°C to approximately 27°C, approximately 15°C to approximately 30°C, approximately 15°C to approximately 50°C, approximately 17°C to approximately 20°C, approximately 17°C to approximately 22°C, approximately 17°C to approximately 25°C, approximately 17°C to approximately 27°C, approximately 17°C to approximately Incubate at temperatures of 30°C, about 17°C to about 50°C, about 20°C to about 22°C, about 20°C to about 25°C, about 20°C to about 27°C, about 20°C to about 30°C, about 20°C to about 50°C, about 22°C to about 25°C, about 22°C to about 27°C, about 22°C to about 30°C, about 22°C to about 50°C, about 25°C to about 27°C, about 25°C to about 30°C, about 25°C to about 50°C, about 27°C to about 30°C, about 27°C to about 50°C, or about 30°C to about 50°C.

[0278] In some implementations, the first time period is at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, or at least about 5 days.

[0279] In some implementations, the first time period is up to about 10 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 48 hours, up to about 36 hours, up to about 24 hours, up to about 12 hours, up to about 10 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 60 minutes, up to about 45 minutes, up to about 30 minutes, up to about 25 minutes, up to about 20 minutes, up to about 15 minutes, up to about 10 minutes, up to about 5 minutes, up to about 4 minutes, up to about 3 minutes, up to about 2 minutes, up to about 1 minute, or up to about 30 seconds.

[0280] In some embodiments, the methods of this disclosure may include beading a sample. Beading can assist in cell lysis by agitating the sample with grinding media or beads. In some embodiments, a lysis matrix may be used for beading. In some embodiments, the lysis matrix may include silica, glass, ceramics, silicon carbide, zirconium silicate, garnet, stainless steel, and / or zirconium oxide.

[0281] In some embodiments, the second temperature of the method of this disclosure is at least about, at most about or about 30°C, 40°C, 50°C, 60°C, 70°C, 75°C, 80°C, 85°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 105°C, 110°C, 115°C, 120°C, 125°C, 130°C, 140°C, at least about 150°C, or a range between any two of these values.

[0282] In some implementations, the second time period is at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, or at least about 5 hours.

[0283] In some implementations, the second time period is up to about 10 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 60 minutes, up to about 45 minutes, up to about 30 minutes, up to about 25 minutes, up to about 20 minutes, up to about 15 minutes, up to about 10 minutes, up to about 5 minutes, up to about 4 minutes, up to about 3 minutes, up to about 2 minutes, up to about 1 minute, or up to about 30 seconds.

[0284] After buffer recovery, the treated sample can be contacted with the reaction mixture. In some embodiments, the reaction mixture contains a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, and / or probes. In some embodiments, In some embodiments, the thermostable enzyme comprises a large fragment of *Bacillus stearothermophilus* polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and / or any mutant thereof. In some embodiments, the thermostable enzyme comprises a DNA polymerase. In some embodiments, the thermostable enzyme comprises Taq DNA polymerase. In some embodiments, the thermostable enzyme comprises a DNA-dependent DNA polymerase. In some embodiments, the thermostable enzyme comprises a strand displacement DNA polymerase. In some embodiments, the reaction mixture stabilizes the enzymatic activity of the thermostable enzyme. The large fragment of *Bacillus stearothermophilus* polymerase is a portion of *Bacillus stearothermophilus* DNA polymerase, containing 5´→3´ polymerase activity but lacking the 5´→3´ exonuclease domain. In some embodiments, the composition is configured to stabilize the enzymatic activity of the thermostable enzyme for use during nucleic acid amplification.

[0285] In some embodiments, the dNTPs of the reaction mixture include dATP, dCTP, dGTP, dTTP, and / or dUTP. In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is at least about 25 µM, at least about 50 µM, at least about 75 µM, at least about 100 µM, at least about 150 µM, at least about 200 µM, at least about 250 µM, at least about 300 µM, at least about 350 µM, at least about 400 µM, at least about 450 µM, at least about 500 µM, at least about 750 µM, at least about 1000 µM, at least about 1500 µM, at least about 2000 µM, at least about 2500 µM, at least about 3000 µM, at least about 3500 µM, at least about 4000 µM, at least about 4500 µM, at least about 5000 µM, at least about 6000 µM, at least about 7000 µM, at least about 8000 µM, at least about 9000 µM, or at least about 10000 µM.

[0286] In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is up to about 10,000 µM, up to about 9,000 µM, up to about 8,000 µM, up to about 7,000 µM, up to about 6,000 µM, up to about 5,000 µM, up to about 4,500 µM, up to about 4,000 µM, up to about 3,500 µM, up to about 3,000 µM, up to about 2,500 µM, up to about 2,000 µM, up to about 1,500 µM, up to about 1,000 µM, up to about 750 µM, up to about 500 µM, up to about 450 µM, up to about 400 µM, up to about 350 µM, up to about 300 µM, up to about 250 µM, up to about 200 µM, up to about 150 µM, up to about 100 µM. µM, up to about 75 µM, up to about 50 µM or up to about 25 µM.

[0287] In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is from about 50 µM to about 7,500 µM. In some embodiments, when mixed with a sample, the concentration of dNTPs in the reaction mixture is approximately 50 µM to approximately 100 µM, approximately 50 µM to approximately 250 µM, approximately 50 µM to approximately 500 µM, approximately 50 µM to approximately 750 µM, approximately 50 µM to approximately 1,000 µM, approximately 50 µM to approximately 1,250 µM, approximately 50 µM to approximately 1,500 µM, approximately 50 µM to approximately 2,000 µM, approximately 50 µM to approximately 4,000 µM, approximately 50 µM to approximately 5,000 µM, approximately 50 µM to approximately 7,500 µM, approximately 100 µM to approximately 250 µM, approximately 100 µM to approximately 500 µM, approximately 100 µM to approximately 750 µM, approximately 100 µM to approximately 1,000 µM, approximately 100 µM to approximately 1,250 µM, approximately 100 µM to approximately 1,500 µM. µM, about 100 µM to about 2,000 µM, about 100 µM to about 4,000 µM, about 100 µM to about 5,000 µM, about 100 µM to about 7,500 µM, about 250 µM to about 500 µM, about 250 µM to about 750 µM, about 250 µM to about 1,000 µM, about 250 µM to about 1,250 µM, about 250 µM to about 1,500 µM, about 250 µM to about 2,000 µM, about 250 µM to about 4,000 µM, about 250 µM to about 5,000 µM, about 250 µM to about 7,500 µM, about 500 µM to about 750 µM, about 500 µM to about 1,000 µM, about 500 µM to about 1,250 µM, about 500 µM approximately 1,500 µM to 2,000 µM, approximately 500 µM to 4,000 µM, approximately 500 µM to 5,000 µM, approximately 500 µM to 7,500 µM, approximately 750 µM to 1,000 µM, approximately 750 µM to 1,250 µM, approximately 750 µM to 1,500 µM, approximately 750 µM to 2,000 µM, approximately 750 µM to 4,000 µM, approximately 750 µM to 5,000 µM, approximately 750 µM to 7,500 µM, approximately 1,000 µM to 1,250 µM, approximately 1,000 µM to 1,500 µM, approximately 1,000 µM to 2,000 µM, approximately 1,000 µM to 4,000 µM µM, approximately 1,000 µM to approximately 5,000 µM, approximately 1,000 µM to approximately 7,500 µM, approximately 1,250 µM to approximately 1,500 µM, approximately 1,250 µM to about 2,000 µM, about 1,250 µM to about 4,000 µM, about 1,250 µM to about 5,000 µM, about 1,250 µM to about 7,500 µM, about 1,500 µM to about 2,000 µM, about 1,500 µM to about 4,000 µM, about 1,500 µM to about 5,000 µM, about 1,500 µM to about 7,500 µM, about 2,000 µM to about 4,000 µM, about 2,000 µM to about 5,000 µM, about 2,000 µM to about 7,500 µM, about 4,000 µM to about 5,000 µM, about 4,000 µM to about 7,500 µM, or about 5,000 µM to about 7,500 µM.

[0288] In some embodiments, the primer or probe may be a nucleotide segment that hybridizes to the target nucleic acid sequence. In some embodiments, the primer length is at least about 3 nucleotides, at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, or at least about 200 nucleotides. In some implementations, the primer length is up to about 200 nucleotides, up to about 150 nucleotides, up to about 100 nucleotides, up to about 90 nucleotides, up to about 80 nucleotides, up to about 70 nucleotides, up to about 60 nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, up to about 35 nucleotides, up to about 30 nucleotides, up to about 25 nucleotides, up to about 20 nucleotides, up to about 15 nucleotides, up to about 10 nucleotides, up to about 5 nucleotides, or up to about 3 nucleotides.

[0289] In some embodiments, the primer length is from about 3 nucleotides to about 100 nucleotides. In some embodiments, the primer length is at most about 100 nucleotides. In some embodiments, the primer length is from about 3 nucleotides to about 5 nucleotides, from about 3 nucleotides to about 10 nucleotides, from about 3 nucleotides to about 20 nucleotides, from about 3 nucleotides to about 30 nucleotides, from about 3 nucleotides to about 40 nucleotides, from about 3 nucleotides to about 50 nucleotides, from about 3 nucleotides to about 60 nucleotides, from about 3 nucleotides to about 70 nucleotides, from about 3 nucleotides to about 80 nucleotides, from about 3 nucleotides to about 90 nucleotides, from about 3 nucleotides to about 100 nucleotides, from about 5 nucleotides to about 10 nucleotides, from about 5 nucleotides to about 20 nucleotides, from about 5 nucleotides to about 30 nucleotides, from about 5 nucleotides to about 40 nucleotides. Nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 60 nucleotides, about 5 nucleotides to about 70 nucleotides, about 5 nucleotides to about 80 nucleotides, about 5 nucleotides to about 90 nucleotides, about 5 nucleotides to about 100 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 60 nucleotides, about 10 nucleotides to about 70 nucleotides, about 10 nucleotides to about 80 nucleotides, about 10 nucleotides to about 90 nucleotides, about 10 nucleotides to about 100 nucleotides Nucleotides, approximately 20 nucleotides to approximately 30 nucleotides, approximately 20 nucleotides to approximately 40 nucleotides, approximately 20 nucleotides to approximately 50 nucleotides, approximately 20 nucleotides to approximately 60 nucleotides, approximately 20 nucleotides to approximately 70 nucleotides, approximately 20 nucleotides to approximately 80 nucleotides, approximately 20 nucleotides to approximately 90 nucleotides, approximately 20 nucleotides to approximately 100 nucleotides, approximately 30 nucleotides to approximately 40 nucleotides, approximately 30 nucleotides to approximately 50 nucleotides, approximately 30 nucleotides to approximately 60 nucleotides, approximately 30 nucleotides to approximately 70 nucleotides, approximately 30 nucleotides to approximately 80 nucleotides, approximately 30 nucleotides to approximately 90 nucleotides, approximately 30 nucleotides Approximately 100 nucleotides, approximately 40 nucleotides to approximately 50 nucleotides, approximately 40 nucleotides to approximately 60 nucleotides, approximately 40 nucleotides to approximately 70 nucleotides, approximately 40 nucleotides to approximately 80 nucleotides, approximately 40 nucleotides to approximately 90 nucleotides, approximately 40 nucleotides to approximately 100 nucleotides, approximately 50 nucleotides to approximately 60 nucleotides, approximately 50 nucleotides to approximately 70 nucleotides, approximately 50 nucleotides to approximately 80 nucleotides, approximately 50 nucleotides to approximately 90 nucleotides, approximately 50 nucleotides to approximately 100 nucleotides, approximately 60 nucleotides to approximately 70 nucleotides, approximately 60 nucleotides to approximately 80 nucleotides, approximately 60 nucleotides to approximately 90 nucleotides.Approximately 60 nucleotides to approximately 100 nucleotides, approximately 70 nucleotides to approximately 80 nucleotides, approximately 70 nucleotides to approximately 90 nucleotides, approximately 70 nucleotides to approximately 100 nucleotides, approximately 80 nucleotides to approximately 90 nucleotides, approximately 80 nucleotides to approximately 100 nucleotides, or approximately 90 nucleotides to approximately 100 nucleotides.

[0290] In some implementations, the primers are labeled with biotin or 6-carboxyfluorescein (FAM) for visualization on lateral flow immunoassay strips.

[0291] In some implementations, the reaction mixture is lyophilized.

[0292] In some embodiments, the methods and compositions of this disclosure can provide a faster time from obtaining a sample to generating a treated sample. In some embodiments, the time from obtaining a sample to generating a treated sample is at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, or at least about 1 hour. In some embodiments, the time from obtaining a sample to generating a treated sample is at most about 1 hour, at most about 45 minutes, at most about 30 minutes, at most about 20 minutes, at most about 15 minutes, at most about 10 minutes, at most about 5 minutes, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, or at most about 30 seconds.

[0293] In some embodiments, the time from obtaining the sample to generating the treated sample is from about 1 minute to about 45 minutes. In some embodiments, the time from obtaining the sample to generating the treated sample is at most about 45 minutes. In some embodiments, the time from obtaining the sample to generating the treated sample is from about 1 minute to about 2 minutes, from about 1 minute to about 3 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 6 minutes, from about 1 minute to about 7 minutes, from about 1 minute to about 8 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 15 minutes, from about 1 minute to about 30 minutes, from about 1 minute to about 45 minutes, from about 2 minutes to about 3 minutes, from about 2 minutes to about 4 minutes, from about 2 minutes to about 5 minutes, from about 2 minutes to about 6 minutes, and so on. 2 minutes to about 7 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 15 minutes, about 2 minutes to about 30 minutes, about 2 minutes to about 45 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 5 minutes, about 3 minutes to about 6 minutes, about 3 minutes to about 7 minutes, about 3 minutes to about 8 minutes, about 3 minutes to about 10 minutes, about 3 minutes to about 15 minutes, about 3 minutes to about 30 minutes, about 3 minutes to about 45 minutes, about 4 minutes to about 5 minutes, about 4 minutes to about 6 minutes, about 4 minutes to about 7 minutes, approximately 4 minutes to approximately 8 minutes, approximately 4 minutes to approximately 10 minutes, approximately 4 minutes to approximately 15 minutes, approximately 4 minutes to approximately 30 minutes, approximately 4 minutes to approximately 45 minutes, approximately 5 minutes to approximately 6 minutes, approximately 5 minutes to approximately 7 minutes, approximately 5 minutes to approximately 8 minutes, approximately 5 minutes to approximately 10 minutes, approximately 5 minutes to approximately 15 minutes, approximately 5 minutes to approximately 30 minutes, approximately 5 minutes to approximately 45 minutes, approximately 6 minutes to approximately 7 minutes, approximately 6 minutes to approximately 8 minutes, approximately 6 minutes to approximately 10 minutes, approximately 6 minutes to approximately 15 minutes, approximately 6 minutes to approximately 30 minutes Clock, about 6 minutes to about 45 minutes, about 7 minutes to about 8 minutes, about 7 minutes to about 10 minutes, about 7 minutes to about 15 minutes, about 7 minutes to about 30 minutes, about 7 minutes to about 45 minutes, about 8 minutes to about 10 minutes, about 8 minutes to about 15 minutes, about 8 minutes to about 30 minutes, about 8 minutes to about 45 minutes, about 10 minutes to about 15 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 45 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes.

[0294] After contact with the reaction mixture, the treated sample can then be subjected to nucleic acid amplification methods. Various nucleic acid amplification methods can be used with the compositions and methods disclosed herein to amplify target sequences in nucleic acid molecules. Methods for nucleic acid amplification include, but are not limited to, polymerase chain reaction (PCR), sequence-based amplification (NASBA), oligonucleotide ligation assay (OLA), transcription-mediated amplification (TMA), oligonucleotide extension and ligation, rolling circle amplification (RCA), and / or strand displacement amplification (SDA). Target sequences treated by the methods provided herein can be used for further downstream applications, such as isothermal amplification. Exemplary isothermal amplification methods that can be used with the compositions and methods provided herein include, but are not limited to, helicase-dependent amplification (HDA), isothermal multiple displacement amplification (IMDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), single primer isothermal amplification (SPIA), or strand displacement amplification (SDA).

[0295] In some embodiments, the nucleic acid amplification of this disclosure includes PCR or isothermal amplification. In some embodiments, the temperature is varied during the nucleic acid amplification method. In some embodiments, nucleic acid amplification includes thermal cycling of the treated sample. In some embodiments, nucleic acid amplification includes maintaining the treated sample at a constant temperature during amplification.

[0296] In some embodiments, the reaction mixture includes a probe that visualizes the amplified nucleic acid product. In some embodiments, the probe includes a strand displacement probe, an embedded fluorophore, a pH-sensitive dye, and / or a pyrophosphate detection product.

[0297] In some cases, the methods and compositions disclosed herein for processing nucleic acid molecular samples produce amplified nucleic acid products in higher yields compared to those produced by different sample processing methods or compositions. In some embodiments, the sample processing methods and compositions produce amplified nucleic acid products in yields at least about 5-fold, at least 10-fold, at least about 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 1,500-fold, at least 2,000-fold, at least 10-fold, or at least 10,000-fold.

[0298] In some embodiments, the sample processing method and composition generate amplified nucleic acid products in yields that are approximately 3 to approximately 1000 times higher than those generated by treating otherwise identical samples with SDS, polysorbate 80, and / or cyclodextrin alone. In some embodiments, the sample processing method and composition generate amplified nucleic acid products in yields of up to approximately 1000 times higher. In some embodiments, the sample processing method and composition generate am...

Claims

1. A composition for sample preparation, said composition comprising: a detergent, a solubilizer, and a cyclodextrin. The composition is configured to stabilize the enzyme during nucleic acid amplification, and The composition is configured to reduce and / or eliminate the activity of degrading nucleases.

2. The composition of claim 1, wherein the composition is configured to stabilize the nucleic acid during the nucleic acid amplification.

3. The composition according to claim 1 or 2, wherein the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

4. The composition according to any one of claims 1-3, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

5. The composition according to any one of claims 1-4, wherein the solubilizer is a nonionic surfactant.

6. The composition according to any one of claims 1-4, wherein the solubilizer is polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol or secondary alcohol polyoxyethylene ether.

7. The composition of claim 5 or 6, wherein the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60 or a functional variant thereof.

8. The composition of any one of claims 1-5, wherein the detergent is part of a lysis buffer.

9. The composition of any one of claims 1-8, wherein the solubilizer and the cyclodextrin are part of a recovery buffer.

10. The composition of claim 9, wherein the lysis buffer and the recovery buffer are in the same mixture.

11. A composition for sample treatment containing a buffer, said composition comprising: a detergent, a solubilizer, and a cyclodextrin. The composition is configured to stabilize the enzyme during nucleic acid amplification, and The composition is configured to inactivate degrading nucleases.

12. The composition of claim 11, wherein the composition is configured to stabilize the nucleic acid during the nucleic acid amplification.

13. The composition of claim 11 or 12, wherein the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

14. The composition of any one of claims 11-13, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

15. The composition of any one of claims 11-14, wherein the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

16. The composition of any one of claims 1-15, wherein the solubilizer and the cyclodextrin are configured to shorten the cycle threshold or result time in nucleic acid amplification compared to the cycle threshold or result time in nucleic acid amplification of a sample otherwise identical to that treated with SDS, polysorbate 80, or cyclodextrin alone.

17. The composition of claim 16, wherein the cycle threshold is at most 40 or the result time is at most 15 minutes.

18. The composition of any one of claims 1-17, wherein the solubilizer and the cyclodextrin are configured to reduce the coefficient of variation.

19. The composition of any one of claims 1-18, wherein the solubilizer and the cyclodextrin are configured to lower the detection limit.

20. The composition of any one of claims 1-19, wherein the degrading nuclease is a ribonuclease.

21. The composition of any one of claims 8-10, wherein the lysis buffer has a pH value of 2 to 9.

22. The composition of any one of claims 8-10 and 21, wherein the lysis buffer further comprises a chelating agent.

23. The composition of claim 22, wherein the chelating agent is deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, or N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).

24. The composition of any one of claims 8-10, 21 and 22, wherein the lysis buffer further comprises a reducing agent.

25. The composition of claim 24, wherein the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or tetrahydropyran (THP).

26. The composition of any one of claims 8-10 and 21-25, wherein the cleavage buffer comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), tetrahydropyran (THP), or any combination thereof.

27. The composition of claim 26, wherein... The final concentration of EGTA in the lysis buffer in the presence of the sample is approximately 0.1 mmol (mM) to 10 mM. The final concentration of EDTA in the lysis buffer in the presence of the sample is approximately 0.1 mM to 5 mM. The final concentration of TCEP in the lysis buffer in the presence of the sample is approximately 1 mM to 20 mM, or The final concentration of Tris in the lysis buffer in the presence of the sample is approximately 1 mM to 60 mM.

28. The composition of any one of claims 1-27, wherein the composition further comprises an agent capable of reducing disulfide bonds.

29. The composition of claim 28, wherein the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME).

30. The composition of any one of claims 1-29, wherein the detergent is present in the composition mixed with the sample at a final concentration that effectively lyses cells.

31. The composition of any one of claims 1-30, wherein the cyclodextrin is present in the composition mixed with the sample at a final concentration that effectively isolates the detergent within the composition.

32. The composition of any one of claims 1-31, wherein the detergent is configured to form a complex with the solubilizer and / or the cyclodextrin to stabilize the enzyme.

33. The composition of claim 32, wherein the cyclodextrin is configured to increase the efficiency of forming the complex.

34. The composition of any one of claims 1-33, wherein the cyclodextrin has a higher binding affinity for the detergent than the solubilizer.

35. The composition of any one of claims 30-34, wherein the final concentration of the detergent is about 0.1% to 10% w / v (g solute / 100 mL solution).

36. The composition of any one of claims 31-35, wherein the final concentration of the cyclodextrin is from about 0.1 mM to 70 mM.

37. The composition of any one of claims 1-36, wherein the cyclodextrin comprises hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, anionic cyclodextrin, or any combination thereof.

38. The composition of any one of claims 1-37, wherein the solubilizer is present in the composition mixed with the sample at a final concentration of about 0.1% to 50% w / v.

39. The composition of claim 38, wherein the final concentration of the solubilizer effectively forms micelles containing the detergent.

40. The composition of claim 9 or 10, wherein the recovery buffer comprises a salt.

41. The composition of claim 9 or 10, wherein the recovery buffer does not contain salt.

42. The composition of any one of claims 9, 10, 40 and 41, wherein the recovery buffer comprises a pH buffer.

43. The composition of any one of claims 9, 10, 40 and 41, wherein the recovery buffer does not contain a pH buffer.

44. The composition of any one of claims 8-10 and 21-39, wherein the lysis buffer is lyophilized.

45. The composition of any one of claims 9, 10 and 40-43, wherein the recovery buffer is lyophilized.

46. ​​The composition of any one of claims 1-45, wherein the composition further comprises a sample.

47. The composition of claim 46, wherein the sample is a biological sample.

48. The composition of claim 47, wherein the biological sample comprises a target nucleic acid molecule for sample processing.

49. The composition of any one of claims 1-48, wherein the composition further comprises a reaction mixture for nucleic acid amplification.

50. The composition of claim 49, wherein the reaction mixture is lyophilized.

51. The composition of claim 49 or 50, wherein the reaction mixture comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, or probes.

52. The composition of claim 51, wherein the composition is configured to stabilize the enzymatic activity of the thermostable enzyme for use during nucleic acid amplification.

53. The composition of claim 52, wherein the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and any mutant thereof.

54. The composition of claim 51, wherein the dNTP comprises dATP, dCTP, dGTP, dTTP or dUTP.

55. The composition of claim 54, wherein the concentration of the dNTP in the reaction mixture is from about 40 micromoles (µM) to 5000 µM.

56. The composition of claim 51, wherein the primer is at least 4 nucleotides in length.

57. A method of processing a sample, the method comprising mixing the sample with a lysis buffer as described in any one of claims 8-10 or 21-56.

58. The method of claim 57, further comprising mixing the sample with the recovery buffer of any one of claims 9, 10, or 40-56.

59. A method for processing a sample, the method comprising: (a) Contact the sample with a lysis buffer containing detergent; as well as (b) The sample is contacted with a recovery buffer containing a solubilizer and cyclodextrin to treat the sample to produce a treated sample in a mixture containing the detergent, the solubilizer and the cyclodextrin.

60. The method of claim 59, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

61. The method of claim 59 or 60, wherein the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

62. The method of any one of claims 59-61, wherein the sample is a biological sample.

63. The method of any one of claims 59-62, wherein the sample is a purified sample.

64. The method of any one of claims 59-63, wherein (a) and (b) occur simultaneously.

65. The method of any one of claims 59-64, wherein the contact of the samples in (a) and (b) is carried out simultaneously in the same mixture.

66. The method of any one of claims 59-65, the method further comprising incubating the sample at room temperature for a sustained period of time.

67. The method of any one of claims 59-66, the method further comprising heating the sample at a constant temperature for a period of time.

68. The method of any one of claims 59-66, the method further comprising heating the sample at a cyclic temperature for a period of time.

69. The method of any one of claims 59-68, the method further comprising sonicating the sample.

70. The method of any one of claims 67 or 69, wherein the ultrasonic treatment of the sample occurs before, after, or simultaneously with heating the sample.

71. A method for processing a sample, the method comprising: (a) Contact the sample with a lysis buffer containing detergent; (b) The sample is incubated at a first temperature or temperature range for a first time period; (c) Heating the sample at a second temperature or temperature range for a second time period; as well as (d) The sample is contacted with a recovery buffer containing a solubilizer and cyclodextrin to treat the sample to produce a treated sample in a mixture containing the detergent, the solubilizer and the cyclodextrin.

72. The method of claim 71, wherein heating the sample in (c) further comprises heating the sample to the second temperature, cooling the sample, and heating the sample to the second temperature after cooling.

73. The method of claim 71 or 72, wherein the method further comprises sonicating the sample.

74. The method of claim 73, wherein the ultrasonic treatment of the sample is performed before, after, or simultaneously with heating the sample.

75. The method of any one of claims 71-74, the method further comprising beading the sample.

76. The method of any one of claims 71-75, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

77. The method of any one of claims 71-76, wherein the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

78. The method of any one of claims 71-77, wherein the sample is a biological sample.

79. The method of any one of claims 71-78, wherein the sample is a purified sample.

80. The method of any one of claims 71-79, wherein the first temperature or temperature range is about 2°C to 25°C.

81. The method of any one of claims 71-80, wherein the second temperature is about 60°C to 100°C.

82. The method of any one of claims 71-81, wherein the first time period is from at least about 1 minute to at least about 48 hours.

83. The method of any one of claims 71-82, wherein the second time period is from at least about 1 minute to at least about 10 minutes.

84. The method of any one of claims 59-83, wherein the lysis buffer further comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

85. The method of any one of claims 59-84, the method further comprising, after contacting the sample with the recovery buffer, contacting the treated sample with the reaction mixture.

86. The method of claim 85, wherein the reaction mixture comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, or probes.

87. The method of claim 86, wherein the reaction mixture stabilizes the enzymatic activity of the thermostable enzyme for use during nucleic acid amplification.

88. The method of claim 87, wherein the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and any mutant thereof.

89. The method of claim 86, wherein the dNTP comprises dATP, dCTP, dGTP, dTTP, or dUTP.

90. The method of claim 89, wherein the concentration of the dNTP in the reaction mixture is from about 40 µM to 5000 µM.

91. The method of claim 86, wherein the primer is at least 4 nucleotides in length.

92. The method of any one of claims 85-91, the method further comprising performing nucleic acid amplification on the treated sample mixed with the reaction mixture.

93. The method of claim 92, wherein the nucleic acid amplification comprises polymerase chain reaction (PCR) or isothermal amplification.

94. The method of claim 92 or 93, wherein the nucleic acid amplification comprises thermal cycling the treated sample.

95. The method of any one of claims 92-94, wherein the nucleic acid amplification comprises maintaining the treated sample at a constant temperature for amplification.

96. The method of any one of claims 59-95, wherein the method further comprises obtaining the sample from the object prior to (a).

97. The method of claim 96, wherein the subject has or is suspected of having a disease, symptom, or infection.

98. The method of any one of claims 59-97, wherein the sample comprises one or more different target nucleic acid molecules.

99. The method of any one of claims 59-98, wherein the sample comprises blood sample, swab sample, saliva sample, urine sample, cerebrospinal fluid sample, pleural fluid sample, rectal sample, vaginal sample, fecal sample, sputum sample, lymph sample, raw emulsion, pasteurized and / or homogenized emulsion, pasteurized and / or treated emulsion, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue sample, cell culture, purified nucleic acid sample, environmental sample, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof.

100. The method of any one of claims 96-99, wherein the time from obtaining the sample to generating the treated sample is equal to or less than about 30 min, 25 min, 20 min, 15 min, 10 min, 5 min, 4 min, 3 min, 2 min, 1 min or less.

101. The method of any one of claims 98-100, wherein the concentration of one or more different target nucleic acid molecules is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, compared to the concentration of one or more different target nucleic acid molecules in a sample otherwise identical to that treated by SDS, polysorbate 80 or cyclodextrin alone.

102. A kit for sample processing, the kit comprising a lysis buffer containing detergent, a recovery buffer containing solubilizer and cyclodextrin, and instructions for use.

103. The kit of claim 102, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

104. The kit of claim 102 or 103, wherein the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60 or a functional variant thereof.

105. The kit according to any one of claims 102-104, wherein the kit further comprises reagents for nucleic acid amplification, said reagents including thermostable enzymes, deoxynucleoside triphosphates (dNTPs), or primers.

106. The kit of claim 105, wherein the thermostable enzyme is selected from large fragments of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, and any mutant thereof.

107. The kit of claim 105, wherein the dNTP comprises dATP, dCTP, dGTP, dTTP or dUTP.

108. The kit of claim 105 or 107, wherein the concentration of the dNTP in the reaction mixture is from about 40 µM to 5000 µM.

109. The kit of claim 105, wherein the primers are at least 4 nucleotides in length.

110. The kit according to any one of claims 102-109, wherein the kit further comprises a probe for detecting the amplification product generated using the kit.

111. The kit according to any one of claims 102-110, wherein the lysis buffer, the recovery buffer, or the reagent is lyophilized.

112. The composition of claim 9, wherein the recovery buffer further comprises cucurbitaureus.

113. The composition of claim 112, wherein the cucurbituril is cucurbit[n]urea, wherein n is an integer of 5, 6, 7, 8 or 10.

114. The composition of any one of claims 49-56, wherein the reaction mixture comprises an excipient.

115. The composition of claim 114, wherein the excipient comprises one or more agents selected from Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, nonylphenyl alcohol ether-9, trehalose, dextran, polysucrose 400 and cyclodextrin.

116. The composition of claim 115, wherein the cyclodextrin comprises hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, anionic cyclodextrin, or any combination thereof.

117. The composition of claim 115 or 116, wherein: In the presence of the sample, the final concentration of Tris in the excipient is from about 0.001 mol (M) to 1.0 M; In the presence of the sample, the final concentration of sodium chloride and / or potassium chloride is approximately 0.0001 M to 0.25 M; In the presence of the sample, the final concentration of EDTA in the excipient is approximately 0.00001 M to 0.1 M; In the presence of the sample, the final concentration of nonoxynol-9 in the excipient is approximately 0.01% v / v to 2.0% v / v; In the presence of the sample, the final concentration of trehalose in the excipient is approximately 0.001 M to 2.0 M; In the presence of the sample, the final concentration of dextran in the excipient is approximately 0.1% w / v to 10% w / v; In the presence of the sample, the final concentration of sucrose 400 in the excipient is approximately 0.01% w / v to 5.0% w / v; and / or In the presence of the sample, the final concentration of the cyclodextrin in the excipient is from about 0.001 M to 5.0 M.

118. The composition of any one of claims 114-117, wherein the excipient further comprises an additional reagent.

119. The composition of claim 118, wherein the additional reagent comprises a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof.

120. The composition of claim 118 or 119, wherein the additional reagent is configured to stabilize the enzyme.

121. The composition of any one of claims 118-120, wherein the additional reagent is configured to reduce the Cq value of nucleic acid amplification.

122. The composition of any one of claims 49-56 and 114-121, wherein the composition further comprises a sample stabilizing buffer.

123. The composition of claim 122, wherein the sample stabilizing buffer comprises one or more agents selected from collapse modifiers, protein stabilizers, and glass transition modifiers.

124. The composition of claim 122 or 123, wherein the sample stabilizing buffer comprises at least one salt.

125. The composition of any one of claims 122-124, wherein the sample stabilizing buffer comprises cyclodextrin.

126. The composition of any one of claims 122-125, wherein the sample stabilizing buffer is configured to reconstruct the lyophilized sample.

127. The composition of any one of claims 122-126, wherein the stabilizing buffer comprises at least one reducing agent.

128. The composition of claim 127, wherein the at least one reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or tetrahydropyran (THP) or any combination thereof.

129. The method of any one of claims 59-70, wherein the total time for performing (a) and (b) is at most 1 minute, at most 50 seconds, at most 40 seconds, or at most 20 seconds.

130. The method of any one of claims 85-95, wherein the time for treating the sample is the period from the contact in (a) to the contact of the treated sample with the reaction mixture, wherein the period is at most 20 seconds.

131. The method of claim 92, wherein the nucleic acid amplification generates an amplified, processed sample.

132. The method of claim 131, wherein the time period for the nucleic acid amplification to generate the amplified processed sample is at most 5 minutes.

133. The method of any one of claims 59-101 and 129-132, wherein the method of processing the sample does not include heating the sample.

134. A composition for sample amplification, said composition comprising: a nonionic surfactant, a cyclodextrin, and a sucrose / epicochlorohydrin polymer. The composition is configured to increase the amplification rate during nucleic acid amplification.

135. The composition of claim 134, wherein the composition is configured to stabilize the enzyme during nucleic acid amplification.

136. The composition of claim 135, wherein the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

137. The composition of claim 136, wherein the reverse transcriptase is avian myeloblastic leukemia virus (AMV) reverse transcriptase or murine leukemia virus (MMLV) reverse transcriptase.

138. The composition of any one of claims 134-136, wherein the nonionic surfactant is nonoxynol-9.

139. The composition of any one of claims 134-138, wherein the final concentration of the cyclodextrin in the composition, in the presence of the sample, is from 0.01% v / v to 2.0% v / v.

140. The composition of any one of claims 134-139, wherein the cyclodextrin comprises hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof.

141. The composition of any one of claims 134-140, wherein the final concentration of the cyclodextrin in the composition, in the presence of the sample, is from about 0.001 M to 10 M.

142. The composition of any one of claims 134-141, wherein the sucrose / epicochlorohydrin polymer is polysucrose 400.

143. The composition of any one of claims 134-142, wherein the final concentration of the sucrose / epicochlorohydrin polymer in the composition in the presence of the sample is about 0.001% to 5% w / v (g solute / 100 mL solution).

144. The composition of any one of claims 134-143, wherein the composition further comprises at least one salt.

145. The composition of claim 144, wherein the final concentration of the at least one salt in the composition, in the presence of the sample, is from about 0.001 mol (M) to 10 M.

146. The composition of claim 144 or 145, wherein the at least one salt is sodium chloride, potassium phosphate, potassium chloride, or any combination thereof.

147. The composition of any one of claims 134-146, wherein the composition comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferasirox, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

148. The composition of claim 147, wherein: (a) The final concentration of EDTA in the composition, in the presence of the sample, is from about 0.01 mmol (mM) to 10 mM, and / or (b) The final concentration of Tris in the composition, in the presence of the sample, is from about 0.1 mM to 25 mM.

149. The composition of any one of claims 134-148, wherein the composition further comprises an agent capable of reducing disulfide bonds.

150. The composition of claim 149, wherein the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME).

151. The composition of any one of claims 134-150, wherein the composition further comprises at least one sugar and / or sugar alcohol.

152. The composition of claim 151, wherein the at least one sugar and / or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof.

153. The composition of claim 151 or 152, wherein the final concentration of the at least one sugar and / or sugar alcohol is about 0.001 M to 10 M or about 0.1% to 10% w / v (g solute / 100 mL solution).

154. The composition of any one of claims 134-153, wherein the composition further comprises an additional reagent.

155. The composition of claim 154, wherein the additional reagent comprises a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof.

156. The composition of any one of claims 134-155, wherein the composition is lyophilized.

157. The composition of any one of claims 134-156, wherein the composition further comprises a sample.

158. The composition of claim 157, wherein the sample is a biological sample.

159. The composition of claim 158, wherein the biological sample comprises a target nucleic acid molecule for sample processing.

160. The composition of any one of claims 134-159, wherein the composition further comprises a thermostable enzyme, a deoxynucleoside triphosphate (dNTP), a primer, a probe, or any combination thereof.

161. The composition of claim 160, wherein the composition is configured to stabilize the enzymatic activity of the thermostable enzyme for use during the nucleic acid amplification.

162. The composition of claim 160 or 161, wherein the thermostable enzyme is selected from the following: large fragment of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, IsoFast... TM Bst and any of its mutants.

163. The composition of any one of claims 160-162, wherein the dNTP comprises dATP, dCTP, dGTP, dTTP or dUTP.

164. The composition of claim 163, wherein the concentration of the dNTP in the composition is from about 40 micromoles (µM) to 5000 µM.

165. The composition of any one of claims 160-164, wherein the primer is at least 4 nucleotides in length.

166. The composition of any one of claims 160-165, wherein the probe is at least 15 nucleotides in length.

167. A composition comprising: A sample processing buffer, the sample processing buffer comprising: detergent, solubilizer and cyclodextrin; A sample amplification buffer, comprising: a nonionic surfactant, cyclodextrin, and a sucrose / epicochlorohydrin polymer; and A sample stabilizing buffer, which is configured to stabilize enzymes during nucleic acid amplification.

168. The composition of claim 167, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

169. The composition of claim 167 or 168, wherein the solubilizer is a nonionic surfactant.

170. The composition of any one of claims 167-169, wherein the solubilizer is polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol or secondary alcohol polyoxyethylene ether.

171. The composition of claim 170, wherein the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

172. The composition of any one of claims 167-171, wherein the detergent is part of a lysis buffer.

173. The composition of any one of claims 167-172, wherein the solubilizer and the cyclodextrin are part of a recovery buffer.

174. The composition of claim 173, wherein the lysis buffer and the recovery buffer are in the same mixture in the sample processing buffer.

175. The composition of any one of claims 167-174, wherein the solubilizer and the cyclodextrin are configured to shorten the cycle threshold or result time in nucleic acid amplification compared to the cycle threshold or result time in nucleic acid amplification of samples that are otherwise identical to those treated with SDS, polysorbate 80, or cyclodextrin alone.

176. The composition of claim 175, wherein the cycle threshold is at most 40 or the result time is at most 15 minutes.

177. The composition of any one of claims 167-176, wherein the solubilizer and the cyclodextrin are configured to reduce the coefficient of variation.

178. The composition of any one of claims 167-177, wherein the solubilizer and the cyclodextrin are configured to lower the detection limit.

179. The composition of claim 172 or 174, wherein the lysis buffer further comprises a chelating agent.

180. The composition of claim 179, wherein the chelating agent is deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, or N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).

181. The composition of any one of claims 172, 174 and 179-180, wherein the lysis buffer further comprises a reducing agent.

182. The composition of claim 181, wherein the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or tetrahydropyran (THP).

183. The composition of any one of claims 172, 174, and 179-182, wherein the cleavage buffer comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

184. The composition of claim 183, wherein The final concentration of EGTA in the lysis buffer in the presence of the sample is approximately 0.1 mmol (mM) to 10 mM. The final concentration of EDTA in the lysis buffer in the presence of the sample is approximately 0.1 mM to 5 mM. The final concentration of TCEP in the lysis buffer in the presence of the sample is approximately 1 mM to 20 mM, or The final concentration of Tris in the lysis buffer in the presence of the sample is approximately 1 mM to 60 mM.

185. The composition of any one of claims 167-184, wherein the sample processing buffer further comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME).

186. The composition of any one of claims 167-185, wherein the detergent is present in the sample processing buffer mixed with the sample at a final concentration that effectively lyses cells.

187. The composition of any one of claims 167-186, wherein the cyclodextrin is present in the sample processing buffer mixed with the sample at a final concentration that effectively isolates the detergent within the composition.

188. The composition of any one of claims 167-187, wherein the detergent is configured to form a complex with the solubilizer and / or the cyclodextrin to stabilize the enzyme.

189. The composition of claim 188, wherein the cyclodextrin is configured to increase the efficiency of forming the complex.

190. The composition of claim 186, wherein the final concentration of the detergent is about 0.1% to 10% w / v (g solute / 100 mL solution).

191. The composition of claim 187, wherein the final concentration of the cyclodextrin is from about 0.1 mM to 70 mM.

192. The composition of any one of claims 167-191, wherein the cyclodextrin comprises hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof.

193. The composition of any one of claims 167-192, wherein the solubilizer is present in the composition mixed with the sample at a final concentration of about 0.1% to 50% w / v.

194. The composition of claim 193, wherein the final concentration of the solubilizer effectively forms micelles containing the detergent.

195. The composition of claim 173 or 174, wherein the recovery buffer comprises a salt.

196. The composition of claim 195, wherein the recovery buffer comprises a pH buffer.

197. The composition of claim 195, wherein the recovery buffer does not contain a pH buffer.

198. The composition of any one of claims 167-197, wherein the sample processing buffer is lyophilized.

199. The composition of any one of claims 167-198, wherein the nonionic surfactant of the sample amplification buffer is nonoxynol-9.

200. The composition of any one of claims 167-199, wherein the final concentration of the cyclodextrin in the sample amplification buffer in the presence of the sample is from 0.01% v / v to 2.0% v / v.

201. The composition of claim 200, wherein the cyclodextrin comprises hydroxypropyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin hydrate, monopropanediamino-β-cyclodextrin, 6-O-α-D-maltosyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, or any combination thereof.

202. The composition of claim 200 or 201, wherein the final concentration of the cyclodextrin in the sample amplification buffer in the presence of the sample is from about 0.001 M to 10 M.

203. The composition of any one of claims 167-202, wherein the sucrose / epicochlorohydrin polymer is polysucrose 400.

204. The composition of claim 203, wherein the final concentration of the sucrose / epicochlorohydrin polymer in the composition, in the presence of the sample, is about 0.001% to 5% w / v (g solute / 100 mL solution).

205. The composition of any one of claims 167-204, wherein the sample amplification buffer further comprises at least one salt.

206. The composition of claim 205, wherein the final concentration of the at least one salt in the composition, in the presence of the sample, is from about 0.001 mol (M) to 10 M.

207. The composition of claim 205 or 206, wherein the at least one salt is sodium chloride, potassium phosphate, potassium chloride, or any combination thereof.

208. The composition of any one of claims 167-207, wherein the sample amplification buffer further comprises etaneric acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), Tris, deferoxone, ethylenediamine, 1,10-phenanthroline, oxalic acid, pentiformic acid, deferoxamine, deferoxamine methanesulfonate, N,N,N′,N′-tetra(2-pyridylmethyl)-1,2-ethylenediamine (TPEN), formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

209. The composition of claim 208, wherein: (a) The final concentration of EDTA in the composition, in the presence of the sample, is from about 0.01 mmol (mM) to 10 mM, and / or (b) The final concentration of Tris in the composition, in the presence of the sample, is from about 0.1 mM to 60 mM.

210. The composition of any one of claims 167-209, wherein the sample amplification buffer further comprises an agent capable of reducing disulfide bonds.

211. The composition of claim 210, wherein the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (βME).

212. The composition of any one of claims 167-211, wherein the sample amplification buffer further comprises at least one sugar and / or sugar alcohol.

213. The composition of claim 212, wherein the at least one sugar and / or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabinitol, xylitol, sorbitol, mannitol, or any combination thereof.

214. The composition of claim 212 or 213, wherein the final concentration of the at least one sugar and / or sugar alcohol is about 0.001 M to 10 M or about 0.1% to 10% w / v (g solute / 100 mL solution).

215. The composition of any one of claims 167-214, wherein the sample amplification buffer further comprises additional reagents.

216. The composition of claim 215, wherein the additional reagent comprises a base, Brij 98, guanidine thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant albumin (rAlbumin), or any combination thereof.

217. The composition of any one of claims 167-216, wherein the sample amplification buffer is lyophilized.

218. The composition of any one of claims 167-217, wherein the sample amplification buffer further comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), primers, probes, or any combination thereof.

219. The composition of claim 218, wherein the sample amplification buffer is configured to stabilize the enzyme activity of the thermostable enzyme for use during the nucleic acid amplification.

220. The composition of claim 218 or 219, wherein the thermostable enzyme is selected from the following: large fragment of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, OmniTaq 2 LA DNA polymerase, IsoFast... TM Bst and any of its mutants.

221. The composition of any one of claims 218-220, wherein the dNTP comprises dATP, dCTP, dGTP, dTTP or dUTP.

222. The composition of claim 221, wherein the concentration of the dNTP in the composition is from about 40 micromoles (µM) to 5000 µM.

223. The composition of any one of claims 218-222, wherein the primer is at least 4 nucleotides in length.

224. The composition of any one of claims 218-223, wherein the probe is at least 15 nucleotides in length.

225. The composition of any one of claims 167-224, wherein the sample stabilizing buffer comprises one or more agents selected from collapse modifiers, protein stabilizers and glass transition modifiers.

226. The composition of any one of claims 167-225, wherein the sample stabilizing buffer comprises at least one salt.

227. The composition of any one of claims 167-226, wherein the sample stabilizing buffer comprises cyclodextrin.

228. The composition of any one of claims 167-227, wherein the sample stabilizing buffer is configured to reconstruct the lyophilized sample.

229. The composition of any one of claims 167-228, wherein the stabilizing buffer comprises at least one reducing agent.

230. The composition of claim 229, wherein the at least one reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, thiosulfate, sodium dithionite, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or tetrahydropyran (THP) or any combination thereof.

231. The composition of any one of claims 167-230, wherein the composition further comprises a sample.

232. The composition of claim 231, wherein the sample is a biological sample.

233. The composition of claim 232, wherein the biological sample comprises a target nucleic acid molecule for sample processing.

234. The composition according to any one of claims 167-233, wherein the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

235. A method for amplifying a sample, the method comprising: (a) Contact the sample with a sample processing buffer to generate a processed sample; (b) Contact the treated sample with a sample amplification buffer to provide conditions for nucleic acid amplification; as well as (c) Perform nucleic acid amplification on the treated sample, and The sample processing buffer is not removed prior to the contact described in (b).

236. The method of 235, the method further comprising contacting the sample with a sample stabilizing buffer for stabilizing the enzyme during the nucleic acid amplification.

237. The method of claim 236, the method further comprising contacting the sample with the sample stabilizing buffer prior to (c).

238. The method of claim 236 or 237, wherein the sample stabilizing buffer and the sample amplification buffer are in the same mixture.

239. The method of any one of claims 236-238, wherein after the sample is contacted with the sample amplification buffer, the sample stabilizing buffer is contacted with the sample.

240. The method of any one of claims 235-239, wherein the method does not include heating the sample.

241. The method of any one of claims 235-240, wherein the sample processing buffer comprises a lysis buffer and / or a recovery buffer.

242. The method of claim 241, wherein the lysis buffer comprises a detergent.

243. The method of claim 242, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

244. The method of any one of claims 241-243, wherein the recovery buffer comprises a solubilizer and cyclodextrin.

245. The method of any one of claims 235-244, wherein the processing time of the sample is a time period from the contact at (a) to the contact at (b), wherein the time period is at most 20 seconds.

246. The method of any one of claims 235-245, wherein the nucleic acid amplification comprises polymerase chain reaction (PCR) or isothermal amplification.

247. The method of any one of claims 235-246, wherein the nucleic acid amplification comprises thermal cycling of the sample.

248. The method of any one of claims 235-247, wherein the nucleic acid amplification generates an amplified sample.

249. The method of claim 248, wherein the time period from the contact with the amplified sample in (a) is at most 5 minutes.

250. The method of any one of claims 235-249, wherein the sample is a biological sample.

251. The method of claim 250, wherein the biological sample comprises one or more different target nucleic acid molecules.

252. The method of claim 251, wherein the concentration of the one or more different target nucleic acid molecules is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher than the concentration of the one or more different target nucleic acid molecules in other aspects of the same sample treated alone with the sample processing buffer.

253. The method of any one of claims 235-252, wherein the sample processing buffer further comprises cucurbita.

254. A method for processing a sample, the method comprising: (a) Contact the sample with a sample processing buffer to generate a processed sample; (b) Contact the treated sample with a sample amplification buffer to provide conditions for nucleic acid amplification; as well as (c) The treated sample is subjected to nucleic acid amplification in the sample amplification buffer. The time period (i) from contact in (a) to the generation of the processed sample before contact with the sample amplification buffer is no more than the time for the sample processing buffer to be pipetted into the sample to mix the sample processing buffer and the sample, or (ii) at most 1 min, at most 50 seconds, at most 40 seconds or at most 20 seconds.

255. The method of claim 254, wherein the sample processing buffer comprises a lysis buffer and / or a recovery buffer.

256. The method of claim 255, wherein the lysis buffer comprises a detergent.

257. The method of claim 256, wherein the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

258. The method of any one of claims 255-257, wherein the recovery buffer comprises a solubilizer and cyclodextrin.

259. The method of any one of claims 254-258, wherein the sample processing buffer comprises a detergent, a solubilizer, and a cyclodextrin, wherein the sample processing buffer is configured to stabilize an enzyme during nucleic acid amplification, and wherein the sample processing buffer is configured to reduce and / or eliminate the activity of the nuclease.

260. The method of any one of claims 254-259, wherein the sample processing buffer is the composition of any one of claims 1-48.

261. The method of any one of claims 254-260, wherein the sample amplification buffer comprises an excipient.

262. The method of any one of claims 254-261, wherein the sample processing buffer is not removed before (b).

263. The method of any one of claims 254-262, wherein the sample amplification buffer comprises a reaction mixture as described in any one of claims 114-128, or the sample amplification buffer is a composition as described in any one of claims 134-164.

264. The method of any one of claims 254-263, wherein the method does not include heating the sample.

265. The method of any one of claims 254-264, wherein the sample is a biological sample.

266. The method of claim 265, wherein the biological sample comprises blood sample, swab sample, saliva sample, urine sample, cerebrospinal fluid sample, pleural fluid sample, rectal sample, vaginal sample, fecal sample, sputum sample, lymph sample, raw emulsion, pasteurized and / or homogenized emulsion, pasteurized and / or treated emulsion, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue sample, cell culture, purified nucleic acid sample, environmental sample, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof.

267. The method of claim 266, wherein the blood sample is obtained from the object.

268. The method of claim 266 or 267, wherein the blood sample is collected in a blood collection tube.

269. The method of claim 268, wherein the blood collection tube contains a stabilizer for stabilizing RNA.

270. The method of claim 269, wherein the stabilizer comprises tetradecyltrimethylammonium oxalate and / or tartaric acid.

271. The method of claim 269 or 270, wherein the blood sample is contacted with the sample processing buffer without removing the stabilizer.

272. The method of any one of claims 266-271, wherein the blood sample is directly contacted with the sample processing buffer without further processing before contacting the sample processing buffer.

273. The method of claim 271 or 272, wherein the blood sample has not been treated by centrifugation or a rotating column before contacting the sample processing buffer.

274. The method of any one of claims 254-273, wherein the sample is lyophilized.

275. The method of any one of claims 254-274, wherein the sample amplification buffer is lyophilized.

276. The method of any one of claims 254-275, wherein the sample processing buffer further comprises cucurbitaureus.

277. A composition for sample preparation, the composition comprising: A sample processing buffer, the sample processing buffer comprising: detergent, solubilizer and cyclodextrin; Stabilizer, said stabilizer comprising tetradecyltrimethylammonium oxalate and / or tartaric acid, and The composition is configured to stabilize the enzyme during nucleic acid amplification, and The composition is configured to reduce and / or eliminate the activity of degrading nucleases.

278. A composition for sample preparation, the composition comprising: The sample amplification buffer comprises: a nonionic surfactant, cyclodextrin, and a sucrose / epicochlorohydrin polymer; Stabilizer, said stabilizer comprising tetradecyltrimethylammonium oxalate and / or tartaric acid, and The composition is configured to increase the amplification rate during nucleic acid amplification.

279. The composition of claim 277 or 278, wherein the composition does not contain ethanol.

280. The composition of any one of claims 277-279, wherein the composition further comprises cucurbitaureus.

281. The composition of claim 280, wherein the cucurbituril comprises cucurbit[n]urea, wherein n is an integer of 5, 6, 7, 8 or 10.

282. The composition of claim 280, wherein the cucurbituril is cucurbituril[7].

283. The composition of any one of claims 277-282, wherein the composition further comprises a sample.

284. The composition of claim 283, wherein the sample is a biological sample.

285. The composition of claim 284, wherein the biological sample comprises blood sample, swab sample, saliva sample, urine sample, cerebrospinal fluid sample, pleural fluid sample, rectal sample, vaginal sample, fecal sample, sputum sample, lymph sample, raw emulsion, pasteurized and / or homogenized emulsion, pasteurized and / or treated emulsion, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, tissue sample, cell culture, purified nucleic acid sample, environmental sample, one or more intact organisms, one or more homogenized organisms, wastewater, or any combination thereof.

286. The composition of claim 285, wherein the blood sample is obtained from the object.

287. The composition of claim 285 or 286, wherein the blood sample is collected in a blood collection tube.

288. The composition of claim 287, wherein the blood collection tube contains a stabilizer for stabilizing RNA.

289. The composition of claim 288, wherein the stabilizer comprises tetradecyltrimethylammonium oxalate and / or tartaric acid.

290. The composition of claim 288 or 289, wherein the blood sample is contacted with the sample processing buffer without removing the stabilizer.

291. The composition of any one of claims 285-290, wherein the blood sample is directly contacted with the sample processing buffer without further treatment before contacting the sample processing buffer.

292. The composition of claim 290 or 291, wherein the blood sample has not been treated by centrifugation or a rotating column prior to contact with the processing buffer.

293. A method for processing a sample, the method comprising: (a) Contact the sample with a pyrolysis buffer containing detergent, wherein the sample contains tetradecyltrimethylammonium oxalate and / or tartaric acid, or wherein the sample is directly from a sample collection tube; and / or (b) The sample is contacted with a recovery buffer containing a solubilizer and cyclodextrin to treat the sample to produce a treated sample in a mixture containing the detergent, the solubilizer and the cyclodextrin.

294. The method of claim 293, the method further comprising contacting the sample with a sample amplification buffer.

295. A method for processing a sample, the method comprising: The sample is brought into contact with a sample amplification buffer comprising: a nonionic surfactant, cyclodextrin, and a sucrose / epicochlorohydrin polymer, wherein the composition is configured to increase the amplification rate during nucleic acid amplification, and wherein the sample comprises tetradecyltrimethylammonium oxalate and / or tartaric acid, or wherein the sample is directly derived from a sample collection tube.

296. The method of claim 295, the method further comprising contacting the sample with a sample processing buffer before contacting the sample with the sample amplification buffer.

297. A method for processing a sample, the method comprising: (a) Contact the sample with a sample processing buffer, the sample processing buffer comprising: detergent, solubilizer and cyclodextrin; (b) Contact the sample with a sample amplification buffer, the sample amplification buffer comprising: a nonionic surfactant, cyclodextrin; and (c) Contact the sample with a sample stabilizing buffer, the sample stabilizing buffer being configured to stabilize the enzyme during nucleic acid amplification. The sample may contain tetradecyltrimethylammonium oxalate and / or tartaric acid, or the sample may be directly derived from a sample collection tube.

298. The method of any one of claims 293-297, wherein the sample has not been treated with an RNA extraction kit.

299. The method of claim 298, wherein the kit comprises a rotating column.

300. The method of claim 298 or 299, wherein the kit comprises washing pills.

301. The method of any one of claims 293-300, wherein the method does not involve contact with a washing buffer.

302. The method of any one of claims 293-301, wherein the method does not include membrane-based extraction.

303. The method of any one of claims 293-302, the method further comprising performing nucleic acid amplification on the sample.

304. The method of claim 303, wherein the nucleic acid amplification comprises polymerase chain reaction (PCR) or isothermal amplification.

305. The method of claim 303 or 304, wherein the nucleic acid amplification comprises thermal cycling of the sample.

306. The method of any one of claims 303-305, wherein the nucleic acid amplification generates an amplified sample.

307. The method of claim 306, wherein the time period from the contact in (a) to the generation of the amplified sample is at most 5 minutes.

308. The method of any one of claims 293-307, wherein the sample processing time is the time period from the contact in (a) to the generation of the processed sample before contact with the amplification buffer, wherein the processing time is at most 1 min, at most 50 seconds, at most 30 seconds, or at most 20 seconds.

309. The method of any one of claims 293-308, wherein the method does not include heating the sample.

310. The method of any one of claims 293-309, wherein the sample is a blood sample.

311. The method of any one of claims 293-310, the method further comprising obtaining the sample from the object and collecting the sample in the sample collection tube.

312. The method of any one of claims 296-311, wherein the sample processing buffer further comprises cucurbita.

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