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WO2025049920A1 - Procédés et compositions pour le traitement d'échantillons - Google Patents

Procédés et compositions pour le traitement d'échantillons Download PDF

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Publication number
WO2025049920A1
WO2025049920A1 PCT/US2024/044691 US2024044691W WO2025049920A1 WO 2025049920 A1 WO2025049920 A1 WO 2025049920A1 US 2024044691 W US2024044691 W US 2024044691W WO 2025049920 A1 WO2025049920 A1 WO 2025049920A1
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WO
WIPO (PCT)
Prior art keywords
sample
composition
cyclodextrin
buffer
minutes
Prior art date
Application number
PCT/US2024/044691
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English (en)
Inventor
Jonathan David HARDINGHAM
Stephen Judice
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Biomeme, Inc.
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Publication date
Application filed by Biomeme, Inc. filed Critical Biomeme, Inc.
Publication of WO2025049920A1 publication Critical patent/WO2025049920A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • Nucleic acid amplification techniques such as polymerase chain reaction (PCR) and various isothermal amplification techniques have become an integral part of nucleic acid-based diagnostics and research techniques.
  • Samples containing target nucleic acid molecules need to be processed before being amplified in a nucleic acid amplification assay.
  • the target nucleic acid molecules need to be extracted. Optimization of sample processing and preparation can improve the efficiency, precision, and yield in nucleic acid amplification techniques.
  • the present disclosure provides a composition for sample processing comprising: a detergent, a solubilizer, and a cyclodextrin, wherein the composition is configured to stabilize an enzyme during a nucleic acid amplification, and wherein the composition is configured to reduce and/or eliminate activity of a degrading nuclease.
  • the composition is configured to stabilize nucleic acids during the nucleic acid amplification.
  • the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • SDS sodium dodecyl sulfate
  • sodium lauryl sulfate sodium lauryl sulfate
  • lithium dodecyl sulfate or a functional variant thereof.
  • the solubilizer is a non-ionic surfactant.
  • the solubilizer is a polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol, or a secondary alcohol ethoxylate.
  • the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.
  • the detergent is part of a lysis buffer.
  • the solubilizer and the cyclodextrin are part of a recovery buffer.
  • the lysis buffer and the recovery buffer are in a same mixture.
  • the present disclosure provides a composition for sample processing comprising a buffer comprising: a detergent, a solubilizer, and a cyclodextrin, wherein the composition is configured to stabilize an enzyme during a nucleic acid amplification, and wherein the composition is configured to inactivate a degrading nuclease.
  • the composition is configured to stabilize nucleic acids during the nucleic acid amplification.
  • the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.
  • the solubilizer and the cyclodextrin are configured to shorten a cycle threshold value or a time to result value in the nucleic acid amplification compared to a cycle threshold value or a time to result value in a nucleic acid amplification of an otherwise identical sample processed by SDS, polysorbate 80, or a cyclodextrin individually.
  • the cycle threshold value is at most 40 or the time to result value is at most 15 minutes.
  • the solubilizer and the cyclodextrin are configured to decrease a coefficient of variation. In some embodiments, the solubilizer and the cyclodextrin are configured to lower a limit of detection.
  • the degrading nuclease is a ribonuclease.
  • the lysis buffer has a pH value of 2 to 9. In some embodiments, the lysis buffer further comprises a chelating agent.
  • the chelating agent is deferiprone, ethylenediamine, 1,10-Phenanthroline, oxalic acid, pentetic acid, deferasirox, deferoxamine, deferoxamine mesylate, or N,N,N',N'-tetrakis(2-pyridinylmethyl)- 1,2-ethanediamine (TPEN).
  • the lysis buffer further comprises a reducing agent.
  • the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, a thiosulfate, sodium hydrosulfite, l,2-bis(o-aminophenoxy)ethane-N,N,N',N'- WSGR Docket No. 52459-726.601 tetraacetic acid (BAPTA), or tetrahydropyran (THP).
  • the lysis buffer comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2- carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10-Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)-l,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate, a sodium hydrosulfite, l,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydropyr
  • a final concentration of EGTA in the lysis buffer in the presence of a sample is about 0.1 millimolar (mM) to 10 mM
  • a final concentration of EDTA in the lysis buffer in the presence of a sample is about 0.1 mM to 5 mM
  • a final concentration of TCEP in the lysis buffer in the presence of a sample is about 1 mM to 20 mM
  • a final concentration of Tris in the lysis buffer in the presence of a sample is about 1 mM to 60 mM.
  • the composition further comprises an agent capable of reducing a disulfide bond.
  • the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoehtanol (0ME).
  • the detergent is present in the composition mixed with a sample at a final concentration that is effective for lysing cells.
  • the cyclodextrin is present in the composition mixed with a sample at a final concentration that is effective for isolating the detergent within the composition.
  • the detergent is configured to form a complex with the solubilizer and/or the cyclodextrin to stabilize the enzyme.
  • the cyclodextrin is configured to increase the efficiency of forming the complex.
  • the cyclodextrin has a higher binding affinity toward the detergent than a binding affinity of the solubilizer.
  • the final concentration of the detergent is about 0.1% to 10% w/v (g of solute / 100 mL of solution). In some embodiments, the final concentration of the cyclodextrin is about 0.1 mM to 70 mM.
  • the cyclodextrin comprises hydroxypropyl 0-cyclodextrin, hydroxypropyl y- cyclodextrin, (2-hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-a- cyclodextrin hydrate, monopropanediamino-0-cyclodextrin, 6-O-alpha-D-Maltosyl-0- cyclodextrin, 2,6-Di-O-methyl-0-cyclodextrin, hydroxyethyl-0-cyclodextrin, 3A-amino-3A- deoxy-(2AS,3AS)-0-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-y-cyclodextrin hydrate, an anionic cyclodextrin, or any combination thereof.
  • the solubilizer is present in the composition mixed with a sample at a final concentration of about 0.1% to 50% w/v. In some embodiments, the final concentration of the solubilizer is effective for forming micelles comprising the detergent.
  • the recovery buffer comprises a salt. In some embodiments, the recovery buffer does not comprise a salt. In some embodiments, the recovery buffer comprises a pH buffer. In some embodiments, the recovery buffer does not comprise a pH buffer. In some embodiments, the lysis buffer is lyophilized. In some embodiments, the recovery buffer is lyophilized.
  • the method further comprises mixing the sample with the recovery buffer described herein.
  • the present disclosure provides a method of processing a sample, the method comprising: (a) contacting the sample with a lysis buffer comprising a detergent; and (b) contacting the sample with a recovery buffer comprising a solubilizer and a cyclodextrin, thereby processing the sample to generate a processed sample in a mixture comprising the detergent, the solubilizer, and the cyclodextrin.
  • a lysis buffer comprising a detergent
  • a recovery buffer comprising a solubilizer and a cyclodextrin
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.
  • the sample is a biological sample.
  • the sample is a purified sample.
  • (a) and (b) occur simultaneously.
  • contacting the sample in (a) and (b) is performed concurrently in the same mixture.
  • the method further comprises incubating the sample at room temperature for a duration of time.
  • the method further comprises heating the sample at a constant temperature for a period of time.
  • the method further comprises heating the sample at a cyclic temperature for a period of time.
  • the method further comprises sonicating the sample. In some embodiments, sonicating the sample occurs prior to, subsequent to, or concurrent to heating the sample.
  • the heating the sample in (c) further comprises heating the sample to the second temperature, cooling down the sample, and heating the sample to the second temperature after cooling down.
  • the method further comprises sonicating the sample.
  • the sonicating the sample is performed prior to, subsequent to, or concurrent to heating the sample.
  • the method further comprises bead beating the sample.
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.
  • the lysis buffer further comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2-carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10-Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)- 1,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate, a sodium hydrosulfite, l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or
  • the thermostable enzyme is selected from the group consisting of a large fragment of a Bacillus stearothermophilus polymerase, a exo-Klenow polymerase, a B st 2.0 polymerase, a B st 3.0 polymerase, a SD DNA polymerase, a phi29 DNA polymerase, a sequencing-grade T7 exo-polymerase, an OmniTaq 2 LA DNA polymerase, and any mutants thereof.
  • the dNTPs comprise dATP, dCTP, dGTP, dTTP, or dUTP.
  • a concentration of the dNTPs in the reaction mixture is about 40 pM to 5000 pM.
  • the primer is at least 4 nucleotides in length.
  • the method further comprises subjecting the processed sample mixed with the reaction mixture to a nucleic acid amplification.
  • the nucleic acid amplification comprises polymerase chain reaction (PCR) or isothermal amplification.
  • the nucleic acid amplification comprises thermocycling the processed sample.
  • the nucleic acid amplification comprises keeping the processed sample at a constant temperature for amplification.
  • the method further comprising, prior to (a), obtaining the sample from a subject.
  • the subject has or is suspected of having a disease, a condition, or an infection.
  • the sample comprises one or more different target nucleic acid molecules.
  • the sample comprises a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool sample, a sputum sample, a lymph sample, raw milk, pasteurized and/or WSGR Docket No.
  • homogenized milk pasteurized and/or processed milk, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, a tissue sample, a cell culture, a purified nucleic acid sample, an environmental sample, one or more whole organisms, one or more homogenized organisms, wastewater, or any combination thereof.
  • a time from obtaining the sample to generating the processed 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.
  • a concentration of the 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 a concentration of one or more different target nucleic acid molecules of an otherwise identical sample processed by SDS, polysorbate 80, or a cyclodextrin individually.
  • the present disclosure provides a kit for sample processing, the kit comprising a lysis buffer comprising a detergent, a recovery buffer comprising a solubilizer and a cyclodextrin, and an instruction for use.
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.
  • the kit further comprises a reagent for nucleic acid amplification comprising a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), or a primer.
  • thermostable enzyme is selected from the group consisting of a large fragment of a Bacillus stearothermophilus polymerase, a exo-Klenow polymerase, a Bst 2.0 polymerase, a Bst 3.0 polymerase, a SD DNA polymerase, a phi29 DNA polymerase, a sequencing-grade T7 exo-polymerase, an OmniTaq 2 LA DNA polymerase, and any mutants thereof.
  • the dNTPs comprise dATP, dCTP, dGTP, dTTP, or dUTP.
  • a concentration of the dNTPs in a reaction mixture is about 40 pM to 5000 pM.
  • the primer is at least 4 nucleotides in length.
  • the kit further comprises a probe for detecting an amplification product generated using the kit.
  • the lysis buffer, the recovery buffer or the reagent is lyophilized.
  • the recovery buffer further comprises a cucurbituril.
  • the cucurbituril is cucurbit[n]uril, wherein n is an integer of 5, 6, 7, 8, or 10.
  • the reaction mixture comprises an excipient.
  • the excipient comprises one or more reagents selected from the group consisting of a Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), WSGR Docket No. 52459-726.601 potassium chloride, nonoxynol-9, trehalose, dextran, polysucrose 400, and a cyclodextrin.
  • the cyclodextrin comprises hydroxypropyl P-cyclodextrin, hydroxypropyl y- cyclodextrin, (2-hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-a- cyclodextrin hydrate, monopropanediamino-P-cyclodextrin, 6-O-alpha-D-Maltosyl-P- cyclodextrin, 2,6-Di-O-methyl-P-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A-amino-3A- deoxy-(2AS,3AS)-P-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-y-cyclodextrin hydrate, an anionic cyclodextrin, or any combination thereof.
  • a final concentration of Tris in the excipient in the presence of the sample is about 0.001 molar (M) to 1.0 M; a final concentration of sodium chloride and/or potassium chloride in the presence of the sample is about 0.0001 M to 0.25 M; a final concentration of EDTA in the excipient in the presence of the sample is about 0.00001 M to 0.1 M; a final concentration of nonoxynol-9 in the excipient in the presence of the sample is about 0.01% v/v to 2.0% v/v; a final concentration of trehalose in the excipient in the presence of the sample is about .001 M to 2.0 M; a final concentration of dextran in the excipient in the presence of the sample is about 0.1% w/v to 10% w/v; a final concentration of poly sucrose 400 in the excipient in the presence of the sample is about 0.01% w/v to 5.0% w/v; and/or a final concentration of the cyclodextr
  • the at least one reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, a thiosulfate, sodium hydrosulfite, l,2-bis(o- WSGR Docket No. 52459-726.601 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydropyran (THP), or any combination thereof.
  • a total time to perform (a) and (b) is at most 1 min, at most 50 seconds, at most 40 seconds, or at most 20 seconds.
  • a time for processing the sample is a time period from the contacting of (a) to contacting the processed sample with a reaction mixture, wherein the time period is at most 20 seconds.
  • the nucleic acid amplification generates an amplified processed sample.
  • a time period of the nucleic acid amplification to generate the amplified processed sample is at most 5 minutes.
  • the method of processing the sample does not comprise heating the sample.
  • the present disclosure provides a composition for sample amplification comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer, wherein the composition is configured to increase a rate of amplification during a nucleic acid amplification.
  • the composition is configured to stabilize an enzyme during a nucleic acid amplification.
  • the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.
  • the reverse transcriptase is an avian myeloblastosis virus (AMV) reverse transcriptase or a murine leukemia virus (MMLV) reverse transcriptase.
  • the nonionic surfactant is nonoxynol-9.
  • a final concentration of the cyclodextrin in the composition in the presence of a sample is 0.01% v/v to 2.0% v/v.
  • the cyclodextrin comprises the cyclodextrin comprises hydroxypropyl P-cyclodextrin, hydroxypropyl y- cyclodextrin, (2-hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-a- cyclodextrin hydrate, monopropanediamino-P-cyclodextrin, 6-O-alpha-D-Maltosyl-P- cyclodextrin, 2,6-Di-O-methyl-P-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A-amino-3A- deoxy-(2AS,3AS)-P-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-y-cyclodextrin hydrate, or any combination thereof.
  • the sucrose/epichlorohydrin polymer is polysucrose 400. In some embodiments, a final concentration of the sucrose/epichlorohydrin polymer in the composition in the presence of a sample is about 0.001% to 5% w/v (g of solute / 100 mL of solution). In some embodiments, the composition further comprises at least one salt. In some embodiments, a final concentration of the at least one salt in the composition in the presence of a sample is about WSGR Docket No. 52459-726.601
  • the at least one salt is sodium chloride, potassium phosphate, potassium chloride, or any combination thereof.
  • the composition comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2-carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10- Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)-l,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate, a sodium hydrosulfite, 1,2- bis(EGTA), an ethylenediaminetetraacetic acid (EDTA),
  • a final concentration of EDTA in the composition in the presence of a sample is about 0.01 millimolar (mM) to 10 mM, and/or a final concentration of Tris in the composition in the presence of a sample is about 0.1 mM to 25 mM.
  • the composition further comprises an agent capable of reducing a disulfide bond.
  • the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoehtanol (PME).
  • the composition further comprises at least one sugar and/or sugar alcohol.
  • the at least one sugar and/or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof.
  • a 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 of solute / 100 mL of solution).
  • the composition further comprises an additional reagent.
  • the additional reagent comprises a base, Brij 98, guanidinium thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant Albumin (rAlbumin), or any combination thereof.
  • the composition is lyophilized.
  • the composition further comprises a sample.
  • the sample is a biological sample.
  • the biological sample comprises a target nucleic acid molecule subject to sample processing.
  • the composition further comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), a primer, a probe, or any combination thereof.
  • the composition is configured to stabilize enzymatic activity of the thermostable enzyme for use during the nucleic acid amplification.
  • the thermostable enzyme is selected from the group consisting of a large fragment of a Bacillus stearothermophilus polymerase, a exo-Klenow polymerase, a Bst 2.0 polymerase, a Bst 3.0 WSGR Docket No.
  • the dNTPs comprise dATP, dCTP, dGTP, dTTP, or dUTP.
  • a concentration of the dNTPs in the composition is about 40 micromolar (pM) to 5000 pM.
  • the primer is at least 4 nucleotides in length.
  • the probe is at least 15 nucleotides in length.
  • the present disclosure provides a composition
  • a composition 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/epichlorohydrin polymer; and a sample stabilization buffer configured to stabilize an enzyme in a nucleic acid amplification.
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the solubilizer is a non-ionic surfactant.
  • the solubilizer is a polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, or a secondary alcohol ethoxylate.
  • the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.
  • the detergent is part of a lysis buffer.
  • the solubilizer and the cyclodextrin are part of a recovery buffer.
  • the lysis buffer and the recovery buffer are in the sample processing buffer as the same mixture.
  • the solubilizer and the cyclodextrin are configured to shorten a cycle threshold value or a time to result value in the nucleic acid amplification compared to a cycle threshold value or a time to result value in a nucleic acid amplification of an otherwise identical sample processed by SDS, polysorbate 80, or a cyclodextrin individually.
  • the cycle threshold value is at most 40 or the time to result value is at most 15 minutes.
  • the solubilizer and the cyclodextrin are configured to decrease a coefficient of variation. In some embodiments, the solubilizer and the cyclodextrin are configured to lower a limit of detection.
  • the lysis buffer further comprises a chelating agent.
  • the chelating agent is deferiprone, ethylenediamine, 1,10-Phenanthroline, oxalic acid, pentetic acid, deferasirox, deferoxamine, deferoxamine mesylate, or N,N,N',N'-tetrakis(2- pyridinylmethyl)-l,2-ethanediamine (TPEN).
  • the lysis buffer further comprises a reducing agent.
  • the reducing agent is oxalic acid, formic WSGR Docket No.
  • the lysis buffer comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2-carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10-Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)- 1,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate, a sodium hydrosulfite, l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydr
  • the composition described herein comprises a final concentration of EGTA in the lysis buffer in the presence of a sample is about 0.1 millimolar (mM) to 10 mM, a final concentration of EDTA in the lysis buffer in the presence of a sample is about 0.1 mM to 5 mM, a final concentration of TCEP in the lysis buffer in the presence of a sample is about 1 mM to 20 mM, or a final concentration of Tris in the lysis buffer in the presence of a sample is about 1 mM to 60 mM.
  • mM millimolar
  • the sample processing buffer further comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoehtanol (0ME).
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • 2-mercaptoehtanol 0ME
  • the detergent is present in the sample processing buffer mixed with a sample at a final concentration that is effective for lysing cells.
  • the cyclodextrin is present in the sample processing buffer mixed with a sample at a final concentration that is effective for isolating the detergent within the composition.
  • the detergent is configured to form a complex with the solubilizer and/or the cyclodextrin to stabilize the enzyme.
  • the cyclodextrin is configured to increase the efficiency of forming the complex.
  • the final concentration of the detergent is about 0.1% to 10% w/v (g of solute / 100 mL of solution). In some embodiments, the final concentration of the cyclodextrin is about 0.1 mM to 70 mM.
  • the cyclodextrin comprises hydroxypropyl P-cyclodextrin, hydroxypropyl y-cyclodextrin, (2-hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy- (2AS,3AS)-a-cyclodextrin hydrate, monopropanediamino-P-cyclodextrin, 6-O-alpha-D- Maltosyl-P-cyclodextrin, 2,6-Di-O-methyl-p-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A- amino-3A-deoxy-(2AS,3AS)-P-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-y- cyclodextrin hydrate, or any combination thereof.
  • the solubilizer is present in the composition mixed with a sample at a final concentration of about 0.1% to 50% w/v. In some embodiments, the final concentration of the solubilizer is effective for forming micelles comprising the detergent.
  • the recovery buffer comprises a salt. In some embodiments, the recovery buffer comprises a pH buffer. In some embodiments, the recovery buffer does not comprise 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.
  • a final concentration of the cyclodextrin in the sample amplification buffer in the presence of a sample is 0.01% v/v to 2.0% v/v.
  • the cyclodextrin comprises the cyclodextrin comprises hydroxypropyl p-cyclodextrin, hydroxypropyl y- cyclodextrin, (2-hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-a- cyclodextrin hydrate, monopropanediamino-P-cyclodextrin, 6-O-alpha-D-Maltosyl-P- cyclodextrin, 2,6-Di-O-methyl-P-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A-amino-3A- deoxy-(2AS,3AS)-P-cyclodextrin
  • the sucrose/epichlorohydrin polymer is polysucrose 400. In some embodiments, a final concentration of the sucrose/epichlorohydrin polymer in the composition in the presence of a sample is about 0.001% to 5% w/v (g of solute / 100 mL of solution). In some embodiments, the sample amplification buffer further comprises at least one salt. In some embodiments, a final concentration of the at least one salt in the composition in the presence of a sample is about 0.001 molar (M) to 10 M. In some embodiments, the at least one salt is sodium chloride, potassium phosphate, potassium chloride, or any combination thereof.
  • the sample amplification buffer further comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2-carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10-Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)- 1,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate, a sodium hydrosulfite, l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA
  • EGTA eg
  • a final concentration of EDTA in the composition in the presence of a sample is about 0.01 millimolar (mM) to 10 mM, and/or a final concentration of Tris in the composition in the presence of a sample is about 0.1 mM to 60 WSGR Docket No. 52459-726.601 mM.
  • the sample amplification buffer further comprises an agent capable of reducing a disulfide bond.
  • the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2- mercaptoehtanol (0ME).
  • the sample amplification buffer further comprises at least one sugar and/or sugar alcohol.
  • the at least one sugar and/or sugar alcohol comprises sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol, or any combination thereof.
  • a 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 of solute / 100 mL of solution).
  • the sample amplification buffer further comprises an additional reagent.
  • the additional reagent comprises a base, Brij 98, guanidinium thiocyanate (GITC), methionine, non-detergent sulfobetaine (NDSB), tRNA, recombinant Albumin (rAlbumin), or any combination thereof.
  • the sample amplification buffer is lyophilized.
  • the sample amplification buffer further comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), a primer, a probe, or any combination thereof.
  • the sample amplification buffer is configured to stabilize enzymatic activity of the thermostable enzyme for use during the nucleic acid amplification.
  • thermostable enzyme is selected from the group consisting of a large fragment of a Bacillus stearothermophilus polymerase, a exo-Klenow polymerase, a Bst 2.0 polymerase, a Bst 3.0 polymerase, a SD DNA polymerase, a phi29 DNA polymerase, a sequencing-grade T7 exo-polymerase, an OmniTaq 2 LA DNA polymerase, a IsoFastTM Bst, and any mutants thereof.
  • the dNTPs comprise dATP, dCTP, dGTP, dTTP, or dUTP.
  • the composition further comprises a sample.
  • the sample is a biological sample.
  • the biological sample comprises a target nucleic acid molecule subject to sample processing.
  • the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.
  • the present disclosure provides a method of 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 a condition for a nucleic acid amplification; and (c) subjecting the processed sample to the nucleic acid amplification, and wherein prior to the contacting of (b), the sample processing buffer is not removed.
  • the method further comprises contacting the sample with a sample stabilization buffer for stabilizing an enzyme in the nucleic acid amplification. In some embodiments, the method further comprises, prior to (c), contacting the sample with the sample stabilization buffer. In some embodiments, the sample stabilization buffer is in a same mixture as the sample amplification buffer. In some embodiments, the sample stabilization buffer is contacted with the sample after contacting the sample with the sample amplification buffer. In some embodiments, the method does not comprise heating the sample. 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.
  • the nucleic acid amplification generates an amplified sample.
  • a time period from the contacting of (a) to the amplified sample is at most 5 minutes.
  • the sample is a biological sample.
  • the biological sample comprises one or more different target nucleic acid molecules.
  • a 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% greater than a concentration of one or more different target nucleic acid molecules of an otherwise identical sample processed by a sample processing buffer alone.
  • the sample processing buffer further comprises a cucurbituril.
  • the present disclosure provides a method of 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 a condition for a nucleic acid amplification; and (c) subjecting the processed sample to the nucleic acid amplification in the sample amplification buffer, wherein a time period from contacting in (a) to generating the processed sample prior to contacting with the sample amplification buffer is (i) no more than a time for pipetting the sample processing buffer into the sample to mix the sample processing buffer and the sample or is (ii) at most 1 min, at most 50 seconds, at most 40 seconds, or at most 20 seconds.
  • the sample processing buffer comprises a lysis buffer and/or a recovery buffer.
  • the lysis buffer comprises a detergent.
  • the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the recovery buffer comprises a solubilizer and a cyclodextrin.
  • the sample processing buffer comprises a detergent, a solubilizer, and a cyclodextrin, wherein the sample processing buffer is configured to stabilize an enzyme during a nucleic acid amplification, and wherein the sample processing buffer is configured to reduce and/or eliminate activity of a degrading nuclease.
  • the sample processing buffer is the composition described herein.
  • the sample amplification buffer comprises an excipient.
  • the sample processing buffer is not removed.
  • the sample amplification buffer comprises the reaction mixture described herein, or the sample amplification buffer is the composition described herein. In some embodiments, the method does not comprise heating the sample.
  • the sample is a biological sample.
  • the biological sample comprises a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool WSGR Docket No.
  • the blood sample is obtained from a subject.
  • the blood sample is collected in a blood collection tube.
  • the blood collection tube comprises a stabilizing agent for stabilizing RNAs.
  • the stabilizing agent comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid.
  • the blood sample is contacted with the sample processing buffer without removing the stabilizing agent.
  • the blood sample is contacted with the sample processing buffer directly without being subject to other processing prior to contacting the sample processing buffer.
  • the blood sample is not processed by centrifugation or a spin column prior to contacting the sample processing buffer.
  • the sample is lyophilized.
  • the sample amplification buffer is lyophilized.
  • the sample processing buffer further comprises a cucurbituril.
  • the present disclosure provides a composition for sample processing comprising: a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; a stabilizing agent comprising tetradecyl trimethyl-ammonium oxalate and/or tartaric acid, and wherein the composition is configured to stabilize an enzyme during a nucleic acid amplification, and wherein the composition is configured to reduce and/or eliminate activity of a degrading nuclease.
  • the present disclosure provides a composition for sample processing comprising: a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer; a stabilizing agent comprising tetradecyl trimethyl- ammonium oxalate and/or tartaric acid, and wherein the composition is configured to increase a rate of amplification during a nucleic acid amplification.
  • a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer
  • a stabilizing agent comprising tetradecyl trimethyl- ammonium oxalate and/or tartaric acid
  • the composition does not comprise ethanol.
  • the composition further comprises cucurbituril.
  • the cucurbituril comprises cucurbit[n]uril, wherein n is an integer of 5, 6, 7, 8, or 10.
  • the cucurbituril is cucurbit[7]uril.
  • the composition further comprises a sample.
  • the sample is a biological sample.
  • the biological sample comprises a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool sample, a sputum sample, a lymph sample, raw milk, pasteurized and/or homogenized milk, pasteurized and/or processed milk, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, a tissue sample, a cell culture, a purified nucleic acid sample, an environmental sample, one or more whole organisms, one or more homogenized organisms, wastewater, or any combination thereof.
  • the blood sample is obtained from a subject.
  • the blood sample is collected in a blood collection tube.
  • the blood collection tube comprises a stabilizing agent for stabilizing RNAs.
  • the stabilizing agent comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid.
  • the blood sample is contacted with the sample processing buffer without removing the stabilizing agent.
  • the blood sample is contacted with the sample processing buffer directly without being subject to other processing prior to contacting the sample processing buffer.
  • the blood sample is not processed by centrifugation or a spin column prior to contacting the processing buffer.
  • the present disclosure provides a method of processing a sample, the method comprising: (a) contacting the sample with a lysis buffer comprising a detergent, wherein the sample comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube; and/or (b) contacting the sample with a recovery buffer comprising a solubilizer and a cyclodextrin, thereby processing the sample to generate a processed sample in a mixture comprising the detergent, the solubilizer, and the cyclodextrin.
  • the method further comprises contacting the sample with a sample amplification buffer.
  • the present disclosure provides a method of processing a sample, the method comprising: (a) contacting the sample with a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer, wherein the composition is configured to increase a rate of amplification during a nucleic acid amplification, and wherein the sample comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube.
  • a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer, wherein the composition is configured to increase a rate of amplification during a nucleic acid amplification, and wherein the sample comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid or wherein the sample is directly from
  • the method further comprises, prior to contacting the sample with the sample amplification buffer, contacting sample with a sample processing buffer.
  • the present disclosure provides a method of processing a sample, the method comprising: (a) contacting the sample with a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; (b) contacting the sample with a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin; and (c) contacting the sample with a sample stabilization buffer configured to stabilize an enzyme in a nucleic acid amplification, wherein the sample comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube.
  • the sample is not processed by an RNA extraction kit.
  • the kit comprises a spin-column.
  • the kit comprises a wash pellet.
  • the method does not comprise contacting with a wash buffer.
  • the method does not comprise membrane-based extraction.
  • the method further comprises subjecting the sample to the nucleic acid amplification.
  • the nucleic acid amplification comprises polymerase chain reaction (PCR) or isothermal amplification.
  • the nucleic acid amplification comprises thermocycling the sample.
  • the nucleic acid amplification generates an amplified sample.
  • a time period from the contacting in (a) to generating the amplified sample is at most 5 minutes.
  • a processing time for the sample is a time period from the contacting in (a) to generating a processed sample prior to contacting 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.
  • the method does not comprise heating the sample.
  • the sample is a blood sample.
  • the method further comprises obtaining the sample from a subject and collecting the sample is the sample collection tube.
  • the sample processing buffer further comprises a cucurbituril.
  • FIG. 1 shows an exemplary schematic depicting Sample Direct preparation method.
  • a sample can be provided and contacted with a lysis buffer.
  • the sample in the lysis buffer can be incubated at room temperature for a period of time.
  • the sample can be incubated at a higher temperature (e.g., 95°C, 98°C, 100°C, etc.).
  • a recovery buffer can be added to the sample in the lysis buffer and mixed, thereby obtaining a processed sample.
  • the recovery buffer can be liquid or lyophilized.
  • the processed sample can be mixed with a reaction mixture for nucleic acid amplification.
  • the recovery buffer may be lyophilized together with the reaction mixture.
  • FIG. 2 shows the amplification results of different swab samples using the Sample Direct preparation. Singleplex reactions amplifying target RNA sequence of Ribonuclease P protein subunit p30 (RPP30).
  • FIG. 3 shows the amplification results of human nasal swab samples using Sample Direct preparation. Neisseria gonorrhoeas culture, Chlamydia trachomatis culture, and RPP30 samples were run through a triplex isothermal reaction.
  • FIG. 6 shows amplification results of synthetic Monkeypox DNA (left) and Pan Orthopox (right) in NP matrix, Sample Direct preparation, and duplex isothermal reaction (e.g., DTECT chemistry) with approximately 2,000 copies/reaction.
  • FIG. 14 shows DTECT assay performance using MMLV reverse transcriptase (MMLV RT). MMLV functioned in the DTECT assay using varying control RNA dilutions.
  • FIG. 15F depicts a polymerase extending off the 3' end of the cut oligo and displacement of the guide.
  • FIG. 15G depicts endonuclease activity on the newly synthesized portion complementary to the target strand.
  • FIG. 15H depicts a polymerase extending off the 3' end of the cut site and displacement of the synthesized complement to the target strand.
  • FIG. 151 depicts the displaced complement acting as a new target for the second complementary strand duplexed oligo complex.
  • FIG. 15J depicts the polymerase displacing the second complementary strand duplexed guide molecule.
  • FIG. 15K depicts the completed extension on the new guide molecule.
  • FIG. 15L depicts endonucleolytic activity on the second complementary strand oligo/extension product complex.
  • FIG. 15M depicts a polymerase extending off the 3’ end of the cut site of the second complementary strand of the oligo/extension product complex.
  • FIG. 15N depicts endonucleolytic activity on the newly synthesized complementary strand of the second complementary strand guide.
  • FIG. 150 depicts the displaced and single stranded synthesized fragments as starting material for a strand displacement amplification reaction.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide may comprise a synthetic nucleotide.
  • a nucleotide may comprise a nucleotide analog.
  • a nucleotide may comprise a synthetic nucleotide analog.
  • Nucleotides may be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • a peptide nucleic acid analog can react as DNA would react in a given environment, and can additionally bind complementary nucleic acid sequences and WSGR Docket No. 52459-726.601 various proteins. Due to the non-natural backbone, PNAs can be insensitive to endonuclease cleavage in situations where an endonuclease would cleave the equivalent DNA/RNA sequence.
  • the term “nucleotide,” as used herein, may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores).
  • Detectable labels may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • polynucleotide oligonucleotide
  • nucleic acid a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form.
  • a polynucleotide may be DNA.
  • a polynucleotide may be RNA.
  • a polynucleotide may comprise one or more nucleotide analogs (e.g., including those with an altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, wyosine, PNAs, and LNAs.
  • fluorophores e.g., rhodamine or fluorescein linked to the sugar
  • thiol containing nucleotides biotin linked nucleotides, fluorescent base analogs, CpG islands,
  • restriction endonuclease As used herein, the term “restriction endonuclease,” “restriction enzyme,” or grammatical equivalents thereof generally refers to an enzyme that originates in bacterial host defense and is understood to recognize a specific sequence on an incoming viral DNA and cleave the DNA either at the recognition sequence or at a distinct sequence site.
  • One group of restriction endonucleases are identified as Type IIS. This group can recognize asymmetric DNA sequences and cleaves the DNA at a site outside the cleavage site that is at a defined distance from the recognition site. In some cases, type IIS restriction endonucleases cleave DNA between 1 and 20 nucleotides from the relevant recognition site.
  • sample refers to a substance (e.g., solid or liquid) that contains a target nucleic acid sequence to be amplified.
  • the target nucleic acid may be DNA.
  • the target nucleic acid molecule may be RNA.
  • a “processed sample” refers to a sample that has been contacted by a lysis buffer and/or a recovery buffer of the present disclosure.
  • template generally refers to a portion of a target RNA or DNA of a sample that is amplified by a DNA polymerase to produce one or more amplified nucleic acid products.
  • amplified product As used herein, “amplified product”, “amplified nucleic acid product”, or “amplicon” generally refers to the end product resulting from a nucleic acid method, such as PCR or isothermal amplification.
  • DNA polymerase generally refers to an enzyme that produces a complementary replicate of a nucleic acid molecule using the nucleic acid as a template strand.
  • 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 a growing chain of nucleic acid.
  • DNA polymerases synthesize complementary DNA molecules from DNA (e.g. DNA-dependent DNA polymerases) or RNA templates (e.g. RNA-dependent DNA polymerases or reverse transcriptases) and RNA polymerases synthesize RNA molecules from DNA templates (e.g. DNA-dependent RNA polymerases which participate in transcription).
  • DNA polymerases generally use a short, preexisting RNA or DNA strand, called a primer, to begin chain growth. Some DNA polymerases replicate single-stranded templates, while other DNA polymerases displace the strand upstream of the site where they add bases to a chain.
  • strand displacing when used in reference to a polymerase, generally refers to an activity that removes a complementary strand from base-pairing with a template strand being read by the polymerase.
  • Example polymerases having strand displacing activity include the large fragment of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, B st 2.0 polymerase, B st 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, and sequencing-grade T7 exo-polymerase.
  • primer generally refers to a linear oligonucleotide that is complementary to and anneals to a target sequence.
  • the lower limit on primer length is determined by ability to hybridize since very short primers (e.g., less than 5 WSGR Docket No. 52459-726.601 nucleotides) do not form thermodynamically stable duplexes under most hybridization conditions.
  • Primers may vary in length from 4 to 50 nucleotides. In some embodiments, the primer is between about 10 and 20 nucleotides in length. In some embodiments, the primer can be more than about 100 nucleotides in length.
  • the primer can be an oligonucleotide that can hybridize with a target nucleic acid sequence.
  • the primer can be a probe.
  • the primer can comprise a guide oligonucleotide.
  • the primer e.g., the guide oligonucleotide
  • the primer can be an oligonucleotide comprising a target binding region that hybridizes to a target polynucleotide sequence and a non-target binding region that does not hybridize to a target sequence.
  • the non-target binding region of the primer can comprise a palindromic sequence.
  • the palindromic sequence may permit recruitment of binding of a restriction enzyme to process the target sequence.
  • amplifying generally refer to any method for replicating a nucleic acid.
  • the replication can be conducted with the use of a primer-dependent polymerase.
  • the replication can be enzyme-free amplification.
  • amplifying or replicating a target nuclei acid strand also comprises replicating or amplifying a complementary strand of the target nucleic acid strand.
  • Amplified products can be subjected to subsequence analyses, including but not limited to melting curve analysis, nucleotide sequencing, single-strand conformation polymorphism assay, allele-specific oligonucleotide hybridization, Southern blot analysis, and restriction endonuclease digestion.
  • subsequence analyses including but not limited to melting curve analysis, nucleotide sequencing, single-strand conformation polymorphism assay, allele-specific oligonucleotide hybridization, Southern blot analysis, and restriction endonuclease digestion.
  • hybridizes and “annealing,” as used herein, 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 the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence sensitive or specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • a first sequence that can be stabilized via hydrogen bonding with the bases of the nucleotide residues of a second sequence can generally be “hybridizable” to the second sequence. In such a case, the second sequence can also be the to be hybridizable to the first sequence.
  • complement generally refer to a sequence that is fully complementary to and hybridizable to the given sequence.
  • a first sequence that is hybridizable to a second sequence or set of second sequences is specifically or selectively hybridizable to the second sequence or set of second sequences, such that hybridization to the second sequence or set of second sequences is used.
  • Hybridizable sequences can share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, 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.
  • the present application provides compositions of sample processing buffers, sample stabilization buffer, and amplification reaction buffers, kits containing one or more of the buffers described herein, and method of using the compositions.
  • the sample processing buffers, sample stabilization buffer, and amplification reaction buffers can be mixed directly at the appropriate steps without the need of washing steps or removing the buffers from any prior steps.
  • the compositions, methods, and kits described herein can be used as part of a framework system to enhance research and development processes of samples (e.g., biological samples).
  • the compositions, methods, and kits described herein, and uses thereof, can be designed to be flexible and adaptable, reducing the time necessary to develop products.
  • the buffer compositions described herein e.g., lysis buffers, recovery buffers, amplifications and/or stabilization buffers, can be versatile and can function with various component concentrations.
  • compositions and methods described herein can be used to process various samples and can function in the presence of any inhibitors that may be present in a sample.
  • compositions described herein can be part of an amplification buffer system, a sample processing buffer system, a stabilization buffer system, or any combination thereof.
  • the amplification buffer e.g., core amplification buffer
  • the sample processing buffer can comprise reagents of lysis and/or recovery buffers described herein.
  • the sample processing buffer can comprise salts and/or buffers which may be adjusted to optimize amplification reactions (e.g., PCR and/or isothermal amplification).
  • the stabilization buffer can comprise cyclodextrin, WSGR Docket No. 52459-726.601 protein stabilizers, cake structure modifiers (Tc, Tg, Tg’), salts, buffers, or any combination thereof.
  • Cake structure modifiers can comprise reagents that modify the glass transition temperature (Tg), the glass transition temperature of the maximally freeze concentrated master mix solute prior to being dried (Tg’), the onset crystallization temperature (Tc), or any combination thereof.
  • One or more cake structure modifiers may increase a critical collapse temperature of a composition described herein (e.g., a sample stabilization buffer).
  • one or more cake structure modifiers may enable a more efficient (e.g., warmer) drying cycle for the composition described herein (e.g., the sample stabilization buffer).
  • the cake structure modifiers may improve structural properties of the dried composition (e.g., dried cake).
  • the enhanced structural properties may make the dried composition (e.g., dried cake) more resistant to crushing, fracture, cracking, or any combination thereof.
  • glass transition temperatures can vary, for example from about 140 °C to 370 °C.
  • the reagents of the stabilization buffer may be optimized for freeze drying.
  • Screening assays in a representative sample matrix can decrease risks of downstream sample-assay integration. Consistent drying cycles can be available for immediate research and development use.
  • the benefits of the compositions, methods, and kits described herein can include (i) reducing the time to develop and integrate assays into a commercially viable shelfstable formulation; (ii) screening and optimizing assays in a representative sample matrix (e.g., matrix-based screening, compositions of the total sample types), (iii) a baked-in excipient that may be lyophilized with a compatible freeze-drying cycle, and (iv) eliminating the distinction between chemistry (e.g., chemical reagents) intended to be run immediately (for example research and development experiments) and chemistry which are intended to be freeze-dried.
  • the unification of the compositions, methods, and kits described herein provide for greater efficiency in sample processing, stabilization, and amplification.
  • sample Direct preparation methods The methods using the compositions described herein can be referred to as “Sample Direct” preparation methods.
  • the methods provided herein can process a sample quickly (e.g., 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, at most about 45 seconds, at most about 30 seconds, at most about 20 seconds, or less) and improve amplification reaction performance.
  • compositions for Sample Processing, Stabilization, and Amplification WSGR Docket No. 52459-726.601
  • the present disclosure provides compositions and methods for processing a sample comprising a target nucleic acid molecule for nucleic acid amplification.
  • the target nucleic acid molecule may be DNA and/or RNA.
  • the present disclosure provides compositions for sample processing for a nucleic acid amplification method.
  • the composition comprises a detergent, a solubilizer, and a cyclodextrin.
  • the composition may be configured to stabilize an enzyme during the nucleic acid amplification.
  • the composition may also assist in reducing the activity of a degrading nuclease during the nucleic acid amplification.
  • the composition may eliminate the activity of a degrading nuclease during the nucleic acid amplification.
  • the composition may degrade or inactivate the function of a nuclease prior to the nucleic acid amplification.
  • the composition may be configured to lyse cell walls and/or nuclear membranes.
  • the enzyme stabilized by the composition provided herein is a polymerase, an endonuclease, a reverse transcriptase, a ligase, a helicase, a recombinase, or any combination thereof.
  • the nuclease is a ribonuclease.
  • the ribonuclease comprises an endoribonuclease or an exoribonuclease.
  • the endoribonuclease includes, but is not limited to, RNAase A, RNAase H, RNAase III, RNAase L, RNAase P, RNAase PhyM, RNAase Tl, RNAase T2, RNAase U2, RNAase V, RNAase E, and RNAase G.
  • the exoribonuclease includes, but is not limited to, RNAase PH, RNAase R, RNAase D, RNAase T, oligoribonuclease, exoribonuclease I, exoribonuclease II, and polynucleotide phosphorylase (e.g., PNPase).
  • the detergent is sodium dodecyl sulfate (SDS).
  • the detergent comprises sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the detergent is an ionic detergent.
  • the detergent is a non-ionic detergent.
  • the detergent is part of a lysis buffer.
  • a lysis buffer is capable of lysing cells yet leaving nucleic acids intact (e.g., not denaturing a nucleic acid chain to the extent that the chain is disrupted to individual nucleic acids).
  • the lysis buffer is capable of handling challenging solid and liquid sample types.
  • the detergent is present at a final concentration when mixed with the sample to be processed in the lysis buffer.
  • the detergent may be present at a final concentration that is effective for lysing cells in the mixture in the presence of the sample.
  • the WSGR Docket No. 52459-726.601 concentration of any agent described herein when mixed with a sample to be processed can be referred to as final concentration.
  • the concentration (e.g., final concentration) of the detergent in the mixture in the presence of the sample is at least about 0.05% w/v (where w/v refers to g of solute / 100 mL of 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, at least about 1.0% w/v (where
  • the concentration (e.g., final concentration) of the 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, at most about 0.5% w/v, at most about 0.45% w/v, at most about 0.4% w/v
  • the concentration (e.g., final concentration) of the detergent in the mixture 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 the 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
  • the lysis buffer further comprises additional agents including, but not limited to, egtazic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2- carboxyethyl)phosphine (TCEP), and/or tris(hydroxymethyl)aminomethane (e.g., Tris).
  • the lysis buffer comprises SDS, egtazic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), and/or tri s(hydroxymethyl)aminom ethane (c.g, Tris).
  • the lysis buffer further comprises lysis buffer comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2- carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10-Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)-l,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate, a sodium hydrosulfite, l,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or t
  • the lysis buffer comprises a chelating agent.
  • the lysis buffer comprises 1, 2, 3, 4, or more chelating agents.
  • the chelating agent comprises is deferiprone, ethylenediamine, 1,10- WSGR Docket No. 52459-726.601
  • Phenanthroline oxalic acid, pentetic acid, deferasirox, deferoxamine, deferoxamine mesylate, or N,N,N',N'-tetrakis(2-pyridinylmethyl)-l ,2-ethanediamine (TPEN).
  • the lysis buffer comprises a reducing agent.
  • the lysis buffer comprises 1, 2, 3, 4, 5, or more reducing agents.
  • the reducing agent comprises oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, a thiosulfate, sodium hydrosulfite, l,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydropyran (THP).
  • EGTA is present at a final concentration in the lysis buffer effective for binding calcium, magnesium, and/or other ions.
  • 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
  • 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, at most about 2.0 mM, at most about 45.0
  • the concentration (e.g., final concentration) of EGTA in the mixture in the presence of the sample is about 0.5 mM to about 20 mM. In some embodiments, the concentration (e.g., final concentration) of EGTA in the mixture in the presence of the sample is at most about 20 mM. In some embodiments, the concentration (e.g., final WSGR Docket No.
  • 52459-726.601 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 m
  • EDTA is present at a final concentration in the lysis buffer effective for binding magnesium, calcium, and/or other ions.
  • 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
  • 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, at most about 1.0 mM, at most about 0.9 mM, at most about 0.8 m
  • the concentration (e.g., final concentration) of EDTA in the mixture in the presence of the sample is about 0.1 mM to about 25 mM. In some embodiments, the concentration (e.g., final concentration) of EDTA in the mixture in the presence of the sample is at most about 25 mM.
  • 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
  • the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).
  • 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
  • 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
  • the concentration (e.g., final concentration) of TCEP in the mixture in the presence of the sample is about 0.5 mM to about 75 mM. In some embodiments, the concentration (e.g., final concentration) of TCEP 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
  • the reducing agent comprises a Tris.
  • the concentration (e.g., final concentration) of a 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
  • the concentration (e.g., final concentration) of a 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 WSGR Docket No.
  • 52459-726.601 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, at most about 2 mM, or at most about 1 mM.
  • a Tris is present at a concentration e.g., final concentration) in the mixture in the presence of the sample of at least about 0.5 mM to about 75 mM.
  • 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
  • 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
  • the lysis buffer can comprise SDS, EGTA, EDTA, TCEP and/or Tris.
  • the lysis buffer can comprise SDS with a final concentration of about 0.01% w/v to 0.4% w/v in the presence of the sample; EGTA with a final concentration of about 0.1 mM to 3 mM in the presence of the sample; EDTA with a final concentration of about 0.01 mM to 1 mM in the presence of the sample; TCEP with a final concentration of about 1.0 mM to 4.0 mM in the presence of the sample; and Tris with a final concentration of about 1.0 mM to 4.5 mM in the WSGR Docket No.
  • the lysis buffer used can comprise 0.2% SDS, 2 mM EGTA, 0.5 mM EDTA, 1 mM TCEP, and 1 mM Tris final concentration for each component in the presence of the sample.
  • the lysis buffer has a pH value sufficient to lyse a desired cell.
  • the lysis buffer has a pH 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.
  • 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.
  • the lysis buffer has a pH of about 1 to about 10. In some embodiments, the lysis buffer has a pH of at most 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 6.5, about 4 to about 7, about 4 to about 8, about 4 to about
  • the lysis buffer does not contain a detergent. In some embodiments, the lysis buffer does not contain a solubilizer.
  • the final volume of the lysis buffer may depend on the type of sample or amplification method. In some embodiments, the final volume of the lysis buffer is at least about 50 microliter WSGR Docket No. 52459-726.601
  • pl at least about 100 pl, at least about 150 pl, at least about 200 pl, at least about 250 pl, at least about 300 pl, at least about 350 pl, at least about 400 pl, at least about 450 pl, at least about 500 pl, at least about 550 pl, at least about 600 pl, at least about 650 pl, at least about 700 pl, at least about 750 pl, at least about 800 pl, at least about 850 pl, at least about 900 pl, at least about 950 pl, at least about 1000 pl, at least about 1100 pl, at least about 1200 pl, at least about 1300 pl, at least about 1400 pl, at least about 1500 pl, at least about 2000 pl, at least about 2500 pl, at least about 3000 pl, at least about 4000 pl, at least about 5000 pl, at least about 7500 pl, or at least about 10,000 pl.
  • the final volume of the lysis buffer is at most about 10,000 pl, at most about 7500 pl, at most about 5000 pl, at most about 4000 pl, at most about 3000 pl, at most about 2500 pl, at most about 2000 pl, at most about 1500 pl, at most about 1400 pl, at most about 1300 pl, at most about 1200 pl, at most about 1100 pl, at most about 1000 pl, at most about 950 pl, at most about 900 pl, at most about 850 pl, at most about 800 pl, at most about 750 pl, at most about 700 pl, at most about 650 pl, at most about 600 pl, at most about 550 pl, at most about 500 pl, at most about 450 pl, at most about 400 pl, at most about 350 pl, at most about 300 pl, at most about 250 pl, at most about 200 pl, at most about 150 pl, at most about 100 pl, or at most about 50 pl.
  • the final volume of the lysis buffer is about 50 pl to about 2,000 pl. In some embodiments, the final volume of the lysis buffer is at most about 2,000 pl. In some embodiments, the final volume of the lysis buffer is about 50 pl to about 100 pl, about 50 pl to about 200 pl, about 50 pl to about 300 pl, about 50 pl to about 400 pl, about 50 pl to about 500 pl, about 50 pl to about 600 pl, about 50 pl to about 750 pl, about 50 pl to about 1,000 pl, about 50 pl to about 1,500 pl, about 50 pl to about 1,750 pl, about 50 pl to about 2,000 pl, about 100 pl to about 200 pl, about 100 pl to about 300 pl, about 100 pl to about 400 pl, about 100 pl to about 500 pl, about 100 pl to about 600 pl, about 100 pl to about 750 pl, about 100 pl to about 1,000 pl, about 100 pl to about 1,500 pl, about 100 pl to about 1,750 pl, about 100 pl to about 2,000 pl, about 200 pl to about 300 pl, about 200 pl to about 400 pl, about 100 pl to about 500 pl, about
  • 1,500 pl about 1,000 pl to about 1,750 pl, about 1,000 pl to about 2,000 pl, about 1,500 pl to about 1,750 pl, about 1,500 pl to about 2,000 pl, or about 1,750 pl to about 2,000 pl.
  • the final volume of the lysis buffer is about 2 ml to about 10 ml.
  • the lysis buffer is lyophilized. In some embodiments, the lysis buffer is not lyophilized.
  • the final volume of the lysis buffer is about 2 ml to about 2.5 ml, about 2 ml to about 3 ml, about 2 ml to about 3.5 ml, about 2 ml to about 4 ml, about 2 ml to about 4.5 ml, about 2 ml to about 5 ml, about 2 ml to about 6 ml, about 2 ml to about 7 ml, about 2 ml to about 8 ml, about 2 ml to about 9 ml, about 2 ml to about 10 ml, about
  • the solubilizer is a non-ionic surfactant.
  • the solubilizer comprises a polysorbate.
  • the polysorbate may be polyoxyethylene (20) sorbitan monooleate (e.g., polysorbate 80), polyoxyethylene (20) sorbitan monolaurate (e.g., polysorbate WSGR Docket No. 52459-726.601
  • the solubilizer is a TergitolTM surfactant, a TritonTM surfactant, or a Igepal® surfactant.
  • the solubilizer is an alkoxylate or a cocamide.
  • the solubilizer is decyl glucoside, alkyl polyglycoside, lauryl glucoside, sorbitan tristearate, or a niosome.
  • the recovery buffer contains one, two, three, four, or more solubilizers.
  • the solubilizer may mix with the detergent of the present composition.
  • the solubilizer is capable of forming micelles comprising the detergent of the present application.
  • the solubilizer is polysorbate 80.
  • 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
  • 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, at most about 20% v/v, at most about 15% v/v, at most about 10% v/v, at most about 70% v/
  • the concentration (e.g., final concentration) of the solubilizer in the mixture in the presence of the sample is about 0.1% v/v to about 80% v/v. In some embodiments, the concentration (e.g., final concentration) of the solubilizer in the mixture in the presence of the sample is about 0.1% v/v to about 5% v/v, about 0.1% v/v to about 10% v/v, about 0.1% v/v to about 15% v/v, about 0.1% v/v to about 20% v/v, about 0.1% v/v to about 25% v/v, about 0.1% v/v to about 30% v/v, about 0.1% v/v to about 40% v/v, about 0.1% v/v to WSGR Docket No.
  • the composition comprises a cyclodextrin.
  • the cyclodextrin is configured to form a complex with the detergent of the present application.
  • the complex formed between the cyclodextrin and detergent assists in stabilizing the enzyme in the composition.
  • the cyclodextrin increases the efficiency of forming the complex.
  • the cyclodextrin can increase the aqueous solubility of poorly soluble drugs and increase bioavailability and stability in solution.
  • the cyclodextrin comprises (2-hydroxypropyl) P-cyclodextrin, (2-hydroxypropyl) y-cyclodextrin, (2-hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-a-cyclodextrin hydrate, monopropanediamino-P-cyclodextrin, 6-O-alpha-D-Maltosyl-P-cyclodextrin, 2,6-Di-O-methyl- P-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-P-cyclodextrin hydrate, 3 A-amino-3A-deoxy-(2AS,3AS)-y-cyclodextrin hydrate, or any combination thereof.
  • the cyclodextrin can comprise an anionic cyclodextrin.
  • the anionic cyclodextrin may comprise WSGR Docket No. 52459-726.601 carboxymethyl-a-cyclodextrin, carboxymethyl-P-cyclodextrin, succinyl-a-cyclodextrin, succinyl-P-cyclodextrin, succinyl-y-cyclodextrin, (2-carboxyl)-a-cyclodextrin, (2-carboxyl)-P- cyclodextrin, a-cyclodextrin phosphate, P-cyclodextrin phosphate, y-cyclodextrin phosphate, sulfobutylated P-cyclodextrin, a-cyclodextrin sulfate, P-cyclodextrin sulfate, y-cyclodextrin sulfate, carb
  • the cyclodextrin in the recovery buffer can comprise two or more different cyclodextrin species described herein.
  • the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) p-cyclodextrin and (2-hydroxypropyl) y-cyclodextrin.
  • the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) a- cyclodextrin and methyl-P-cyclodextrin.
  • the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) P-cyclodextrin and methyl-P-cyclodextrin.
  • altering the molar substitution ratio of a particular modified cyclodextrin species may improve reaction performance such as shortening time to result values, Ct values, or Cq values.
  • the recovery buffer does not comprise a component in the lysis buffer.
  • the recovery buffer may not comprise a detergent or a reducing agent.
  • the recovery buffer may not comprise one or more agent selected from the group consisting of an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2- carboxyethyl)phosphine (TCEP), a Tris, a deferiprone, a ethylenediamine, 1,10-Phenanthroline, an oxalic acid, a pentetic acid, a deferasirox, a deferoxamine, a deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)-l,2-ethanediamine (TPEN), a formic acid, a lithium aluminum hydride, a sodium borohydride, a thiosulfate,
  • EGTA egtazi
  • the cyclodextrin is present at a final concentration in the presence of the sample effective for isolating the detergent within the composition of the present invention.
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture 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 WSGR Docket No.
  • 52459-726.601 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, at least about 175.0 mM, at least about 200.0 mM, at least about 250.0 mM, or at least about 300.0 mM.
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is at most about 300.0 mM, at most about 250.0 mM, at most about 200.0 mM, at most about 175.0 mM, at most about 150.0 mM, at most about 125.0 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, at most about 30.0 mM, at most about 30.0 mM, at most about 30.0 mM, at most about 30.0 mM
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is about 10 mM to about 300 mM. In some embodiments, the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is at least about 10 mM. In some embodiments, the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is at most about 300 mM.
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is about 10 mM to about 20 mM, about 10 mM to about 25 mM, about 10 mM to about 30 mM, about 10 mM to about 32.5 mM, about 10 mM to about 35 mM, about 10 mM to about 37.5 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 10 mM to about 100 mM, about 10 mM to about 200 mM, about 10 mM to about 300 mM, about 20 mM to about 25 mM, about 20 mM to about 30 mM, about 20 mM to about 32.5 mM, about 20 mM to about 35 mM, about 20 mM to about 37.5 mM, about 20 mM to about 40 mM, about 20 mM to about 50 mM,
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is about 0.1 mM to about 100 mM. In some embodiments, the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is at most about 100 mM.
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is about 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 35 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 75 mM, about 0.1 mM to about 100 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM to about 35 mM, about 1 mM to about 40 mM, about 1 mM
  • the recovery buffer can comprise a cyclodextrin with a final concentration of about 6 mM to 11 mM in the presence of the sample and polysorbate 80 with a final concentration of about 0.1% v/v to 3.0% v/v in the presence of the sample.
  • the recovery buffer can comprise 2 mM cyclodextrin and 1.5% v/v polysorbate 80 final concentration for each component in the presence of the sample.
  • the cyclodextrin has a higher binding affinity toward the detergent than a binding affinity of the solubilizer towards the detergent.
  • the binding affinity of the cyclodextrin to the detergent can be an association constant.
  • the binding affinity of the cyclodextrin to the detergent has an association constant (K a ) of at least about 2.5xl0 3 M’ 1 , at least about 3xl0 3 M’ 1 , at least about 3.5xl0 3 M’ 1 , at least about 4xl0 3 M’ 1 , at least about 5xl0 3 M’ 1 , at least about IxlO 4 M’ 1 , at least about 2xl0 4 M’ 1 , at least about 3xl0 4 M’ 1 , at least about 4xl0 4 M’ 1 , at least about 5xl0 4 M’ 1 , at least about IxlO 5 M’ 1 , at least about 5xl0 5 M’ 1 , or at least about IxlO 6 M' 1 to the detergent.
  • K a association constant
  • the binding affinity of the cyclodextrin to the detergent has an association constant (K a ) of at most about IxlO 6 M’ 1 , at most about 5xl0 5 M’ 1 , at most about IxlO 5 M’ 1 , at most about 5xl0 4 M’ 1 , at most about 4xl0 4 M’ 1 , at most about 3xl0 4 M’ 1 , at most about 2xl0 4 M’ 1 , at most about IxlO 4 M’ 1 , at most about 5xl0 3 M’ 1 , at most about 4xl0 3 M’ 1 , at most about 3xl0 3 M’ 1 , or at most about 2.5xl0 3 M' 1 .
  • K a association constant
  • the solubilizer and the cyclodextrin are configured to shorten a cycle threshold value or time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an nucleic acid amplification of an otherwise identical sample processed by SDS, polysorbate 80, or a cyclodextrin individually.
  • cycle threshold refers to the number of cycles used to amplify a target nucleic acid molecule to a detectable level (e.g., a signal exceeding a background threshold level). A lower cycle threshold value can indicate a greater amount of target nucleic acid in a sample.
  • time to result value can also be used and it refers to the time used to amplify a target nucleic acid molecule to a detectable level.
  • the solubilizer and/or cyclodextrin described herein are configured to shorten a cycle threshold value to at most about 60, at most about 50, at most about 40, at most about 30, at most about WSGR Docket No. 52459-726.601
  • the solubilizer and/or cyclodextrin described herein are configured to shorten a time to result 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.
  • the solubilizer and/or the cyclodextrin are configured to decrease a coefficient of variation in a nucleic acid amplification compared to a coefficient of variation in a nucleic acid amplification of an otherwise identical sample processed by SDS, polysorbate 80, or a cyclodextrin individually.
  • coefficient of variation refers to a measure of precision of an amplification method.
  • the solubilizer and/or the cyclodextrin are configured to decrease a 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%.
  • the solubilizer and/or the cyclodextrin are configured to lower a limit of detection of a nucleic acid amplification compared to a limit of detection in a nucleic acid amplification of an otherwise identical sample processed by SDS, polysorbate 80, or a cyclodextrin individually.
  • the “limit of detection” refers to the lowest quantity of a component in a sample that be reliably detected in an amplification method.
  • the solubilizer and/or the cyclodextrin are configured to lower a limit of detection 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.
  • the solubilizer and the cyclodextrin are part of a recovery buffer.
  • the recovery buffer comprises a salt.
  • the recovery buffer does not comprise a salt.
  • the salt comprises a sodium salt.
  • the recovery buffer comprises a pH buffer.
  • the recovery WSGR Docket No. 52459-726.601 buffer does not comprise a pH buffer.
  • 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.
  • 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.
  • the recovery buffer is lyophilized.
  • the recovery buffer can be liquid.
  • the recovery buffer can be lyophilized together with a reaction buffer / reaction mixture for nucleic acid amplifications.
  • the lysis buffer and the recovery buffer are in the same mixture.
  • the mixing of the lysis buffer and the recovery buffer is performed by hand.
  • the mixing of the lysis buffer and the recovery buffer is performed by a vortex.
  • the mixing of the lysis buffer and the recovery buffer is performed by an automated instrument, a consumable, or a microfluidic system.
  • the mixing of the lysis buffer and the recovery buffer is performed until the lysis buffer and the recovery buffer are mixed to homogeneity.
  • the present disclosure provides for a composition for sample processing comprising a buffer comprising: (i) a detergent), (ii) a solubilizer, and (iii) a cyclodextrin.
  • the buffer stabilizes an enzyme during a nucleic acid amplification.
  • the buffer is configured to inactivate a degrading enzyme.
  • the enzyme is a ribonuclease.
  • the composition of the present disclosure further comprises an agent capable of reducing a disulfide bond.
  • the agent capable of reducing said disulfide bond comprises dithiothreitol (DTT), hydroxylamine, hydroxylamine-HCl, 2- mercaptoethanol (BME), or TCEP.
  • the agent capable of reducing said disulfide bond comprises a compound in a monothiol class, a dithiol class, or a phosphine class.
  • the composition further comprises a sample, (e.g., a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool sample, a sputum sample, and/or a lymph sample), raw milk, pasteurized and/or homogenized milk, pasteurized and/or processed milk, one or more Bacillus anthracis spores, one or more Bacillus anthracis vegetative cells, a tissue sample, a cell culture, a purified nucleic acid sample, an environmental sample, one or more WSGR Docket No.
  • a sample e.g., a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool sample, a sputum
  • the swab sample comprises a vaginal swab, an oral swab, and/or a rectal swab.
  • the sample is a solid sample.
  • the sample is a liquid sample.
  • the sample is obtained from a subject.
  • the subject has a disease, a condition, or an infection.
  • the sample comprises a biological sample.
  • the sample comprises a purified sample.
  • the biological sample comprises a target nucleic acid molecule subject to sample processing.
  • the composition further comprises a reaction mixture for nucleic acid amplification.
  • the reaction mixture is lyophilized. In some embodiments, the reaction mixture is not lyophilized.
  • the reaction mixture comprises (i) a thermostable enzyme, (ii) deoxynucleoside triphosphates (dNTPs), (iii) a primer, and/or (iv) a probe.
  • the dNTPs of the reaction mixture comprise dATP, dCTP, dGTP, dTTP, and/or dUTP.
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is at least about 25 micromolar (pM), at least about 50 pM, at least about 75 pM, at least about 100 pM, at least about 150 pM, at least about 200 pM, at least about 250 pM, at least about 300 pM, at least about 350 pM, at least about 400 pM, at least about 450 pM, at least about 500 pM, at least about 750 pM, at least about 1000 pM, at least about 1500 pM, at least about 2000 pM, at least about 2500 pM, at least about 3000 pM, at least about 3500 pM, at least about 4000 pM, at least about 4500 pM, at least about 5000
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is at most about 10000 pM, at most about 9000 pM, at most about 8000 pM, at most about 7000 pM, at most about 6000 pM, at most about 5000 pM, at most about 4500 pM, at most about 4000 pM, at most about 3500 pM, at most about 3000 pM, at most about 2500 pM, at most about 2000 pM, at most about 1500 pM, at most about 1000 pM, at most about 750 pM, at most about 500 pM, at most about 450 pM, at most about 400 pM, at most about 350 pM, at most about 300 pM, at most about 250 pM, at most about 200 pM, at most about 150 pM, at most about 100 pM, at most about 75 pM, at most about 50 pM, or at most about 25
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is about 50 pM to about 7,500 pM. In some embodiments, a concentration of dNTPs in the reaction mixture when mixed with the sample is about 50 pM to about 100 pM, about 50 pM to about 250 pM, about 50 pM to about 500 pM, about 50 pM to about 750 pM, about 50 pM to about 1,000 pM, about 50 pM to about 1,250 pM, about 50 pM to about 1,500 pM, about 50 pM to about 2,000 pM, about 50 pM to about 4,000 pM, about 50 pM to about 5,000 pM, about 50 pM to about 7,500 pM, about 100 pM to about 250 pM, about 100 pM to about 500 pM, about 100 pM to about 750 pM, about 100 pM to about 1,000 pM, about 100
  • the primer or probe can be a stretch of nucleotides that hybridizes with a target nucleic acid sequence.
  • the primer 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 length.
  • the primer is at most about 200 nucleotides, at most about 150 nucleotides, at most about 100 nucleotides, at most about 90 nucleotides, at most about 80 nucleotides, at most about 70 nucleotides, at most about 60 nucleotides, at most about 50 nucleotides, at most about 45 nucleotides, at most about 40 nucleotides, at most about 35 nucleotides, at most about 30 nucleotides, at most about 25 nucleotides, at most about 20 nucleotides, at most about 15 nucleotides, at most about 10 nucleotides, at most about 5 nucleotides, or at most about 3 nucleotides in length.
  • the primer is about 3 nucleotides to about 100 nucleotides in length. In some embodiments, the primer is at most about 100 nucleotides. In some embodiments, the primer is about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 20 nucleotides, about 3 nucleotides to about 30 nucleotides, about 3 nucleotides to about 40 nucleotides, about 3 nucleotides to about 50 nucleotides, about 3 nucleotides to about 60 nucleotides, about 3 nucleotides to about 70 nucleotides, about 3 nucleotides to about 80 nucleotides, about 3 nucleotides to about 90 nucleotides, about 3 nucleotides to about 100 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides
  • nucleotides to about 90 nucleotides about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 90 nucleotides, about 20 nucleotides to about 100 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 60 nucleotides, about 30 nucleotides to about 70 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 50 nucleotides, about
  • the reaction mixture includes probes to visualize amplified nucleic acid products.
  • the probes comprise strand displacement probes, intercalating fluorophores, pH-sensitive dyes, and/or detecting pyrophosphate products.
  • the reaction mixture described herein can comprise an excipient.
  • the excipient can comprise a saccharide (e.g., a monosaccharide, a disaccharide, a polysaccharide, or any combination thereof).
  • the excipient can comprise a surfactant (e.g., nonoxynol-9).
  • the excipient comprises a polymer comprising a cross-linking sucrose with epichlorohydrin.
  • the polymer may be polysucrose 400.
  • the excipient comprises Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, nonoxynol-9, trehalose, dextran, polysucrose 400, a cyclodextrin, or any combination thereof.
  • the excipient may comprise dithiothreitol (DTT).
  • the excipient may comprise a final concentration of Tris, sodium chloride and/or potassium chloride, EDTA, nonoxynol-9, one or more saccharides (e.g., dextran and/or trehalose), polysucrose 400, and/or cyclodextrin.
  • the concentration (e.g., final concentration) of dithiothreitol (DTT) 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, at most about 11 mM
  • the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient can be between about 0.0001 M to about 5 M. In some embodiments, the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient can be between WSGR Docket No. 52459-726.601 at most about 5 M.
  • the concentration (e.g., final concentration) of dithiothreitol (DTT) in the excipient can be between 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, 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
  • 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
  • 52459-726.601 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.
  • the concentration (e.g., final concentration) of EDTA in the excipient can be between about 0.01 mM to about 5 mM. In some embodiments, the concentration (e.g., final concentration) of EDTA in the excipient can be between at most about 5 mM.
  • the concentration (e.g., final concentration) of EDTA in the excipient can be between about 0.01 mM to about 0.05 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.75 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 1.25 mM, about 0.01 mM to about 1.5 mM, about 0.01 mM to about 1.75 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 5 mM, about 0.05 mM to about 0.1 mM, about 0.05 mM to about 0.5 mM, about 0.05 mM to about 0.75 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 1.25 mM, about 0.05 mM to about 1.5
  • the concentration (e.g., final concentration) of nonoxynol-9 in the excipient is at least about 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.
  • the concentration (e.g., final concentration) of nonoxynol-9 in the excipient 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.
  • the concentration (e.g., final concentration) of nonoxynol-9 in the excipient can be between about 0.01 % v/v to about 5 % v/v. In some embodiments, the concentration (e.g., final concentration) of nonoxynol-9 in the excipient can be between at most about 5 % v/v.
  • the concentration (e.g., final concentration) of nonoxynol- 9 in the excipient can be between about 0.01 % v/v to about 0.05 % v/v, about 0.01 % v/v to about 0.1 % v/v, about 0.01 % v/v to about 0.5 % v/v, about 0.01 % v/v to about 0.75 % v/v, about 0.01 % v/v to about 1 % v/v, about 0.01 % v/v to about 1.25 % v/v, about 0.01 % v/v to about 1.5 % v/v, about 0.01 % v/v to about 1.75 % v/v, about 0.01 % v/v to about 2 % v/v, about 0.01 % v/v to about 3 % v/v, about 0.01 % v/v to about 5 % v/v, about 0.05 % v/v/v,
  • the concentration (e.g., final concentration) of trehalose 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,
  • the concentration (e.g., final concentration) of trehalose in the excipient is at most about 5000 mM, at most about 4000 mM, at most about 3000 mM, at most about 2000 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 WSGR Docket No.
  • the concentration (e.g., final concentration) of trehalose in the excipient can be between about 0.001 M to about 5 M.
  • the concentration (e.g., final concentration) of trehalose in the excipient can be between 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 0.05 M, about 0.001 M to about 0.075 M, about 0.001 M to about 0.1 M, about 0.001 M to about 0.25 M, about 0.001 M to about 0.5 M, about 0.001 M to about 0.75 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 0.05 M, about 0.005 M to about 0.075 M, about 0.005 M to about 0.1 M, 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 1 M, about 0.005 M to about
  • the concentration (e.g., final concentration) of dextran in the excipient is at least about 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 WSGR Docket No.
  • the concentration (e.g., final concentration) of dextran in the excipient is at most about 15% w/v, at most about 10% w/v, at most about 7.5% w/v, at most about 5% w/v, at most about 4% w/v, at most about 3% w/v, at most about 2% w/v, at most about 1% w/v, at most about 0.5% w/v, at most about 0.1% w/v, at most about 0.05% w/v, at most about 0.01% w/v, at most about 0.005% w/v, at most about 0.001% w/v, or less than about 0.001% w/v.
  • the concentration (e.g., final concentration) of dextran in the excipient can be between about 0.1 % w/v to about 8 % w/v. In some embodiments, the concentration (e.g., final concentration) of dextran in the excipient can be between at most about 8 % w/v.
  • the concentration (e.g., final concentration) of dextran in the excipient can be between about 0.1 % w/v to about 0.5 % 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 0.1 % w/v to about 2.5 % w/v, about 0.1 % w/v to about 3 % w/v, about 0.1 % w/v to about 3.5 % w/v, about 0.1 % w/v to about 4 % w/v, about 0.1 % w/v to about 5 % w/v, about 0.1 % w/v to about 6 % w/v, about 0.1 % w/v to about 8 % w/v, about 0.5 % w/v to about 1 % w
  • the concentration (e.g., final concentration) of poly sucrose 400 in the excipient is at least about 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 of solute / 100 mL of solution).
  • the concentration (e.g., final concentration) of polysucrose 400 in the excipient is at most about 15% w/v, at most about 10% w/v, at most about 7.5% w/v, at most about 5% w/v, at most about 4% w/v, at most about 3% w/v, at most about 2% w/v, at most about 1% w/v, at most about 0.5% w/v, at most about 0.1% w/v, at most about 0.05% w/v, at most about 0.01% w/v, at most about 0.005% w/v, at most about 0.001% w/v, or less than about 0.001% w/v (g of solute / 100 mL of solution).
  • the concentration (e.g., final concentration) of polysucrose 400 in the excipient can be between about 0.001 % w/v to about 5 % w/v. In some embodiments, the concentration (e.g., final concentration) of polysucrose 400 in the excipient can be between at most about 5 % w/v.
  • the concentration (e.g., final concentration) of polysucrose 400 in the excipient can be between about 0.001 % w/v to about 0.01 % w/v, about 0.001 % w/v to about 0.1 % w/v, about 0.001 % w/v to about 0.2 % w/v, about 0.001 % w/v to about 0.3 % w/v, about 0.001 % w/v to about 0.4 % w/v, about 0.001 % w/v to about 0.5 % w/v, about 0.001 % w/v to about 0.75 % w/v, about 0.001 % w/v to about 1 % w/v, about 0.001 % w/v to about 2 % w/v, about 0.001 % w/v to about 3 % w/v, about 0.001 % w/v to about 5 % w
  • 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 .
  • the concentration (e.g., final concentration) of cyclodextrin in the excipient is at most about 5000 mM, at most about 4000 mM, at most about 3000 mM, at most about 2000 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
  • the concentration (e.g., final concentration) of cyclodextrin in the excipient can be between about 0.001 M to about 5 M. In some embodiments, the concentration (e.g., final concentration) of cyclodextrin in the excipient can be between at most about 5 M.
  • the concentration (e.g., final concentration) of cyclodextrin in the excipient can be between 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, 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 3 M, about 0.005 M to about 0.05 M,
  • the excipient may comprise at least one additional reagent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more additional reagents).
  • the additional reagent can comprise a base, Brij 98, guanidinium 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 an enzyme.
  • the additional reagent may lower a Cq value of a nucleic acid amplification.
  • a nucleic acid amplification can WSGR Docket No. 52459-726.601 have a lower Cq value following a composition comprising an excipient described herein, compared to a Cq value of a nucleic acid amplification that does not comprise a composition comprising the excipient.
  • a composition described herein may further comprise a sample stabilization buffer.
  • the sample stabilization buffer can comprise one or more reagents.
  • the one or more reagents may be a collapse modifier, a protein stabilizer, a glass transition modifier, or any combination thereof.
  • the sample stabilization buffer can comprise at least one salt (e.g., 1, 2, 3, 4, 5, or more salts).
  • the sample stabilization buffer may comprise a cyclodextrin, wherein the cyclodextrin can be a cyclodextrin and/or a concentration of a cyclodextrin as described herein.
  • the one or more reagents of the sample stabilization buffer may be optimized for freeze drying.
  • the sample stabilization buffer may be configured to reconstitute a lyophilized sample.
  • Application of the sample stabilization buffer may reconstitute a lyophilized sample and provide for an improved nucleic acid amplification of the sample.
  • the sample stabilization buffer comprises one or more reducing agents.
  • the one or more reducing agents can be oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, a thiosulfate, sodium hydrosulfite, 1 ,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydropyran (THP), or any combination thereof.
  • composition provided herein may stabilize nucleic acids during the nucleic acid amplification which may improve the precision and/or efficiency of the amplification.
  • a composition e.g., a recovery buffer described herein may comprise one or more cucurbituril.
  • Cucurbiturils are macrocyclic molecules made of glycoluril monomers linked by methylene bridges. Cucurbiturils may form host guest complexes in a composition described herein. Without wishing to be bound by theory, cucurbiturils may be advantageous in a sample preparation as they host one or more inhibitory substances in samples (e.g., PAX samples) so that the samples may be run directly without purification.
  • the PAX sample refers to a sample collected in a PAXgene® tube (or PAX tube as used here).
  • compositions and/or methods described herein comprising one or more of the buffers described herein can improve stabilization of genetic material (e.g., RNA) compared to that of the PAXgene® Blood RNA system.
  • the use of the compositions and/or methods described herein may enhance stabilization of genetic material (e.g., RNA) to improve quality of an amplified genetic product, reduce time to produce an amplified product, or any combination thereof.
  • the cucurbituril may be noted as cucurbit[n]uril, where n is an integer designating the number of glycoluril units.
  • the composition e.g., a recovery buffer
  • the composition may comprise cucurbit[l]uril, cucurbit[2]uril, cucurbit[3]uril, cucurbit[4]uril, cucurbit[5]uril, cucurbit[6]uril, cucurbit[7]uril, cucurbit[8]uril, cucurbit[9]uril, or cucurbit[10]uril.
  • a composition for sample processing may comprise a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; a stabilizing agent comprising tetradecyl trimethyl-ammonium oxalate and/or tartaric acid, and wherein the composition is configured to stabilize an enzyme during a nucleic acid amplification, and wherein the composition is configured to reduce and/or eliminate activity of a degrading nuclease.
  • 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.
  • the cyclic temperature is at most about 115°C. In some embodiments, the cyclic 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
  • 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.
  • the sample may be contacted with a recovery buffer.
  • the recovery buffer may add to the enzyme stability and/or robustness.
  • the sample is contacted with a recovery buffer comprising a solubilizer and a cyclodextrin.
  • the solubilizer is polysorbate 80.
  • 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% WSGR Docket No. 52459-726.601
  • 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, at most about 20% v/v, at most about 15% v/v, at most about 10% v/v, at most about 70% v/
  • the cyclodextrin is present at a final concentration mixed with the sample that is effective for isolating the detergent within the composition of the present invention.
  • the concentration (e.g., final concentration) of the cyclodextrin in the mixture in the presence of the sample is at least about, 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
  • the final volume of the recovery buffer is at least about, at most about, or about 100 pl, 200 pl, 300 pl, 400 pl, 450 pl, 500 pl, 550 pl, 600 pl, 650 pl, 700 pl, 750 pl, 800 pl, 900 pl, 1000 pl, or a range between any two of these values.
  • the lysis buffer may be frozen at a temperature of about -10°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -5°C. In some embodiments, the lysis buffer may be frozen at a temperature of about 0°C.
  • the buffer may be thawed and mixed with the sample.
  • the efficiency of the thawed lysis buffer is tested and compared with the efficiency of an unfrozen lysis buffer.
  • the efficiency of the thawed lysis buffer is similar to the efficiency of the unfrozen lysis buffer.
  • the buffer may be thawed and mixed with the sample.
  • the efficiency of the thawed recovery buffer is tested and compared with the efficiency of an unfrozen recovery buffer.
  • the efficiency of the thawed recovery buffer is similar to the efficiency of the unfrozen recovery buffer.
  • the sample may be a processed sample.
  • the sample processing methods of the present disclosure may reduce the total preparation time of an unprocessed sample to a processed sample.
  • the sample processing of the present disclosure shortens the processing time of a sample 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 the processing time of a sample by a different sample processing method (e.g., a sample processing method with SDS, polysorbate 80, or a cyclodextrin used individually).
  • a different sample processing method e.g., a sample processing method with SDS, polysorbate 80, or a cyclodextrin used individually.
  • sample processing methods can comprise alternative reagents, including, but not limited to, isopropanol and/or ethanol.
  • the sample processing of the present disclosure shortens the processing time of a sample by at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, WSGR Docket No. 52459-726.601 at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or at most about 3% compared to the processing time of a sample by a different sample processing method.
  • the sample processing of the present disclosure shortens the processing time of a sample by about 2% to about 75%. In some embodiments, the sample processing of the present disclosure shortens the processing time of a sample by at most about 75%.
  • the sample processing of the present disclosure shortens the processing time of a sample by about 2% to about 3%, about 2% to about 5%, about 2% to about 7%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 60%, about 2% to about 75%, about 3% to about 5%, about 3% to about 7%, about 3% to about 10%, about 3% to about 15%, about 3% to about 20%, about 3% to about 30%, about 3% to about 40%, about 3% to about 50%, about 3% to about 60%, about 3% to about 75%, about 5% to about 7%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 75%, about 7% to about 10%, about 7% to about 15%, about 5%
  • the present disclosure provides a method of processing a sample, the method comprising: (a) contacting the sample with a lysis 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 processed sample in a mixture comprising the detergent, the solubilizer and the cyclodextrin.
  • a lysis buffer comprising a detergent
  • heating the sample in (c) further comprises heating the sample to the second temperature, cooling down the sample, and heating the sample to the second temperature after cooling down.
  • the sample may be cooled down to room temperature. In some embodiments, the sample may be cooled down to a temperature below room temperature.
  • 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.
  • the sample in (b) may be incubated at a temperature of at most about 50°C, at most about 45°C, at most about 40°C, at most about 35°C, at most about 30°C, at most about 28°C, at most about 26°C, at most about 24°C, at most about 22°C, at most about 20°C, at most about 18°C, at most about 16°C, at most about 14°C, at most about 12°C, at most about 10°C, at most about 5°C, at most about 0°C, at most about -5°C, or at most about -10°C.
  • the sample in (b) may be incubated at a temperature of at about - 10°C to about 50°C. In some embodiments, the sample in (b) may be incubated at a temperature at about -10°C to about 0°C, about -10°C to about 10°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 0°C to about 10°C, about 0°C to about 12°C, about 0°C to about 15°C, about 0°C to about 17°C, about 0°C to about 20°C, about 0°C to about 22°C, about 0°C
  • 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.
  • the first time period is at most about 10 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 12 hours, at most about 10 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 60 minutes, at most about 45 minutes, at most about 30 minutes, at most about 25 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.
  • a method of the present disclosure may comprise bead beating the sample.
  • Bead beating can assist in cell lysis to agitate a sample with grinding media or beads.
  • a lysing matrix may be used for bead beating.
  • the lysing matrix may comprise silica, glass, ceramic, silicon carbide, zirconium silicate, garnet, stainless steel, and/or zirconium oxide.
  • the second temperature of the method of the present 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.
  • 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.
  • the second time period is at most about 10 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 60 minutes, at most about 45 minutes, at most about 30 minutes, at most about 25 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.
  • the processed sample may be contacted with a reaction mixture.
  • the reaction mixture comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), a primer, and/or a probe.
  • dNTPs deoxynucleoside triphosphates
  • the thermostable enzyme comprises a large fragment of a Bacillus stearothermophilus polymerase, an exo-Klenow polymerase, a Bst 2.0 polymerase, a Bst 3.0 polymerase, a SD DNA polymerase, a phi29 DNA polymerase, a sequencing-grade T7 exo-polymerase, an OmniTaq 2 LA DNA polymerase, and/or any mutants thereof.
  • the thermostable enzyme comprises a DNA polymerase.
  • the thermostable enzymes comprises a Taq DNA polymerase.
  • the thermostable enzyme comprises a DNA-dependent DNA polymerase.
  • thermostable enzyme comprises a strand-displacing DNA polymerase.
  • reaction mixture stabilizes enzymatic activity of the thermostable enzyme.
  • a large fragment of a Bacillus stearothermophilus polymerase is the portion of the Bacillus stearothermophilus
  • the composition is configured to stabilize enzymatic activity of the thermostable enzyme for use during a nucleic acid amplification.
  • the dNTPs of the reaction mixture comprise dATP, dCTP, dGTP, dTTP, and/or dUTP.
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is at least about 25 pM, at least about 50 pM, at least about 75 pM, at least about 100 pM, at least about 150 pM, at least about 200 pM, at least about 250 pM, at least about 300 pM, at least about 350 pM, at least about 400 pM, at least about 450 pM, at least about 500 pM, at least about 750 pM, at least about 1000 pM, at least about 1500 pM, at least about 2000 pM, at least about 2500 pM, at least about 3000 pM, at least about 3500 pM, at least about 4000 pM, at least about 4500 pM, at least about 5000 pM, at least about 6000 pM, at least about 7000 pM, at least about 8000 pM, at least about 9000 pM, or at least about 10000 pM.
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is at most about 10000 pM, at most about 9000 pM, at most about 8000 pM, at most about 7000 pM, at most about 6000 pM, at most about 5000 pM, at most about 4500 pM, at most about 4000 pM, at most about 3500 pM, at most about 3000 pM, at most about 2500 pM, at most about 2000 pM, at most about 1500 pM, at most about 1000 pM, at most about 750 pM, at most about 500 pM, at most about 450 pM, at most about 400 pM, at most about 350 pM, at most about 300 pM, at most about 250 pM, at most about 200 pM, at most about 150 pM, at most about 100 pM, at most about 75 pM, at most about 50 pM, or at most about 25
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is about 50 pM to about 7,500 pM. In some embodiments, a concentration of dNTPs in the reaction mixture when mixed with the sample is about 50 pM to about 100 pM, about 50 pM to about 250 pM, about 50 pM to about 500 pM, about 50 pM to about 750 pM, about 50 pM to about 1,000 pM, about 50 pM to about 1,250 pM, about 50 pM to about 1,500 pM, about 50 pM to about 2,000 pM, about 50 pM to about 4,000 pM, about 50 pM to about 5,000 pM, about 50 pM to about 7,500 pM, about 100 pM to about 250 pM, about 100 pM to about 500 pM, about 100 pM to about 750 pM, about 100 pM to about 1,000 pM, about
  • the primer or probe can be a stretch of nucleotides that hybridizes with a target nucleic acid sequence.
  • the primer 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 length.
  • the primer is at most about 200 nucleotides, at most about 150 nucleotides, at most about 100 nucleotides, at most about 90 nucleotides, at most about 80 nucleotides, at most about 70 nucleotides, at most about 60 nucleotides, at most about 50 nucleotides, at most about 45 nucleotides, at most about 40 nucleotides, at most about 35 nucleotides, at most about 30 nucleotides, at most about 25 nucleotides, at most about 20 nucleotides, at most about 15 nucleotides, at most about 10 nucleotides, at most about 5 nucleotides, or at most about 3 nucleotides in length.
  • the primer is about 3 nucleotides to about 100 nucleotides in length. In some embodiments, the primer is at most about 100 nucleotides. In some embodiments, the primer is about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 20 nucleotides, about 3 nucleotides to about 30 nucleotides, about 3 nucleotides to about 40 nucleotides, about 3 nucleotides to about 50 nucleotides, about 3 nucleotides to about 60 nucleotides, about 3 nucleotides to about 70 nucleotides, about 3 nucleotides to about 80 nucleotides, about 3 nucleotides to about 90 nucleotides, about 3 nucleotides to about 100 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleot
  • nucleotides to about 90 nucleotides about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 90 nucleotides, about 20 nucleotides to about 100 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 60 nucleotides, about 30 nucleotides to about 70 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 50 nucleotides, about
  • the primer is tagged with biotin or 6-carboxyfluorescein (FAM) for visualization on a lateral flow immunoassay strip.
  • FAM 6-carboxyfluorescein
  • the reaction mixture is lyophilized.
  • the methods and compositions of the present disclosure can provide for a faster time from obtaining a sample to generating a processed sample.
  • a time from obtaining the sample to generating the processed 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.
  • a time from obtaining the sample to generating the processed 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 WSGR Docket No. 52459-726.601 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.
  • 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), nucleic acid 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).
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence based amplification
  • OVA oligonucleotide ligation assay
  • TMA transcription mediated amplification
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • the target sequence processed by the methods provided herein can be used for further downstream applications, e.g., isothermal amplifications.
  • Exemplary isothermal amplification methods that can be used with the compositions and methods provided herein include, but are not limited to, helicase-dependent amplification (HD A), isothermal multiple displacement amplification (IMDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), single primer isothermal amplification (SPIA), or strand displacement amplification (SDA).
  • HD A helicase-dependent amplification
  • IMDA isothermal multiple displacement amplification
  • LAMP loop-mediated isothermal amplification
  • RPA recombinase polymerase amplification
  • SPIA single primer isothermal amplification
  • SDA strand displacement amplification
  • the nucleic acid amplification of the present disclosure comprises PCR or isothermal amplification.
  • a temperature is changed over the course of the nucleic acid amplification method.
  • the nucleic acid amplification comprises thermocycling the processed sample.
  • the nucleic acid amplification comprises keeping the processed sample at a constant temperature during the amplification.
  • the reaction mixture includes probes to visualize amplified nucleic acid products.
  • the probes comprise strand displacement probes, intercalating fluorophores, pH-sensitive dyes, and/or detecting pyrophosphate products.
  • the methods and compositions for processing nucleic acid molecule samples disclosed herein generate a higher yield of amplified nucleic acid products than a yield of amplified nucleic acid products generated by different sample processing methods or compositions.
  • the sample processing methods and compositions generate a yield of amplified nucleic acid products that is at least about 5 times, at least 10 times, at least about 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 1,500 times, at least 2,000 times, at least 10 times, or at least 10,000 times greater than a WSGR Docket No. 52459-726.601 yield of amplified nucleic acid products from an otherwise identical sample processed by SDS, polysorbate 80, and/or a cyclodext
  • the sample processing methods and compositions generate a yield of amplified nucleic acid products that is about 3 times to about 1,000 times greater than a yield of amplified nucleic acid products from an otherwise identical sample processed by SDS, polysorbate 80, and/or a cyclodextrin individually. In some embodiments, the sample processing methods and compositions generate a yield of amplified nucleic acid products that is at most about 1,000 times.
  • the sample processing methods and compositions generate a yield of amplified nucleic acid products that is about 3 times to about 5 times, about 3 times to about 10 times, about 3 times to about 25 times, about 3 times to about 50 times, about 3 times to about 100 times, about 3 times to about 150 times, about 3 times to about 200 times, about 3 times to about 250 times, about 3 times to about 500 times, about 3 times to about 750 times, about 3 times to about 1,000 times, about 5 times to about 10 times, about 5 times to about 25 times, about 5 times to about 50 times, about 5 times to about 100 times, about 5 times to about 150 times, about 5 times to about 200 times, about 5 times to about 250 times, about 5 times to about 500 times, about 5 times to about 750 times, about 5 times to about 1,000 times, about 10 times to about 25 times, about 10 times to about 50 times, about 10 times to about 100 times, about 10 times to about 150 times, about 10 times to about 200 times, about 10 times to about 250 times, about 10 times to about 500 times, about 10 times to about
  • a total time to produce a processed sample using the composition and/or methods described herein may be 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, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, at most about 50 seconds, at most about 40 seconds, at most about 30 seconds, at most about 20 seconds, at most about 15 seconds, at most about 10 seconds, or less than about 10 seconds.
  • a time for processing a sample can comprise a time period from contacting the sample with a reaction mixture to generating a processed sample.
  • the time period from contacting the sample with a reaction mixture to generating a processed sample may be 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, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, at most about 50 seconds, at most about 40 seconds, at most about 30 seconds, at most about 20 seconds, at most about 15 seconds, at most about 10 seconds, or less than about 10 seconds.
  • the methods described herein may not comprise heating a sample. In some cases, the methods described herein may comprise heating a sample.
  • a sample may be heated from about 50°C to about 120°C. In some embodiments, a sample may be heated from about 50°C to about 60°C, about 50°C to about 70°C, about 50°C to about 80°C, about 50°C to about 85°C, about 50°C to about 90°C, about 50°C to about 95°C, about 50°C to about 100°C, about 50°C to about 105°C, about 50°C to about 110°C, about 50°C to about 115°C, about 50°C to about 120°C, about 60°C to about 70°C, about 60°C to about 80°C, about 60°C to about 85°C, about 60°C to about 90°C, about 60°C to about 95°C, about 60°C to about 100°C, about 60°C to about 105°C, about 60°C to about 110
  • the method can comprise contacting a sample with a composition.
  • the composition may be a sample processing buffer described herein.
  • Contacting the sample with the sample processing buffer can generate a processed sample.
  • the method can comprise contacting the processed sample with a composition (e.g., a sample amplification buffer).
  • Contacting the processed sample with the sample amplification buffer can provide a condition for an amplification (e.g., a nucleic acid amplification) for the sample.
  • the method can comprise subjected the processed sample to an amplification (e.g., a nucleic acid amplification).
  • the sample processing buffer may not be removed.
  • prior to contacting the processed sample with the sample amplification buffer the sample processing buffer may be removed.
  • a method of amplifying a sample can comprise: (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 a condition for a nucleic acid amplification; and (c) subjecting the processed sample to the nucleic acid amplification, and wherein prior to the contacting of (b), the sample processing buffer is not removed.
  • the sample stabilization buffer may stabilize an enzyme in the nucleic acid amplification.
  • the sample amplification buffer may comprise a reaction mixture described herein.
  • the sample amplification buffer may comprise an excipient described herein.
  • the sample may be contacted with the sample stabilization buffer prior to subjecting the processed sample to the nucleic acid amplification.
  • the sample may be contacted with the sample stabilization buffer after subjecting the processed WSGR Docket No. 52459-726.601 sample to the nucleic acid amplification.
  • the sample stabilization buffer may be in the same mixture as the sample amplification buffer.
  • the sample stabilization buffer may not be in the same mixture as the sample amplification buffer.
  • a sample is contacted, in order, by: the sample processing buffer, the sample stabilization buffer, and the sample amplification buffer. In some embodiments, a sample is contacted, in order, by: the sample processing buffer, the sample amplification buffer, and the sample stabilization buffer. In some embodiments, the sample may be contacted by the sample processing buffer, and then the sample amplification buffer and the sample stabilization buffer simultaneously (e.g., the sample amplification buffer and the sample stabilization buffer may be in a mixture).
  • the sample processing buffer can comprise a lysis buffer described herein, a recovery buffer described herein, or any combination thereof. In some embodiments, the sample processing buffer can comprise one or more cucurbituril.
  • the cucurbituril of the sample processing buffer described herein may comprise at least about 1 glycoluril unit, at least about 2 glycoluril units, at least about 3 glycoluril units, at least about 4 glycoluril units, at least about 5 glycoluril units, at least about 6 glycoluril units, at least about 7 glycoluril units, at least about 8 glycoluril units, at least about 9 glycoluril units, at least about 10 glycoluril units, or greater than about 10 glycoluril units.
  • the cucurbituril may be noted as cucurbit[n]uril, where n is an integer designating the number of glycoluril units.
  • the sample processing buffer described herein may comprise cucurbit[l]uril, cucurbit[2]uril, cucurbit[3]uril, cucurbit[4]uril, cucurbit[5]uril, cucurbit[6]uril, cucurbit[7]uril, cucurbit[8]uril, cucurbit[9]uril, or cucurbit[10]uril.
  • a processing time for a sample may comprise a time period from contacting a sample with the sample processing buffer to contacting a sample with the sample stabilization buffer.
  • a processing time for a sample may comprise a time period from contacting a sample with the sample processing buffer to contacting a sample with the sample amplification buffer.
  • a processing time for a sample may be at least about 5 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 45 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, or greater than about 5 minutes.
  • a processing time for a sample may be 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, at most about 45 seconds, at most about 30 seconds, at most about 20 seconds, at most about 10 seconds, at most about 5 seconds, or less than about 5 seconds.
  • a nucleic acid amplification may comprise any amplification described herein (e.g., PCR, thermocycling, isothermal amplification, or any combination thereof).
  • the time period from contacting the sample with the sample processing buffer to generating an amplified sample may be 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, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, at most about 50 seconds, at most about 40 seconds, at most about 30 seconds, at most about 20 seconds, at most about 15 seconds, at most about 10 seconds, or less than about 10 seconds.
  • the sample can comprise a biological sample.
  • the biological sample can comprise one or more target nucleic acid molecules (e.g., one or more different target nucleic acid molecules).
  • the one or more different target nucleic acid molecules may be at least about 1%, at least about 2%, at least about 3%, at least about 4%, 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 60%, or 75% greater than a concentration of one or more different target nucleic acid molecules of an otherwise identical sample processed by a sample processing buffer alone.
  • a sample may be contacted by a sample processing buffer (e.g., a lysis buffer, a recovery buffer, or any combination thereof), a sample stabilization buffer, and/or a sample amplification buffer, in any order.
  • a sample can be contacted by the sample amplification buffer to provide a condition for an amplification (e.g., a nucleic acid amplification).
  • the sample may be subjected to nucleic acid amplification in the sample amplification buffer.
  • the sample may be subjected to nucleic acid amplification not in the sample amplification buffer.
  • a processing time for a sample may comprise a time period from contacting a sample with the sample processing buffer.
  • a time period for generating a processed sample prior to contacting the sample with a sample amplification buffer can comprise at most a duration of time for pipetting the sample processing buffer into the sample and mixing the sample processing buffer with the sample.
  • This duration of time may comprise at most about 30 seconds, at most about 25 seconds, at most about 20 seconds, at most about 15 seconds, at most about 10 seconds, at most about 5 seconds, at most about 4 seconds, at most about 3 seconds, at most about 2 seconds, at most about 1 second, or less than about 1 second.
  • a time period for generating a processed sample prior to contacting the sample with a sample amplification buffer can be at least about 5 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 45 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, or greater than about 5 minutes.
  • a time period for generating a processed sample prior to contacting the sample with a sample amplification buffer can be 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, at most about 45 seconds, at most about 30 seconds, at most about 20 seconds, at most about 10 seconds, at most about 5 seconds, or less than about 5 seconds.
  • a buffer e.g., a lysis buffer
  • the lysis buffer can comprise a detergent (e.g., sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof).
  • the sample can comprise tetradecyl trimethyl -ammonium oxalate.
  • the sample can comprise tartaric acid.
  • the sample can comprise tetradecyl trimethyl-ammonium oxalate and tartaric acid.
  • the method may comprise contacting the sample with a recovery buffer.
  • the recovery buffer can comprise a solubilizer described herein.
  • the recovery buffer may comprise a cyclodextrin (e.g., hydroxypropyl P-cyclodextrin, hydroxypropyl y-cyclodextrin, (2- hydroxypropyl)-a-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-a-cyclodextrin hydrate, monopropanediamino-P-cyclodextrin, 6-O-alpha-D-Maltosyl-P-cyclodextrin, 2,6-Di-O-methyl- P-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-P-cyclodextrin hydrate, 3 A-amino-3A-deoxy-(2AS,3 AS)-y-cyclodextrin hydrate, an ani
  • a method of processing a sample can comprise: (a) contacting the sample with a lysis buffer comprising a detergent, wherein the sample comprises tetradecyl WSGR Docket No. 52459-726.601 trimethyl -ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube; and/or (b) contacting the sample with a recovery buffer comprising a solubilizer and a cyclodextrin, thereby processing the sample to generate a processed sample in a mixture comprising the detergent, the solubilizer, and the cyclodextrin.
  • contacting the sample with the lysis buffer can process the sample and generate a processed sample. In some embodiments, contacting the sample with the recovery buffer can process the sample and generate a processed sample. In some embodiments, contacting the sample with the lysis buffer and the recovery buffer can process the sample and generate a processed sample. In some embodiments, the sample is only contacted with the lysis buffer described herein. In some embodiments, the sample is only contacted with the recovery buffer described herein. In some embodiments, the sample can be contacted with a sample amplification buffer.
  • a sample may be contacted by the lysis buffer described herein, generating a processed sample, and then may be contacted by the sample amplification buffer.
  • a sample may be contacted by the recovery buffer described herein, generating a processed sample, and then may be contacted by the sample amplification buffer.
  • the sample may be contacted by the lysis buffer described herein prior to the recovery buffer, generating a processed sample, and then may be contacted by the sample amplification buffer.
  • the sample may be contacted by the recovery buffer described herein prior to the lysis buffer, generating a processed sample, and then may be contacted by the sample amplification buffer.
  • the sample amplification buffer can comprise a nonionic surfactant, a cyclodextrin, a sucrose/epichlorohydrin polymer, or any combination thereof.
  • the sample amplification buffer may increase a rate of amplification of the sample.
  • the sample amplification buffer may be configured to increase a rate of amplification of sample by at least about 1%, at least about 2%, at least about 3%, at least about 4%, 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 75%, at least about 100%, or greater than about 100% compared to a rate of amplification of a sample not contacted by the sample amplification buffer.
  • the sample amplification buffer may be configured to increase a rate of amplification of sample by at most about 100%, at most about 75%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most WSGR Docket No. 52459-726.601 about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% compared to a rate of amplification of a sample not contacted by the sample amplification buffer.
  • a method of processing a sample can comprise: contacting the sample with a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer, wherein the composition is configured to increase a rate of amplification during a nucleic acid amplification, and wherein the sample comprises tetradecyl trimethyl -ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube.
  • a sample amplification buffer comprising: a nonionic surfactant, a cyclodextrin, and a sucrose/epichlorohydrin polymer, wherein the composition is configured to increase a rate of amplification during a nucleic acid amplification, and wherein the sample comprises tetradecyl trimethyl -ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube.
  • a sample Prior to contacting a sample with the sample amplification buffer, a sample may be contacted by a sample processing buffer (e.g., a lysis buffer, a recovery buffer, or any combination thereof).
  • a sample processing buffer e.g., a lysis buffer, a recovery buffer, or any combination thereof.
  • a sample may be contacted by the sample processing buffer to generate a processed sample, and the processed sample may be contacted by the sample amplification buffer.
  • sample processing buffer comprising contacting a sample with a sample processing buffer described herein, a sample amplification buffer described herein, a sample stabilization buffer described herein, or any combination thereof.
  • the sample may be contacted by one or more compositions (e.g., buffers) in any order.
  • the sample may be contacted by the sample processing buffer prior to being contacted by the sample amplification buffer.
  • the sample may be contacted by the sample amplification buffer prior to being contacted by the sample stabilization buffer.
  • sample may be contacted by the sample stabilization buffer prior to being contacted by the sample amplification buffer.
  • the sample may be contacted by the sample processing buffer, followed by the sample amplification buffer, and then the sample stabilization buffer.
  • the sample may be contacted by the sample processing buffer, followed by the sample stabilization buffer, and then the sample amplification buffer.
  • the sample may be collected directly from a sample collection tube. In some embodiments, the sample may not be collected directly from a sample collection tube.
  • the sample can comprise tetradecyl trimethyl-ammonium oxalate, tartaric acid, or any combination thereof.
  • a method of processing a sample may comprise: (a) contacting the sample with a sample processing buffer comprising: a detergent, a solubilizer, and a cyclodextrin; (b) contacting the sample with a sample amplification buffer comprising: a WSGR Docket No. 52459-726.601 nonionic surfactant, a cyclodextrin; and (c) contacting the sample with a sample stabilization buffer configured to stabilize an enzyme in a nucleic acid amplification, wherein the sample comprises tetradecyl trimethyl-ammonium oxalate and/or tartaric acid or wherein the sample is directly from a sample collection tube.
  • the sample may not be processed by an RNA extraction kit.
  • the sample may be processed by an RNA extraction kit.
  • the kit e.g., RNA extraction kit
  • the kit can comprise a spin-column.
  • the kit e.g., RNA extraction kit
  • the methods described herein may not comprise contacting the sample with a wash buffer.
  • the methods described herein may comprise contacting the sample with a wash buffer.
  • the methods described herein may not comprise membrane-based extraction.
  • the methods described herein may comprise membrane-based extraction.
  • Membrane extraction can refer to a process of using a membrane as an intermediate between two phases. The membrane may allow for the extraction of one or more analytes from one phase to another based on physical properties (e.g., mass, weight, charge, or any combination thereof).
  • the processed sample may be subjected to amplification (e.g., nucleic acid amplification) to generate an amplified sample.
  • amplification e.g., nucleic acid amplification
  • compositions and methods provided herein can be used for processing samples that are used in various down-stream operations, including nucleic acid amplifications.
  • Various nucleic acid amplifications can be used including polymerase chain reactions and isothermal amplifications.
  • the nucleic acid amplification described herein may generate an amplified processed sample.
  • a time period of a nucleic acid amplification described herein to generate an amplified processed sample can be 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, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, at most about 50 seconds, at most about 40 seconds, at most about 30 seconds, at most about 20 seconds, at most about 15 seconds, at most about 10 seconds, or less than about 10 seconds.
  • the nucleic acid amplification described herein can be a Polymerase Chain Reaction (PCR).
  • a sample e.g., DNA
  • the sample can then be cooled and mixed with specific oligonucleotide primers, allowing annealing of the primers to the singlestranded DNA template.
  • a thermostable DNA polymerase in a reaction mixture can be added to the sample, along with free dNTPs that are linked by the polymerase to the replicating nucleic acid strand.
  • the products can again be heated to separate the strands and subjected to another round of primer hybridization and polymerase replication.
  • This process can be repeated any number of times. Since each nucleic acid product of a given cycle of this process can serve as a template for production of two new nucleic acid molecules (one from each parent strand), the PCR process can result in an exponential increase in the concentration of the target sequence. Thus, in a well-controlled, high-fidelity PCR process, as few as 20 cycles can result in an over one million fold amplification of the target nucleic acid sequence.
  • thermostable DNA polymerases can be used to synthesize a new DNA strand complementary to the DNA template strand.
  • the thermostable DNA polymerase most commonly used in PCR is Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus. Taq polymerase can function at temperatures of 70- 80°C, and can maintain substantial activity upon repeated exposure to temperatures of 92°-95°C.
  • Taq polymerase In addition to Taq polymerase, other thermostable polymerases may be used.
  • Taq polymerase in PCR
  • Isothermal amplification can rapidly and efficiently copy nucleic acids without temperature changing cycles. Instead, isothermal amplification can use specific DNA polymerases, and specially designed primer sets to exponentially amplify a target sequence. Different isothermal amplification methods can utilize different DNA polymerases.
  • Helicase- WSGR Docket No. 52459-726.601 dependent amplification (HD A) can use helicase to unwind DNA, allowing primers to bind.
  • Two accessory proteins, MutL and single-stranded DNA-binding protein (SSB) can be used to prevent complimentary strands from associating.
  • Isothermal multiple displacement amplification (IMDA) can use strand-displacing DNA polymerase and multiple primer sets.
  • Loop-mediated isothermal amplification can use 4-6 primers and a strand-displacing DNA polymerase.
  • LAMP is a highly efficient amplification method that can synthesize a large quantity of DNA in a short period of time.
  • Recombinase polymerase amplification RPA
  • RPA Recombinase polymerase amplification
  • Recombinase may be complexed with the primer and the complex then can use strand exchange to bind to doublestranded DNA. After the strand exchange an SSB T4 gp32 can stabilize the displaced strand.
  • Rolling circle amplification can synthesize long single-stranded DNA using a short, circular single-stranded DNA template and a single primer.
  • RCA can use a strand-displacing DNA polymerase called phi 29.
  • Single primer isothermal amplification may use only one DNA-RNA chimeric primer along with RNAase H and a strand-displacing DNA polymerase.
  • Strand displacement amplification may rely on a restriction enzyme (Hindi) and an exonuclease-deficient DNA polymerase. HinCII can nick the target DNA, and the DNA polymerase can then extend the 3’ end.
  • Hindi restriction enzyme
  • HinCII can nick the target DNA, and the DNA polymerase can then extend the 3’ end.
  • the methods, compositions, and kits described herein can be used for processing target nucleic acid molecules.
  • the present disclosure provides for methods of amplification of nucleic acids (e.g., isothermal amplification). Such a method can involve a cycle of steps such as that depicted in FIGs. 15A through 150.
  • the compositions and methods as shown in FIGs. 15A through 150 can be referred to as differential targeted endonuclease cutting technology (DTECT).
  • DTECT differential targeted endonuclease cutting technology
  • the methods provided herein can offer higher amplification efficiency and easier optimization procedure compared with existing amplifications (e.g. isothermal amplifications). Additional details of the isothermal application methods described herein are disclosed in the International Application No. PCT/US2023/079306, which is incorporated herein by reference in its entirety.
  • the processed target nucleic acid molecules can be used in various amplification reactions not limited to the amplification or processing methods described herein.
  • the DTECT method described herein can start with the formation of a structure such as that depicted in FIG. 15A, in which a guide nucleic acid complex (or a guide complex) is formed to direct a restriction enzyme to a predetermined site in a nucleic acid.
  • FIG. 15A depicts WSGR Docket No. 52459-726.601 a nucleic acid strand (e.g., a single-stranded DNA strand or ssDNA strand) (100) comprising a target nucleic acid sequence (101).
  • the ssDNA strand can be generated by reverse transcribing a target RNA sequence.
  • the ssDNA strand can be generated by denaturing a double-stranded DNA (dsDNA) sequence.
  • a type Ils restriction enzyme (120) is directed to the vicinity of the target site via formation of a guide complex.
  • This guide nucleic acid complex is constituted via self-annealing of single copies of a guide polynucleotide which comprise: a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme (117), a target binding region configured to hybridize to the target sequence (115), and a blocked 3' end non-extendable by a polymerase (116). Note that in FIG. 15A, self-annealing of the two copies of the guide polynucleotide forms a double-stranded palindromic region that permits binding of the type II restriction enzyme in the vicinity of the target site.
  • the DTECT method described herein can continue in a second stage with the process depicted in FIG. 15B and FIG. 15C.
  • the type Ils restriction enzyme (120) is directed to the vicinity of the target site (101) by the double-stranded palindromic region (two copies of 117) formed by self-annealing of the guide polynucleotides
  • the type Ils restriction enzyme is able to, characteristic to its activity, cleave single-stranded locations (130, 135) distal to its binding site (FIG. 15B).
  • One of these cleavable single- stranded locations (135) is on the nucleic acid strand (101) that comprises the target nucleic acid sequence (101).
  • the other cleavable single-stranded location (130) is located on the guide polynucleotide itself (130). If selective enzymatic conditions, an engineered polymerase, or BspD6I is used, cleavage at one of the sites (e.g. the single-stranded site on the nucleic acid strand (101) that comprises the target nucleic acid sequence (101)) can be favored. Cleavage at the single-stranded site on the nucleic acid strand (101) that comprises the target nucleic acid sequence (101) generates a free 3' hydroxyl that can then be extended by a strand-displacing polymerase present in the reaction.
  • the DTECT method described herein can continue in a third stage with the process depicted in FIG. 15D through FIG. 15F.
  • Extension of the free 3' hydroxyl by the stranddisplacing polymerase (140, FIG. 15C) produces a region (160) of the nucleic acid strand (101) that comprises the target nucleic acid sequence (101) that is complementary to the restriction endonuclease recognition sequence for the type Ils restriction enzyme (117) from the guide polynucleotide (FIG. 15D).
  • Extension of the nucleic acid (100) displaces the second copy of the guide polynucleotide (116/117, lower molecule), that previously formed half of the guide complex.
  • cleavage at the single-stranded site (135) that contains the target nucleic acid site (100) causes the strand (100) to merely be extended again by the polymerase
  • cleavage at the single-stranded site (130) allows for a new procedure to commence (FIG. 15E).
  • cleavage at site 130 of FIG. 15E on the annealed guide polynucleotide removes the sequence containing the blocked 3' end (116) and allows the guide polynucleotide to be extended to comprise a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101) (FIG. 15F).
  • the DTECT method described herein can continue in a fourth stage with the process depicted in FIG. 15G and FIG. 15H.
  • the double-stranded structure of FIG. 15G no longer comprises a blocked 3' end, repeated cleavage at site 130 of FIG. 15G liberates a single strand comprising a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101), and then allows extension of a new strand (171) to replace it.
  • the liberated strand (170) can further serve as a new template analogously to the strand 100 of FIG. 15A (FIG. 151), which allows for strand 170 to be further cleaved and repeatedly extended as in FIG. 15H (FIG. 15J).
  • FIG. 15K depicts an exemplary completed extension on the new guide molecule.
  • the method can continue, as seen in FIG. 15L, wherein endonucleolytic activity can occur on the second complementary strand oligo/extension product complex (170).
  • FIG. 15M depicts a polymerase (140) extending of the 3’ end of the cut site of the second complementary strand of the oligo/extension product complex. Endolytic activity on the newly synthesized strand (130) occurs (FIG. 15N) and the displaced, single-stranded synthesized fragment (42) of FIG. 150 can serve as starting material for additional strand displacement amplification reactions.
  • methods according to the disclosure do not involve amplification and utilize the structure depicted in FIG. 15A to direct cleavage of a single-stranded nucleic acid molecule (100) containing a target site (101) at a specified position (135, FIG. 15B).
  • the DTECT amplification methods described herein can utilize DNA polymerases with high strand-displacement activity and specially designed primer sets to exponentially amplify a target sequence.
  • the combination of the DTECT amplification methods with the sample processing, stabilizing, and/or amplifying compositions described herein may provide a faster WSGR Docket No. 52459-726.601 time to amplify a target nucleic acid molecule compared to a time with an existing amplification and/or sample processing method.
  • the DTECT method can comprise contacting a singlestranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes to the single- stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme (e.g., a restriction enzyme).
  • the restriction enzyme can be a type Ils restriction enzyme.
  • the guide polynucleotide can further comprise (ii) a target binding region configured to hybridize to the target sequence.
  • the guide polynucleotide can further comprise (iii) a blocked 3' end non-extendable by a polymerase.
  • the guide polynucleotide further comprises (i), (ii), and (iii) in 5' to 3' order.
  • the non-target binding region can be located at the 5' end of the guide polynucleotide.
  • the target binding region can be located at the 3' end of the guide polynucleotide.
  • the non-target binding region further comprises a sequence containing a reverse complement of the restriction endonuclease recognition sequence for the type Ils restriction enzyme 3' to the restriction endonuclease recognition sequence for a type Ils restriction enzyme and 5' to the target binding region configured to hybridize to the target sequence.
  • the cut exposes an extendable 3' end of the target sequence.
  • the method further comprises reverse-transcribing the singlestranded nucleic acid molecule from an RNA.
  • the nucleic acid target processed (e.g., nicked or cut mediated by the guide complex or enzyme) by the methods described herein may be used as an initial template to be used with any existing isothermal amplification.
  • the isothermal amplifications can be performed at a constant temperature, for example, at about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61 °C, about 62°C, about 63 °C, about 64°C, about 65 °C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71 °C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, or about 80°C.
  • Amplification products of any amplification reactions described herein can be detected by various methods.
  • the amplification products may be detected by gel electrophoresis, thus detecting reaction products having a specific length.
  • the nucleotides may, for example, be labeled, such as, for example, with biotin.
  • Biotin-labeled amplified sequences may be captured using avidin bound to a signal generating enzyme, for example, peroxidase.
  • Nucleic acid detection methods may employ the use of dyes that specifically stain double-stranded DNA. WSGR Docket No. 52459-726.601
  • Intercalating dyes that exhibit enhanced fluorescence upon binding to DNA or RNA can be used.
  • Dyes may be, for example, DNA or RNA intercalating fluorophores and may include but are not limited to the following examples: Acridine orange, ethidium bromide, Hoechst dyes, PicoGreen, propidium iodide, SYBRI (an asymmetrical cyanine dye), SYBRII, TOTO (a thiaxole orange dimer) and YOYO (an oxazole yellow dimer), and the like. Dyes can provide an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with various detection methods and may have varying optimal usage parameters.
  • Nucleic acid detection methods may also employ the use of labeled nucleotides incorporated directly into the target sequence or into probes containing complementary or substantially complementary sequences to the target of interest. Such labels may be radioactive and/or fluorescent in nature. Labeled nucleotides, which can be detected but otherwise function as native nucleotides, can be to be distinguished from modified nucleotides, which do not function as native nucleotides.
  • the production or presence of target nucleic acids and nucleic acid sequences may be detected and monitored by Molecular Beacons.
  • the production or presence of target nucleic acids and nucleic acid sequences may also be detected and monitored by Fluorescence resonance energy transfer (FRET).
  • FRET Fluorescence resonance energy transfer
  • fluorophores and/or dyes may be used in the methods described herein according to the present disclosure.
  • Available fluorophores include coumarin; fluorescein; tetrachlorofluorescein; hexachlorofluorescein; Lucifer yellow; rhodamine; BODIPY; tetramethylrhodamine; Cy3; Cy5; Cy7; eosine; Texas red; SYBR Green I; SYBR Gold; 5-FAM (also called 5 -carboxy fluorescein; also called Spiro(isobenzofuran-1(3H), 9'-(9H)xanthene)-5- carboxylic acid, 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein ([4,7,2',4',5',7'-hexachloro-(3 ',6'-dipivaloyl-fluorescein); 5-
  • Combination fluorophores such as fluorescein-rhodamine dimers may also be suitable. Fluorophores may be chosen to absorb and emit in the visible spectrum or outside the visible spectrum, such as in the ultraviolet or infrared ranges. Suitable quenchers may also include DABCYL and variants thereof, such as DABSYL, DAB MI and Methyl Red. Fluorophores may also be used as quenchers, because they tend to quench fluorescence when touching certain other fluorophores. In some cases, quenchers may be chromophores such as DABCYL or malachite green, or fluorophores that may not fluoresce in the detection range when the probe is in the open conformation.
  • kits comprising a lysis buffer and a recovery buffer as described herein, and an instruction for use.
  • the kit comprises a lysis buffer comprising a detergent and a recovery buffer comprising a solubilizer and a cyclodextrin.
  • the detergent is sodium dodecyl sulfate (SDS).
  • the detergent comprises sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.
  • the detergent is an ionic detergent.
  • the detergent is a non-ionic detergent.
  • the solubilizer is a non-ionic surfactant.
  • the solubilizer comprises a polysorbate.
  • the polysorbate may be polyoxyethylene (20) sorbitan monooleate (e.g., polysorbate 80), polyoxyethylene (20) sorbitan monolaurate (e.g., polysorbate 20), polyoxyethylene (20) sorbitan monopalmitate (e.g., polysorbate 40), polyoxyethylene (20) sorbitan monostearate (e.g., polysorbate 60), or a functional variant thereof.
  • the solubilizer is a TergitolTM surfactant, a TritonTM surfactant, or a Igepal® surfactant.
  • the solubilizer is an alkoxylate or a cocamide.
  • the solubilizer is decyl glucoside, alkyl polyglycoside, lauryl glucoside, sorbitan tristearate, or a niosome.
  • the cyclodextrin comprises cyclodextrin comprises hydroxypropyl P-cyclodextrin, hydroxypropyl y-cyclodextrin, (2-hydroxypropyl)-a- cyclodextrin, 3 A-amino-3 A-deoxy-(2AS,3AS)-a-cyclodextrin hydrate, monopropanediamino-P- cyclodextrin, 6-O-alpha-D-Maltosyl-P-cyclodextrin, 2,6-Di-O-methyl-P-cyclodextrin, hydroxyethyl-P-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-P-cyclodextrin,
  • the kit further comprises a reagent for nucleic acid amplification comprising a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), and/or a primer.
  • the kit further comprises a thermostable enzyme compatible with the primers and nucleic acid molecule samples as described herein.
  • the kit further comprises a strand-displacing polymerase.
  • a large fragment of a Bacillus stearothermophilus polymerase is the portion of the Bacillus stearothermophilus DNA polymerase that contains the 5' — > 3' polymerase activity, but lacks the 5' — >3' exonuclease domain.
  • the composition is configured to stabilize enzymatic activity of the thermostable enzyme for use during a nucleic acid amplification.
  • the dNTPs of the reaction mixture comprise dATP, dCTP, dGTP, dTTP, and/or dUTP.
  • a concentration of dNTPs in the reaction mixture when mixed with the sample is at least about 25 pM, at least about 50 pM, at least about 75 pM, at least about 100 pM, at least about 150 pM, at least about 200 pM, at least about 250 pM, at least about 300 pM, at least about 350 pM, at least about 400 pM, at least about 450 pM, at least about 500 pM, at least about 750 pM, at least about 1000 pM, at least about 1500 pM, at least about 2000 pM, at least about 2500 pM, at least about 3000 pM, at least about 3500 pM, at least about 4000 pM, at least about 4500 pM, at least about 5000 pM
  • the primer in the kit 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 length.
  • the primer is at most about 200 nucleotides, at most about 150 nucleotides, at most about 100 nucleotides, at WSGR Docket No. 52459-726.601 most about 90 nucleotides, at most about 80 nucleotides, at most about 70 nucleotides, at most about 60 nucleotides, at most about 50 nucleotides, at most about 45 nucleotides, at most about 40 nucleotides, at most about 35 nucleotides, at most about 30 nucleotides, at most about 25 nucleotides, at most about 20 nucleotides, at most about 15 nucleotides, at most about 10 nucleotides, at most about 5 nucleotides, or at most about 3 nucleotides in length.
  • the kit further comprises a probe for detecting an amplification product generated using the kit.
  • the probe comprises a fluorescent tag or dye.
  • the lysis buffer, the recovery buffer, and/or the reagent of the kit may be stable for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 10 days, at least about 15 days, at least about 30 days, at least about 2 months, at least about 3 months, at least about 6 months, at least about one year, at least about two years, at least about three years, at least about four years, or at least about five years when stored at room temperature.
  • the lysis buffer, the recovery buffer, and/or the reagent of the kit may be stable for at most about 5 years, at most about 4 years, at most about 3 years, at most about 2 years, at most about 1 year, at most about 6 months, at most about 3 months, at most about 2 months, at most about 30 days, at most about 15 days, at most about 10 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, or at most about 1 day when stored at room temperature.
  • the lysis buffer, the recovery buffer, and/or the reagent of the present kit may comprise dry agents.
  • the instruction for use comprises optimal reaction temperatures for sample processing and nucleic acid amplification methods, or optimal buffer conditions for the same.
  • the instructions may be in physical (e.g., printed) or electronic form.
  • the instructions may be in print media.
  • the instructions may be accessible by a user on the Internet, such as through a uniform resource locator.
  • FIG. 1 An exemplary schematic of the Sample Direct process is depicted in FIG. 1.
  • the lysis buffer used in this example included sodium dodecyl sulfate (SDS), egtazic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), and Tris
  • the recovery buffer used included cyclodextrin (containing (2-hydroxypropyl) P-cyclodextrin and (2-hydroxypropyl) y- cyclodextrin) and polysorbate 80.
  • Singleplex reactions amplified target RNA sequence of Ribonuclease P protein subunit p30 (RPP30). Reagents and concentrations for the singleplex reactions of Example 1 are shown in Table 1.
  • BST denotes the polymerase
  • Nt.BstNBI denotes the endonuclease
  • avian myeloblastosis virus reverse transcriptase AMV RT was the reverse transcriptase.
  • bCD denotes beta-cyclodextrin.
  • the concentrations of each component in the lysis buffer can vary. SDS was present in the presence of the sample at a concentration of about 0.01% w/v to 0.4% w/v; EGTA was present in the presence of the sample at a concentration of about 0.1 mM to 3 mM; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 1 mM; TCEP was present in the presence of the sample at a concentration of about 1.0 mM to 4.0 mM; and Tris was present in the presence of the sample at a concentration of about 1.0 mM to 4.5 mM.
  • the concentrations of each component in the recovery buffer can vary.
  • Cyclodextrin was present in the presence of the sample at a concentration of about 6 mM to 11 mM; and polysorbate 80 was present in the presence of the sample at a concentration of about 0.1% v/v to 3.0% v/v.
  • the lysis buffer used can comprise 0.2% SDS, 2 mM EGTA, 0.5 mM EDTA, 1 mM WSGR Docket No. 52459-726.601
  • the recovery buffer used can comprise 2 mM cyclodextrin and 1.5% v/v polysorbate 80 final concentration for each component in the presence of the sample.
  • Results of the amplification are shown in FIG. 2. Both cheek and nose swab samples showed superior amplification compared to that from a control swab. Amplification reactions were conducted using the differential targeted endonuclease cutting technology (DTECT) methods described herein.
  • DTECT differential targeted endonuclease cutting technology
  • Example 2 Triplex Isothermal Amplification of Samples Prepared Using Sample Direct
  • human nasal swabs were prepared using the Sample Direct preparation methods described herein and then triplex isothermal reaction was run to see the effect on the resulting amplification of target products.
  • the lysis buffer used in this example included sodium dodecyl sulfate (SDS), egtazic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2- carboxyethyl)phosphine (TCEP), and Tris
  • the recovery buffer used included cyclodextrin and polysorbate 80.
  • the triplex reaction was conducted on RPP30 (grey cross), Neisseria gonorrhoeae RNA (open circle), and Chlamydia trachomatis RNA (open triangle). Reagents and concentrations for the amplification reactions of Example 2 are shown in Table 2.
  • BST denotes the polymerase
  • Nt.BstNBI denotes the endonuclease
  • avian myeloblastosis virus reverse transcriptase AMV RT was the reverse transcriptase.
  • the 2X lyophilized reagent contained trehalose at 5%, dextran (40K molecular weight) at 5%, and glycine at 2.5%.
  • the 5x Reaction Buffer contained Tris base, Tris-HCl, MgSCU, ISfeSCU, WSGR Docket No. 52459-726.601
  • the Tris base was present at a reaction concentration of about 15-40 mM
  • MgSCh was present at a reaction concentration of about 3-12 mM
  • Na2SO4 was present at a reaction concentration of about 8-20 mM
  • (NEU ⁇ SCU was present at a reaction concentration of about 8-20 mM.
  • the concentrations of each component in the lysis buffer can vary. SDS was present in the presence of the sample at a concentration of about 0.01% w/v to 0.4% w/v; EGTA was present in the presence of the sample at a concentration of about 0.1 mM to 3 mM; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 1 mM; TCEP was present in the presence of the sample at a concentration of about 1.0 mM to 4.0 mM; and Tris was present in the presence of the sample at a concentration of about 1.0 mM to 4.5 mM.
  • the concentrations of each component in the recovery buffer can vary.
  • Cyclodextrin was present in the presence of the sample at a concentration of about 6 mM to 11 mM; and polysorbate 80 was present in the presence of the sample at a concentration of about 0.1% v/v to 3.0% v/v.
  • the lysis buffer used can comprise 0.2% SDS, 2 mM EGTA, 0.5 mM EDTA, 1 mM TCEP, and 1 mM Tris final concentration for each component in the presence of the sample.
  • the recovery buffer used can comprise 2 mM cyclodextrin and 1.5% v/v polysorbate 80 final concentration for each component in the presence of the sample.
  • Example 3 Isothermal Amplification of Culture Samples Using Sample Direct Preparation
  • the lysis buffer used in this example included sodium dodecyl sulfate (SDS), egtazic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), and Tris
  • the recovery buffer used included cyclodextrin and polysorbate 80.
  • the triplex isothermal reaction was run on Neisseria gonorrhoeas, Chlamydia trachomatis, and RPP30. 1000 IFU per reaction for C. trachomatis titration (black lines) and 1000 CFU per reaction for N. gonorrhoeae WSGR Docket No. 52459-726.601
  • Example 3 black crossed lines cultures spiked into human nasal swab.
  • the reagents and experimental setup for Example 3 are the same as those for Example 2.
  • the concentrations of each component in the lysis buffer can vary. SDS was present in the presence of the sample at a concentration of about 0.01% w/v to 0.4% w/v; EGTA was present in the presence of the sample at a concentration of about 0.1 mM to 3 mM; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 1 mM; TCEP was present in the presence of the sample at a concentration of about 1.0 mM to 4.0 mM; and Tris was present in the presence of the sample at a concentration of about 1.0 mM to 4.5 mM.
  • the concentrations of each component in the recovery buffer can vary. Cyclodextrin was present in the presence of the sample at a concentration of about 6 mM to 11 mM; and polysorbate 80 was present in the presence of the sample at a concentration of about 0.1% v/v to 3.0% v/v.
  • the lysis buffer used can comprise 0.2% SDS, 2 mM EGTA, 0.5 mM EDTA, 1 mM TCEP, and 1 mM Tris final concentration for each component in the presence of the sample.
  • the recovery buffer used can comprise 2 mM cyclodextrin and 1.5% v/v polysorbate 80 final concentration for each component in the presence of the sample.
  • FIG. 4A The results of the amplification reaction are shown in FIG. 4A.
  • NTC no template control
  • This experiment used the method of Example 1 and then ran isothermal strand displacement amplification (SDA) to see the effect on the resulting amplification of target products.
  • SDA isothermal strand displacement amplification
  • Attenuated SARS-CoV-2 virus was amplified in an NP matrix (e.g., nasopharyngeal material released from the sample swab) direct amplification reaction, Sample Direct preparation, and triplex isothermal reaction.
  • NP matrix e.g., nasopharyngeal material released from the sample swab
  • the lysis buffer used in this example included sodium dodecyl sulfate (SDS), egtazic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), and Tris, and the recovery buffer used included cyclodextrin and polysorbate 80. Reagents and concentrations for the amplification reaction of Example 4 are shown in Table 4.
  • BST denotes the polymerase
  • Nt.BstNBI denotes the endonuclease
  • avian myeloblastosis virus reverse transcriptase AMV RT was the reverse transcriptase.
  • the 2X lyophilized reagent (2X Lyoph) contained trehalose at 5%, dextran (40K molecular weight) at 5%, and glycine at 2.5%.
  • the 5x Reaction Buffer contained Tris base, Tris-HCl, MgSC , Na2SO4, (NH4)2SO4, and H2O at a total volume of 1000 pl.
  • the Tris base was present at a reaction concentration of about 15-40 mM
  • MgSC was present at a reaction concentration of about 3-12 mM
  • Na2SO4 was present at a reaction concentration of about 8-20 mM
  • (NH4)2SO4 was present at a reaction concentration of about 8-20 mM.
  • the terms “spk” (SARS- CoV-2 Spike), “nsp” (SARS-CoV-2 NSP2), and “rpp” (RPP30) denote amplification targets.
  • concentrations of each component in the lysis buffer can vary.
  • SDS was present in the presence of the sample at a concentration of about 0.01% w/v to 0.4% w/v; EGTA was present in the presence of the sample at a concentration of about 0.1 mM to 3 mM; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 1 mM; TCEP was present in the presence of the sample at a concentration of about 1.0 mM to 4.0 mM; and Tris was present in the presence of the sample at a concentration of about 1.0 mM to 4.5 mM.
  • the concentrations of each component in the recovery buffer can vary.
  • Cyclodextrin was present in the presence of the sample at a concentration of about 6 mM to 11 mM; and polysorbate 80 was present in the presence of the sample at a concentration of about 0.1% v/v to 3.0% v/v.
  • the lysis buffer used can comprise 0.2% SDS, 2 mM EGTA, 0.5 mM EDTA, 1 mM TCEP, and 1 mM Tris final concentration for each component in the presence of the sample.
  • the recovery buffer used can comprise 2 mM cyclodextrin and 1.5% v/v polysorbate 80 final concentration for each component in the presence of the sample.
  • Example 5 Isothermal Amplification of Culture Samples Using Sample Direct Preparation
  • SDS sodium dodecyl sulfate
  • EGTA egtazic acid
  • EDTA ethylenediaminetetraacetic acid
  • TCEP tris(2-carboxyethyl)phosphine
  • Tris Tris
  • the recovery buffer used included cyclodextrin and polysorbate 80.
  • Synthetic Monkeypox DNA and Pan Orthopox DNA was amplified in an NP matrix direct amplification reaction, Sample Direct preparation, and duplex isothermal reaction. Reagents and concentrations for the amplification reaction of Example 5 are shown in Table 6.
  • BST denotes the polymerase
  • Nt.BstNBI denotes the endonuclease
  • avian myeloblastosis virus reverse transcriptase AMV RT was the reverse transcriptase.
  • the 3X lyophilized reagent (3X Lyoph) contained trehalose at 5%, dextran (40K molecular weight) at 5%, and glycine at 2.5%.
  • the 5x Reaction Buffer contained Tris base, Tris-HCl, MgSCh, Na2SO4, (NH4)2SO4, and H2O at a total volume of 1000 pl.
  • the Tris base was present at a reaction concentration of about 15-40 mM
  • MgSCk was present at a reaction concentration of about 3-12 mM
  • Na2SO4 was present at a reaction concentration of about 8-20 mM
  • (NH4)2SO4 was present at a reaction concentration of about 8-20 mM.
  • MPV WSGR Docket No. 52459-726.601
  • the concentrations of each component in the lysis buffer can vary. SDS was present in the presence of the sample at a concentration of about 0.01% w/v to 0.4% w/v; EGTA was present in the presence of the sample at a concentration of about 0.1 mM to 3 mM; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 1 mM; TCEP was present in the presence of the sample at a concentration of about 1.0 mM to 4.0 mM; and Tris was present in the presence of the sample at a concentration of about 1.0 mM to 4.5 mM.
  • BST denotes the polymerase
  • Nt.BstNBI denotes the endonuclease
  • avian myeloblastosis virus reverse transcriptase AMV RT was the reverse transcriptase.
  • MM denotes the total amount of master mix (MM) for the reaction mixture. The results of the amplification are show in FIG. 8. Functional capability was demonstrated with Triplex DTECT RNA assays (wet and lyophilized) and qPCR DNA assay.
  • PAX denotes the sample
  • recovery denotes the recovery buffer as described herein
  • CB denotes the cucurbit[7]urils
  • y-CD denotes gamma-cyclodextrin
  • MM denotes DTECT formulation.
  • the term “No amplif. ” in the last row signifies that there WSGR Docket No. 52459-726.601 was no amplification seed via the probe signal.
  • ACNF refers to Anionic cellulose nanofibrils.
  • CCB was diluted 1 :5 with water and the concentration of y-CD was 266 mM. Water (H2O) was added at a concentration of 55 M.
  • BST denotes the polymerase
  • Nt.BstNBI denotes the endonuclease
  • avian myeloblastosis virus reverse transcriptase AMV RT was the reverse transcriptase.
  • IL1RN, 18S, and MCTP1 were the targets to be amplified.
  • IL1RN fam, MCTP1 hex, and 18S cy5 denote the fhiorophores used for the amplification targets.
  • the excipient contained Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, dithiothreitol (DTT), nonoxynol-9, trehalose, dextran, polysucrose 400, and a cyclodextrin.
  • concentrations of each component in the excipient can vary.
  • Tris was present in the presence of the sample at a concentration of about 0.002 molar (M) to 0.1 M; sodium chloride and/or potassium chloride was present in the presence of the sample at a concentration of about 0.01 M to 0.3 M; DTT was present in the presence of the sample at a concentration of about 0.001 M to 0.02 M; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 0.1 mM; nonoxynol-9 was WSGR Docket No.
  • 52459-726.601 present in the presence of the sample at a concentration of about 0.05% v/v to 0.5% v/v; trehalose was present in the presence of the sample at a concentration of about 0.01 M to 0.5 M; dextran was present in the presence of the sample at a concentration of about 0.5% w/v to 5% w/v; poly sucrose 400 was present in the presence of the sample at a concentration of about 0.08% w/v to 1.0% w/v; and cyclodextrin was present in the presence of the sample at a concentration of about 0.01 M to 0.5 M.
  • the recovery buffer used in this Example included cyclodextrin, EDTA, polysorbate 80, magnesium sulfate (MgSCU), sodium sulfate (NaSCU), ammonium sulfate (NH4SO4), and Tris (Tris 2A:8B).
  • concentrations of the reagents of the recovery buffer can vary.
  • Cyclodextrin was present in recovery buffer at a concentration of about 100 mM to 150 mM; EDTA was present in the recovery buffer at a concentration of about 0.5 mM to 5 mM; polysorbate 80 was present in the recovery buffer at a concentration of about 3% v/v to 6% v/v; MgSO4 was present in the recovery buffer at a concentration of about 40 mM to 70 mM; NaSCU was present in the recovery buffer at a concentration of about 45 mM to 80 mM; NH4SO4 was present in the recovery buffer at a concentration of about 40 mM to 70 mM; and Tris was present in the recovery buffer at a concentration of about 210 mM to 265 mM.
  • FIGs. 9-11 The results of the experiment are shown in FIGs. 9-11.
  • amplification was assessed for different samples between cucurbit[7]urils alone (CCB; shown in column 1 in Table 10), cucurbit[7]urils with gamma-cyclodextrin (CCB+gamma; shown in column 2 in Table 10), and gammacyclodextrin alone (gamma; shown in column 3 in Table 10).
  • Amplification was quantified by cycle threshold, wherein the number of reaction cycles to reach “positive” threshold is the cycle threshold (Ct) value. The lower the Ct value (meaning, the fewer cycles needed to turn a test positive), the greater the amount of genetic material is present in the original sample. All three gene targets were from the human gene sequence.
  • the amplification program was 58°C for 15 minutes with data collection performed approximately every 12 seconds.
  • FIG. 10 shows amplification results using a IL1RN RNA sample.
  • Cucurbit[7]urils alone (CCB) showed improvement in amplification compared to that of gamma-cyclodextrin alone and cucurbit[7]urils with gamma-cyclodextrin.
  • FIG. 12 shows a summary of the three experiments, from left to right: 18s RNA, IL1RN RNA, and MCTP1 RNA.
  • a human RNase P assay within the sexually transmitted infection (STI) tetraplex panel was performed using the range of titrated Tris concentrations and purified human control RNA (commercial source, Life Technologies) was used as template.
  • the amplification program was 58 °C, and the recovery buffer contained components as described herein. The amounts and/or concentrations of all other components was kept constant with the only alteration being the Tris concentration. [00378] Reagents and concentrations for the Tris titration experimental setup of this Example are shown in Table 12.
  • the excipient contained Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, dithiothreitol (DTT), nonoxynol-9, trehalose, dextran, polysucrose 400, and a cyclodextrin.
  • concentrations of each component in the excipient can vary.
  • Tris was present in the presence of the sample at a concentration of about 0.002 molar (M) to 0.1 M; sodium chloride and/or potassium chloride was present in the presence of the sample at a concentration of about 0.01 M to 0.3 M; DTT was present in the presence of the sample at a concentration of about 0.001 M to 0.02 M; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 0.1 mM; nonoxynol-9 was present in the presence of the sample at a concentration of about 0.05% v/v to 0.5% v/v; trehalose was present in the presence of the sample at a concentration of about 0.01 M to 0.5 M; dextran was present in the presence of the sample at a concentration of about 0.5% w/v to 5% w/v; poly sucrose 400 was present in the presence of the sample at a concentration of about 0.08% w/v to 1.0% w/v; and cyclodextrin was present in the presence of
  • the amplification reactions can tolerate high concentrations of Tris and can function at various Tris concentrations.
  • the results show that decreasing Tris concentrations (e.g., final concentration in amplification reaction) resulted in decreasing cycle threshold (Ct) values.
  • Concentrations of 35 mM, 52.5 mM, and 17.5 mM showed the lowest Ct values compared to other tested Tris concentrations.
  • MMLV Murine leukemia virus reverse transcriptase function in DTECT chemistry
  • MMLV RT MMLV reverse transcriptase
  • Purified human control RNA (commercial source, Life Technologies) was used as template and the amplification program was 58 °C.
  • the reaction comprised additional reagents including Isofast® Bst polymerase, dNTP, and Nt.BstNBI endonuclease.
  • concentration of MMLV RT was 0.12 u/pl.
  • Recovery buffer components are as described herein.
  • the excipient contained Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, dithiothreitol (DTT), nonoxynol-9, trehalose, dextran, polysucrose 400, and a cyclodextrin.
  • concentrations of each component in the excipient can vary.
  • Tris was present in the presence of the sample at a concentration of about 0.002 molar (M) to 0.1 M; sodium chloride and/or potassium chloride was present in the presence of the sample at a concentration of about 0.01 M to 0.3 M; DTT was present in the presence of the sample at a concentration of about 0.001 M to 0.02 M; EDTA was present in the presence of the sample at a concentration of about 0.01 mM to 0.1 mM; nonoxynol-9 was present in the presence of the sample at a concentration of about 0.05% v/v to 0.5% v/v; trehalose was present in the presence of the sample at a concentration of about 0.01 M to 0.5 M; dextran was present in the presence of the sample at a concentration of about 0.5% w/v to 5% w/v; poly sucrose 400 was present in the presence of the sample at a concentration of about 0.08% w/v to 1.0% w/v; and cyclodextrin was present in the presence of
  • MMLV functioned with the DTECT chemistry as shown in the assay performance. Across all control RNA dilutions from the stock, MMLV showed similar Ct values demonstrating its effectiveness in the amplification assay and as a component of the DTECT protocol.

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  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des méthodes et des compositions pour le traitement d'une séquence d'acide nucléique cible. Les procédés et les compositions de l'invention comprennent un détergent, un agent de solubilisation et une cyclodextrine conçue pour stabiliser une enzyme et des acides nucléiques. La séquence d'acide nucléique cible traitée peut être en outre mise en contact avec un mélange réactionnel utilisé dans une amplification d'acide nucléique (par exemple, amplification isotherme).<i /> Un échantillon d'acide nucléique cible traité peut être mis en contact avec un tampon de stabilisation conçu pour stabiliser une ou plusieurs enzymes dans une amplification d'acide nucléique. L'échantillon d'acide nucléique cible traité peut en outre être mis en contact avec un tampon d'amplification conçu pour augmenter un taux d'amplification pendant une amplification d'acide nucléique (par exemple, amplification isotherme).
PCT/US2024/044691 2023-09-01 2024-08-30 Procédés et compositions pour le traitement d'échantillons WO2025049920A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130052721A1 (en) * 2010-02-26 2013-02-28 Qiagen Gmbh Method for isolating rna from a rna and dna containing sample
US20170152546A1 (en) * 2012-10-24 2017-06-01 Ge Healthcare Uk Limited Direct Nucleic Acid Amplification Kit, Reagent and Method
WO2019143812A1 (fr) * 2018-01-18 2019-07-25 Biomeme, Inc. Procédés de dosage pour déceler la présence de micro-organismes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130052721A1 (en) * 2010-02-26 2013-02-28 Qiagen Gmbh Method for isolating rna from a rna and dna containing sample
US20170152546A1 (en) * 2012-10-24 2017-06-01 Ge Healthcare Uk Limited Direct Nucleic Acid Amplification Kit, Reagent and Method
WO2019143812A1 (fr) * 2018-01-18 2019-07-25 Biomeme, Inc. Procédés de dosage pour déceler la présence de micro-organismes

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