WO2025207576A1

WO2025207576A1 - Methods and compositions for amplification and analysis of nucleic acids

Info

Publication number: WO2025207576A1
Application number: PCT/US2025/021265
Authority: WO
Inventors: Stephen Judice; Jonathan David HARDINGHAM
Original assignee: Biomeme Inc
Current assignee: Biomeme Inc
Priority date: 2024-03-25
Filing date: 2025-03-25
Publication date: 2025-10-02
Anticipated expiration: 2026-09-25

Abstract

Provided herein are methods, systems, compositions, and kits for analyzing a target nucleic acid sequence. The methods, systems, compositions, and kits provided herein use relative quantitation for determining the abundance of a target nucleic acid molecule. The methods, systems, compositions, and kits for analyzing a target nucleic acid sequence can offer robust target quantification, limited test optimization, and rapid time to result. The methods, systems, compositions, and kits provided herein for analyzing a target nucleic acid molecule can achieve rapid testing by reducing time required from sample input to result and may not require the generation of a standard curve at the time of the test. The methods, systems, compositions, and kits described herein can be used for point-of-care tests.

Description

METHODS AND COMPOSITIONS FOR AMPLIFICATION AND ANALYSIS OF NUCLEIC ACIDS

CROSS- REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/569,579, filed on March 25, 2024, the entire content of which is entirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Nucleic acid amplification techniques such as polymerase chain reaction (PCR) and various isothermal amplification techniques, and the analysis of the amplification products generated thereby, have become an integral part of nucleic acid-based diagnostics and research techniques. Point-of-care tests can produce rapid, reliable results that aid in identification and monitoring of infections or chronic diseases. Real-time amplification of nucleic acid targets associated with infections or chronic diseases still has certain limitations such as low sensitivity and insufficient rapidness for point-of-care tests. Especially, due to the need for temperature cycling, real-time PCR (RT-PCR) may not be adaptable for point-of-care detection. In this regard, isothermal amplifications may be a better option. In either PCR or isothermal amplifications, the real-time amplifications may typically involve using a standard curve or an internal reference gene (or a normalizer, endogenous control or house-keeping gene) to quantify the amplification products, which may complicate the quantification process and make it even less adaptable to point-of-care tests.

SUMMARY OF THE INVENTION

[0003] Recognized herein is a need for improved methods and compositions for analyzing target nucleic acid molecules with robust quantification, less optimization, and rapid time to result. The methods and compositions may not require a standard curve or require the standard curve to be performed together with a test. The methods and compositions for analyzing the target nucleic acid molecules can achieve rapid testing by reducing time required from sample input to the result. The methods and compositions described herein can be used for point-of-care tests.

[0004] In an aspect, the present disclosure provides a method of analyzing a sample having or suspected of having a target nucleic acid molecule, the method comprising: (a) providing a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; (b) subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents, and wherein during the nucleic acid amplification reaction a reaction launch time of the target nucleic acid molecule varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule; (c) detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value; and (d) determining a relative time to response value of the first time to response value and the second time to response value, wherein the relative time to response value is indicative of a presence or absence of the target nucleic acid molecule in the sample.

[0005] In some embodiments, provided herein is a method of analyzing a sample having or suspected of having a target nucleic acid molecule, the method comprising: (a) providing a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; (b) subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents; (c) detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value; and (d) determining a relative time to response value of the first time to response value and the second time to response value, wherein the relative time to response value is indicative of a presence or absence of the target nucleic acid molecule in the sample, wherein the second time to response value of the reference nucleic acid molecule varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule.

[0006] In some embodiments, a method provided herein further comprises, subsequent to (d), determining based on the relative time to response value whether the target nucleic acid is present in the sample. In some embodiments, a method provided herein further comprises, subsequent to (d), determining based on the relative time to response value an amount of the target nucleic acid molecule in the sample.

[0007] In some embodiments, the conditions of a method provided herein comprise a sub-optimal condition for the nucleic acid amplification reaction, and wherein the sub-optimal condition comprises less than 100%, less than 95%, less than 90%, less than 85%, or less than 80% amplification efficiency of the target nucleic acid and/or the reference nucleic acid molecule.

[0008] In some embodiments, the reaction launch time is a time period from subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction to initiation of the nucleic acid amplification by a polymerase of the target nucleic acid molecule. In certain embodiments, reaction launch time is measured by a fluorescence detection method or an electrochemical method. In certain embodiments, the fluorescence detection method comprises molecular beacon. In certain embodiments, the electrochemical method comprises detecting an electrochemical signal of the nucleic acid amplification reaction.

[0009] In some embodiments of a method provided herein comprises changing compositions or concentrations of one or more reagents of the reagents changes the relative time to response value. In certain embodiments, the one or more reagents comprises a salt, a surfactant, a polyol, a polymer, a sugar, a polyamine, or any combinations thereof. In certain embodiments, the salt comprises a carbonate salt, a bicarbonate salt, a sulfate salt, a guanidine salt, a chloride salt, a lithium salt, or any combinations thereof. In certain embodiments, the carbonate salt comprises ammonium carbonate, magnesium carbonate, or any combination thereof. In certain embodiments, the bicarbonate salt comprises sodium bicarbonate. In certain embodiments, the sulfate salt comprises sodium sulfate, magnesium sulfate, ammonium sulfate, or any combination thereof. In certain embodiments, the guanidine salt comprises guanidine hydrochloride, guanidine thiocyanate, guanidine sulfate, guanidine carbonate, or any combination thereof. In certain embodiments, chloride salt comprises sodium chloride, potassium chloride, magnesium chloride, or any combination thereof. In certain embodiments, the lithium salt comprises lithium chloride. In certain embodiments, the surfactant comprises a cationic surfactant, an anionic surfactant, and nonionic surfactant, an amphoteric surfactant, a sulfate, a sulfonate, a carboxylate, a poloxamer, a zwitterionic surfactant, a Gemini surfactant, a polymeric surfactant, a co-block polymer surfactant, or any combination thereof. In certain embodiments, the sugar is a non-reducing sugar. In certain embodiments, the non-reducing sugar is trehalose, sucrose, raffinose, or any combination thereof. In certain embodiments, the polyol is mannitol. In certain embodiments, the polymer is dextran, ficoll, or any combination thereof. In certain embodiments, the polyamine is a linear polyamine, a branched polyamine, or any combination thereof. In certain embodiments, the polyamine is spermine, spermidine, (bis)aminopropylspermidine, Tetrakis(3-aminopropyl)-l,4-butanediamine, or any combination thereof. In some embodiments, a method provided herein further comprises changing compositions or concentrations of the one or more reagents of the reagents. In some embodiments, the target nucleic acid molecule comprises two or more different target nucleic acid molecules in the reaction mixture, and changing the compositions or concentrations of the one or more reagents of the reagents changes a relative time to response value between two or more different target nucleic acid molecules.

[0010] In some embodiments, the method further comprises, prior to (a), processing the sample with a sample processing buffer. In some embodiments, the sample processing buffer comprises a lysis buffer. In some embodiments, the lysis buffer comprises sodium acetate, 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), tetrahydropyran (THP), or any combination thereof.

[0011] In some embodiments, the lysis buffer comprises sodium acetate. In some embodiments, the sodium acetate is present in the lysis buffer mixed with the sample at a final concentration of about 20 mM to 80 mM. In some embodiments, the sodium acetate is present at a pH from about 3 to 6. In some embodiments, the sodium acetate is configured to improve a freeze-thaw stability of the sample in the nucleic acid amplification reaction.

[0012] In some embodiments, the lysis buffer further comprises a chelating agent. In some embodiments, 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). In some embodiments, the lysis buffer further comprises a reducing agent. In some embodiments, the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, a thiosulfate, sodium hydrosulfite, l,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

[0013] In some embodiments, the sample processing buffer comprises a recovery buffer. In some embodiments, the recovery buffer comprises a cyclodextrin, an ethylenediaminetetraacetic acid (EDTA), a solubilizer, a Tris, magnesium sulfate, ammonium sulfate, sodium sulfate, or any combination thereof. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof. In some embodiments, 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.

[0014] In some embodiments, the first time to response value of the target nucleic acid molecule at a concentration of higher than 1 copy/reaction is at least 2-fold less than the first time to response value of the target nucleic acid molecule at a concentration of less than 1 copy/reaction. In some embodiments, the time to response value is a Ct value. In some embodiments, the nucleic acid amplification reaction is a real-time nucleic acid amplification reaction.

[0015] In some embodiments, the nucleic acid amplification reaction is an isothermal amplification. In certain embodiments, the isothermal amplification reaction comprises subjecting the reaction mixture to a constant temperature. In certain embodiments, the reaction mixture of the isothermal amplification reaction further comprises a guide polynucleotide comprising a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and a target binding region configured to hybridize to a target sequence. In certain embodiments, the enzyme exhibits at least two differential enzymatic activity rates. In certain embodiments, the at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites. In certain embodiments, one of the two differential endonuclease activity rates comprises cutting the target sequence of the target nucleic acid molecule with low frequency. In certain embodiments, the cutting at the low frequency is a rate limiting step for determining the reaction launch time.

[0016] In some embodiments, a method provided herein further comprises changing a temperature during the nucleic acid amplification or subsequent to the nucleic acid amplification. In certain embodiments, changing the temperature during the nucleic acid amplification comprises maintaining the temperature at a first temperature for a first period of time and changing the temperature to a second temperature different from the first temperature for a second period of time during the nucleic acid amplification. In certain embodiments, a method provided herein comprises changing the temperature back to the first temperature during the nucleic acid amplification. In certain embodiments, changing the temperature subsequent to the nucleic acid amplification comprises maintaining the temperature at a first temperature for a first period of time until completion of the nucleic acid amplification, and changing the temperature to a second temperature different from the first temperature. In certain embodiments, changing a temperature during the nucleic acid amplification or subsequent to the nucleic acid amplification changes the relative time to response value. In certain embodiments, the target nucleic acid molecule comprises two or more different target nucleic acid molecules in the reaction mixture, and wherein changing a temperature during the nucleic acid amplification or subsequent to the nucleic acid amplification changes a relative time to response value between two or more different target nucleic acid molecules.

[0017] In some embodiments of a method provided herein, the nucleic acid amplification reaction is polymerase chain reaction (PCR). [0018] In some embodiments, a method provided herein further comprises in (d) comparing the relative time to response value with a standard value, wherein the standard value comprises a standard relative time to response value of a time to response value associated with amplifying a known concentration of the target nucleic acid molecule and a time to response value associated with amplifying the reference nucleic acid molecule in a single reaction. In certain embodiments, a method provided herein further comprises generating the standard value. In certain embodiments, the standard value comprises two or more standard relative time to response values, each being a standard relative time to response value of a time to response value associated with amplifying a known concentration of the target nucleic acid molecule and a time to response value associated with amplifying the reference nucleic acid molecule in a single reaction, and wherein the two or more standard relative time to response values are generated using two or more different known concentrations of the target nucleic acid molecule. In certain embodiments, generating the standard value comprises: (i) providing a plurality of reaction mixtures, each comprising a reference nucleic acid molecule at a first concentration and a target nucleic acid molecule at a second concentration, wherein the first concentration is constant across the plurality of reaction mixtures, and wherein the second concentration varies across the plurality of reaction mixtures; (ii) subjecting the plurality of reaction mixtures to the nucleic acid amplification reaction; (iii) detecting signals for the plurality of reaction mixtures to obtain a standard relative time to response value of a time to response value of the target nucleic acid molecule and a time to response value of the reference nucleic acid molecule for each reaction mixture of the plurality of reaction mixtures, thereby generating the standard value. In certain embodiments, the time to response value of the reference nucleic acid molecule varies depending on the second concentration of the target nucleic acid molecule. In certain embodiments, a reaction launch time of the reference nucleic acid molecule varies depending on the second concentration of the target nucleic acid molecule. In certain embodiments, the standard value is not generated concurrently with the relative time to response value. In certain embodiments, the standard value is generated prior to, concurrently with, or subsequent to obtaining the relative time to response value.

[0019] In some embodiments the target nucleic acid molecule comprises two or more different target nucleic acid molecules in the reaction mixture. In certain embodiments, the two or more different target nucleic acid molecules in the reaction mixture is detected by a same signal.

[0020] In certain embodiments, a method provided herein further comprises for each target nucleic acid molecule, calculating a relative time to response value of a first value of the target nucleic acid molecule and a reference value of the reference nucleic acid molecule. In certain embodiments, a method provided herein further comprises for each target nucleic acid molecule, calculating a ratio of a first value of the target nucleic acid molecule and a reference value of the reference nucleic acid molecule to obtain a relative time to response value.

[0021] In some embodiments, provided herein is a method wherein the reference nucleic acid molecule is an additional target nucleic acid molecule different from the target nucleic acid molecule. In certain embodiments, the plurality of reaction mixtures comprises the target nucleic acid at a concentration from about 0.01 to about 100,000 RFU/reaction.

[0022] In certain embodiments, the second concentration comprises a series dilution of the target nucleic acid molecule. In certain embodiments, the plurality of reaction mixtures further comprises the refence nucleic acid molecule at a concentration of about 50 pg/reaction to about 500 pg/ reaction. In certain embodiments, the plurality of reaction mixtures further comprises the refence nucleic acid molecule at a concentration of about 100 pg/reaction to about 400 pg/ reaction. In certain embodiments, the plurality of reaction mixtures further comprises the refence nucleic acid molecule at a concentration of about 200 pg/reaction to about 300 pg/ reaction. In certain embodiments, the plurality of reaction mixtures further comprises the reference nucleic acid molecule at a concentration of about 250 pg/reaction. In certain embodiments, the reference nucleic acid molecule comprises a human deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In certain embodiments, the human DNA or RNA comprises a sequence encoding ribonuclease P/MRP subunit p30 (RPP30).

[0023] In some embodiments of a method provided herein, the target nucleic acid molecule comprises a nucleic acid sequence from a pathogen. In certain embodiments, the target nucleic acid molecule comprises a bacterial DNA or RNA. In certain embodiments, the bacterial DNA or RNA is from a bacterial species associated with an infection. In certain embodiments, the bacterial species is Neisseria gonorrhoeae. In certain embodiments, the bacterial species is Chlamydia trachomatis. In certain embodiments, the target nucleic acid molecule comprises a parasitic DNA or RNA. In certain embodiments, the parasitic DNA or RNA is from a parasitic species associated with an infection. In certain embodiments, the parasitic species is Trichomonas vaginalis. In certain embodiments, the target nucleic acid molecule comprises a viral DNA or RNA. In certain embodiments, the viral DNA or RNA is from a viral species associated with an infection.

[0024] In some embodiments of a method provided herein, the target nucleic acid molecule comprises a mutation associated with a disease or a condition. In certain embodiments, the disease or the condition comprises a cancer. In some embodiments, a method provided herein further comprises obtaining the sample. In some embodiments, the sample is from a human or a nonhuman animal. In some embodiments, the sample comprises a blood sample, a fecal sample, a urine sample, a tissue sample, a vaginal swab, an oral swab, or a rectal swab. [0025] In some embodiments, the human or the non-human animal has, is diagnosed with having, is suspected of having, or is at risk of having an infection of a pathogen comprising the target nucleic acid molecule.

[0026] In some embodiments, provided herein is a method of analyzing a sample having or suspected of having a target nucleic acid molecule, the method comprising: (a) providing a plurality of reaction mixtures comprising a first reaction mixture and a second reaction mixture, wherein the first reaction mixture comprises the target nucleic acid molecule at a first concentration and a reference nucleic acid molecule, and wherein the second reaction mixture comprises the target nucleic acid at a second concentration and the reference nucleic acid molecule, wherein the first concentration and the second concentration are different; (b) subjecting the plurality of reaction mixtures to a nucleic acid amplification reaction; (c) detecting from the first reaction mixture (i) a first signal of the target nucleic acid molecule to obtain a first value, and (ii) a first reference signal of the reference nucleic acid molecule to obtain a first reference value; (d) detecting from the second reaction mixture (i) a second signal of the target nucleic acid molecule to obtain a second value, and (ii) a second reference signal of the reference nucleic acid molecule to obtain a second reference value; (e) determining a first relative value based on the first value and the first reference value and a second relative value based on the second value and the second reference value to obtain a standard value; (f) subjecting the sample to the nucleic acid amplification reaction in the presence of the reference nucleic acid molecule; (g) detecting a signal of the target nucleic acid molecule to obtain a value, and a reference signal of the reference nucleic acid molecule to obtain a reference value, and determining a relative value of the value and the reference value; and (h) using the relative value, the first relative value and the second relative value to determine the presence or absence of the target nucleic acid molecule in the sample. In some embodiments, the target nucleic acid molecule and the reference nucleic acid molecule compete for reaction reagents of the nucleic acid amplification reaction and a reaction launch time of the target nucleic acid varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule. In some embodiments, using the relative value, the first relative value and the second relative value in (g) comprises comparing the relative value to the first relative value and the second relative value.

[0027] In some embodiments, obtaining the standard value in (a)-(d) is not performed concurrently with (e)-(g). In some embodiments, obtaining the standard value in (a)-(d) is performed at least 7 days, 14 days, 21 days, 1 month, 2 months, 5 months, 10 months, 1 year, 2 years or more prior to (e)-(g). In some embodiments, a method provided herein further comprises analyzing a different sample using the same standard value. In some embodiments, the standard value is not generated each time a sample is analyzed.

[0028] In some embodiments, a concentration of the reference nucleic acid molecule is constant in the first reaction mixture and the second reaction mixture. In some embodiments, the plurality of reaction mixtures comprises a third reaction mixture comprising the target nucleic acid molecule at a third concentration different from the first concentration and the second concentration and a reference nucleic acid molecule. In some embodiments, the plurality of reaction mixtures comprises the target nucleic acid molecule at a concentration from about 0.01 to about 100,000 RFU/reaction. In some embodiments, the plurality of reaction mixtures further comprise the refence nucleic acid molecule at a concentration of about 50 pg/reaction to about 500 pg/ reaction. In some embodiments, the plurality of reaction mixtures further comprises the refence nucleic acid molecule at a concentration of about 100 pg/reaction to about 400 pg/ reaction. In some embodiments, the plurality of reaction mixtures further comprises the refence nucleic acid molecule at a concentration of about 200 pg/reaction to about 300 pg/ reaction. In some embodiments, the plurality of reaction mixtures further comprises the reference nucleic acid molecule at a concentration of about 250 pg/reaction.

[0029] In some embodiments, provided herein is a method wherein the reference nucleic acid molecule comprises a human deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In certain embodiments, the human DNA or RNA comprises a sequence encoding ribonuclease P/MRP subunit p30 (RPP30).

[0030] In some embodiments, provided herein is a method wherein the target nucleic acid molecule comprises a nucleic acid sequence from a pathogen. In certain embodiments, the target nucleic acid molecule comprises a bacterial DNA or RNA. In certain embodiments, the bacterial DNA or RNA is from a bacterial species associated with an infection. In certain embodiments, the target nucleic acid molecule comprises a parasitic DNA or RNA. In certain embodiments, the parasitic DNA or RNA is from a parasitic species associated with an infection. In certain embodiments, the parasitic species is Trichomonas vaginalis. In certain embodiments, the target nucleic acid molecule comprises a viral DNA or RNA. In certain embodiments, the viral DNA or RNA is from a viral species associated with an infection.

[0031] In some embodiments, a method provided herein further comprises, prior to (e), obtaining the sample. In some embodiments, the sample is from a human or a non-human animal. In some embodiments, wherein the sample comprises a blood sample, a fecal sample, a urine sample, a tissue sample, a vaginal swab, an oral swab, or a rectal swab. In some embodiments, the human or the non-human animal has, is diagnosed with having, is suspected of having, or is at risk of having an infection of a pathogen comprising the target nucleic acid molecule.

[0032] In some embodiments, provided herein is a method of analyzing a sample having or suspected of having a target nucleic acid molecule, the method comprising: (a) providing a reaction mixture comprising the sample and a reference nucleic acid molecule; (b) subjecting the reaction mixture to a nucleic acid amplification reaction; (c) detecting a first signal of the target nucleic acid molecule to obtain a first time to response value, and a reference signal of the reference nucleic acid molecule to obtain a reference time to response value; and (d) determining a relative time to response value of the first time to response value and the reference time to response value, wherein the relative time to response value indicates the presence or absence of the target nucleic acid molecule in the sample; wherein a duration of analyzing the sample from (a) to (d) is equal to, at most, or about 30 min or less. In certain embodiments, the target nucleic acid molecule and the reference nucleic acid molecule compete for reaction reagents of the nucleic acid amplification reaction and a reaction launch time of the target nucleic acid varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule. In certain embodiments, the duration of the analyzing is equal to, at most, or about 15 min or less, 12 min or less, 10 min or less, 8 min or less, 7 min or less, 6 min or less, or 5 min or less.

[0033] In some embodiments, provided herein is a method wherein the sample is processed to extract genetic materials from the sample prior to subjecting the sample to nucleic acid amplification reaction. In certain embodiments, the sample is processed by a sample extraction device or by heating.

[0034] In some embodiments, provided herein is a method wherein the method does not comprise calibrating a volume of the reaction mixture.

[0035] In some embodiments, provided herein is a kit comprising a reference nucleic acid molecule of a method provided herein, one or more reaction reagents for nucleic acid amplification, and an instruction for user to perform a method provided herein. In certain embodiments, a kit provided herein further comprises the standard value in a method provided herein.

[0036] In some embodiments, provided herein is a system for analyzing a sample having or suspected of having a target nucleic acid molecule, the system comprising: an analytic unit configured to receive a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit, wherein the method comprises: (a) subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents, and wherein during the nucleic acid amplification reaction a reaction launch time of the target nucleic acid molecule varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule; (b) detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value; and (c) determining a relative time to response value of the first time to response value and the second time to response value, wherein the relative time to response value is indicative of a presence or absence of the target nucleic acid molecule in the sample.

[0037] In some embodiments, provided herein is a system of analyzing a sample having or suspected of having a target nucleic acid molecule, the system comprising: an analytic unit configured to receive a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit, wherein the method comprises: (a) subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents; (b) detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value; and (c) determining a relative time to response value of the first time to response value and the second time to response value, wherein the relative time to response value is indicative of a presence or absence of the target nucleic acid molecule in the sample, wherein the second time to response value of the reference nucleic acid molecule varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule.

[0038] In some embodiments, provided herein is a system of analyzing a sample having or suspected of having a target nucleic acid molecule, the system comprising: an analytic unit configured to receive a plurality of reaction mixtures comprising a first reaction mixture and a second reaction mixture, wherein the first reaction mixture comprises the target nucleic acid molecule at a first concentration and a reference nucleic acid molecule, and wherein the second reaction mixture comprises the target nucleic acid at a second concentration and the reference nucleic acid molecule, wherein the first concentration and the second concentration are different; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit, wherein the method comprises: (a) subjecting the plurality of reaction mixtures to a nucleic acid amplification reaction; and (b) detecting from the first reaction mixture (i) a first signal of the target nucleic acid molecule to obtain a first value, and (ii) a first reference signal of the reference nucleic acid molecule to obtain a first reference value; (c) detecting from the second reaction mixture (i) a second signal of the target nucleic acid molecule to obtain a second value, and (ii) a second reference signal of the reference nucleic acid molecule to obtain a second reference value; (d) determining a first relative value based on the first value and the first reference value and a second relative value based on the second value and the second reference value to obtain a standard value; (d) subjecting the sample to the nucleic acid amplification reaction in the presence of the reference nucleic acid molecule; (f) detecting a signal of the target nucleic acid molecule to obtain a value, and a reference signal of the reference nucleic acid molecule to obtain a reference value, and determining a relative value of the value and the reference value; and (g) using the relative value, the first relative value and the second relative value to determine the presence or absence of the target nucleic acid molecule in the sample.

[0039] In some embodiments, provided herein is a system of analyzing a sample having or suspected of having a target nucleic acid molecule, the system comprising: an analytic unit configured to receive a reaction mixture comprising the sample and a reference nucleic acid molecule; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit, wherein the method comprises: (a) subjecting the reaction mixture to a nucleic acid amplification reaction; (b) detecting a first signal of the target nucleic acid molecule to obtain a first time to response value, and a reference signal of the reference nucleic acid molecule to obtain a reference time to response value; and (c) determining a relative time to response value of the first time to response value and the reference time to response value, wherein the relative time to response value indicates the presence or absence of the target nucleic acid molecule in the sample; wherein a duration of analyzing the sample from (a) to (d) is equal to, at most, or about 30 min or less.

[0040] In some aspects, provided herein is 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, wherein the composition is configured to reduce and/or eliminate activity of a degrading nuclease, and wherein the detergent is part of a lysis buffer, and wherein the lysis buffer has a pH of less than 8.0. [0041] In some embodiments, the pH is less than 7.0, less than 6.0, or less than 5.5. In some embodiments, the pH is 5.0 to 6.0. In some embodiments, the pH is 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

[0042] In some embodiments, the lysis buffer comprises sodium acetate. In some embodiments, the sodium acetate has a pH of less than 8.0, less than 7.0, less than 6.0, or less than 5.5, or wherein the sodium acetate has a pH 5.0 to 6.0, or wherein the sodium acetate has pH 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the composition is configured to stabilize nucleic acids during the nucleic acid amplification. In some embodiments, the enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof. In some embodiments, the detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the solubilizer is a non-ionic surfactant. In some embodiments, the solubilizer is a polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol, or a secondary alcohol ethoxylate. In some embodiments, the solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

[0043] In some embodiments, the solubilizer and the cyclodextrin are part of a recovery buffer. In some embodiments, the lysis buffer and the recovery buffer are in a same mixture. In some embodiments, 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 recovery buffer is lyophilized. In some embodiments, the lysis buffer is lyophilized.

[0044] In some embodiments, 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. In some embodiments, the cycle threshold value is at most 40 or the time to result value is at most 15 minutes. In some embodiments, 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. In some embodiments, the degrading nuclease is a ribonuclease.

[0045] In some embodiments, the lysis buffer further comprises a chelating agent. In some embodiments, 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). In some embodiments, the lysis buffer further comprises a reducing agent. In some embodiments, the reducing agent is oxalic acid, formic acid, lithium aluminum hydride, sodium borohydride, a thiosulfate, sodium hydrosulfite, l,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or tetrahydropyran (THP). In some embodiments, the lysis buffer comprises an egtazic acid (EGTA), an ethylenedi aminetetraacetic acid (EDTA), a tris(2-carboxyethyl)phosphine (TCEP), 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), tetrahydropyran (TEIP), or any combination thereof. In some embodiments, the composition further comprises an agent capable of reducing a disulfide bond. In some embodiments, the agent capable of reducing the disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2- mercaptoehtanol (PME).

[0046] In some embodiments, the detergent is present in the composition mixed with a sample at a final concentration that is effective for lysing cells. In some embodiments, 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 cyclodextrin is present in the composition mixed with a sample at a final concentration that is effective for isolating the detergent within the composition. In some embodiments, the final concentration of the cyclodextrin is about 0.1 mM to 70 mM. In some embodiments, the detergent is configured to form a complex with the solubilizer and/or the cyclodextrin to stabilize the enzyme. In some embodiments, the cyclodextrin is configured to increase the efficiency of forming the complex. In some embodiments, the cyclodextrin has a higher binding affinity toward the detergent than a binding affinity of the solubilizer. In some embodiments, 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. In some embodiments, 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.

[0047] In some embodiments, the composition further comprises a sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample comprises a target nucleic acid molecule subject to sample processing. In some embodiments, the composition further comprises a reaction mixture for nucleic acid amplification. In some embodiments, the reaction mixture is lyophilized. In some embodiments, the reaction mixture comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), a primer, or a probe. In some embodiments, the composition is configured to stabilize enzymatic activity of the thermostable enzyme for use during a nucleic acid amplification. In some embodiments, 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. In some embodiments, the dNTPs comprise dATP, dCTP, dGTP, dTTP, or dUTP. In some embodiments, a concentration of the dNTPs in the reaction mixture is about 40 micromolar (pM) to 5000 pM. In some embodiments, the primer is at least 4 nucleotides in length.

INCORPORATION BY REFERENCE

[0048] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0050] FIGs. 1A-1C show the results of triplex isothermal amplification reactions. FIG. 1A depicts the results from a triplex isothermal amplification reaction using Neisseria gonorrhoeae, Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). These data show amplification of C. trachomatis RNA titrations (dark circles) in 250 picograms per reaction of purified human RNA (light circles); the concentration of C. trachomatis RNA was varied in culture fluid, while the concentration of human RNA was held constant. FIG. IB depicts the results from a triplex isothermal amplification reaction using Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of N. gonorrhoeae RNA titrations (dark circles) in 250 picograms per reaction of purified human RNA (light circles); the concentration of A. gonorrhoeae RNA was varied in culture fluid, while the concentration of human RNA was held constant. FIG. 1C depicts the results from a triplex isothermal amplification reaction using Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show co-amplification of N. gonorrhoeae (triangles) and C. trachomatis (squares) RNA titrations in 250 picograms per reaction of purified human RNA (circles); the concentrations of N. gonorrhoeae RNA and C. trachomatis RNA were varied in culture fluid, while the concentration of human RNA was held constant.

[0051] FIGs. 2A-2G show the results of tetraplex isothermal amplification reactions. FIG. 2A depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). These data show amplification of Ml purified T. vaginalis RNA titrations in 250 picograms per reaction of purified human RNA; the concentration of T. vaginalis RNA was varied in culture fluid, while the concentration of human RNA was held constant. FIG. 2B depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of 250 picograms per reaction of purified human RNA in titration series; the concentration of T. vaginalis RNA was varied in culture fluid, while the concentration of human RNA was held constant. FIG. 2C depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of Ml purified T. vaginalis RNA titrations in 250 picograms per reaction of purified human RNA; the concentration of T. vaginalis RNA was varied in culture fluid, while the concentration of human RNA was held constant. FIG. 2D depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of Ml purified T. vaginalis, N. gonorrhoeae, and C. trachomatis RNA in concentrations of 10,000 cells/IFU/CFU per reaction in 250 picograms per reaction of purified human RNA. FIG. 2E depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of Ml purified T. vaginalis, N. gonorrhoeae, and C. trachomatis RNA in concentrations of 1,000 cells/IFU/CFU per reaction in 250 picograms per reaction of purified human RNA. FIG. 2F depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of Ml purified T. vaginalis, N. gonorrhoeae, and C. trachomatis RNA in concentrations of 100 cells/IFU/CFU per reaction in 250 picograms per reaction of purified human RNA. FIG. 2G depicts the results from a tetraplex isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30. These data show amplification of Ml purified T. vaginalis, N. gonorrhoeas, and C. trachomatis RNA in concentrations of 0 cells/IFU/CFU per reaction (no T. vaginalis, N. gonorrhoeas, or C. trachomatis RNA present in the amplification reaction) in 250 picograms per reaction of purified human RNA.

[0052] FIG. 3 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0053] FIG. 4 shows a flow chart of an example method of preparing a sample using a sample preparation device or system of the present disclosure.

[0054] FIGs. 5A-5B show individual value plots depicting number of dropouts (FIG. 5A) and coefficient of variation (FIG. 5B) for two targets using either Tris (Tris SDS) or sodium acetate (NaOAc_SDS) in the lysis buffer.

[0055] FIGs. 6A-6C show individual plots depicting time to detection in minutes. FIG. 6A shows the time to detection for IL1RN across 4 different samples using lysis buffer with either Tris (Tris SDS) or sodium acetate (NaOAc SDS). FIG. 6B shows the time to detection for MCTP1 across 4 different samples using lysis buffer with either Tris (Tris SDS) or sodium acetate (NaOAc SDS). FIG. 6C shows a boxplot summary of the data from FIG. 6A, depicting the time to detection for target IL1RN for all samples between timepoint TO and T2H for lysis buffer with either Tris (Tris SDS) or sodium acetate (NaOAc SDS). TO corresponds to “time zero”, which refers to the sample assayed after being lysed. T2H corresponds to a timepoint at which the sample is lysed, then assayed after 2 hour room temperature (~22°C) incubation.

[0056] FIGs 7A-7B show plots depicting time to detection in minutes for targets IL1RN or MCTP1. Time to detection was measured across four timepoints (TO, T0_F/Txl, T2H, and T2H_F/Txl) using lysis buffer with either Tris (Tris SDS) or sodium acetate (NaOAc SDS). TO corresponds to “time zero”, which refers to the sample assayed after being lysed. T0_F/Txl corresponds to a timepoint at which the sample is frozen (e.g., frozen at -80°C) immediately after lysis, thawed, then assayed. T2H corresponds to a timepoint at which the sample is lysed, then assayed after 2 hour room temperature (~22°C) incubation. T2H_F/Txl corresponds to a timepoint at which the sample is lysed, incubated at room temperature for 2 hours, frozen at -80°C, thawed, then assayed. FIG. 7A shows the data depicted as boxplots. FIG. 7B shows the data depicted as individuals values. The plots depict the time to detection for all patients combined.

DETAILED DESCRIPTION OF THE INVENTION

[0057] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. It is appreciated that although the vial caps are described in the Figures as having a configuration comprising three void filling caps filling three vials in linear arrangement, that such description is merely illustrative as the inventive concepts described herein contemplate various configurations and numbers of void filling caps.

[0058] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0059] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0060] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

Definitions

[0061] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)) (which is entirely incorporated by reference herein).

[0062] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0063] The term “nucleotide,” as used herein, 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)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [aS]dATP, 7- deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. Synthetic nucleotide analogs may include locked nucleic acids (LNAs), bridged nucleic acids (BNAs), fluorinated nucleic acids (also known as fluoromodified nucleic acids), and peptide nucleic acids (PNAs). As used herein, the term “locked nucleic acid” (“LNA”), generally refers to a nucleic acid analog wherein the ribose ring is “locked” with an extra bridge connecting the 2'-oxygen atom with the 4'-carbon atom of the nucleotide such as a methylene bridge (see e.g., WO 99/14226, which is incorporated by reference in its entirety herein). As used herein, the term “bridged nucleic acid (BNA),” generally refers to constrained or inaccessible nucleic acid molecules which have a fixed bridge structure at the 2'- or 4'-position. As used herein, “fluorinated nucleic acids” generally refer to nucleic acids which have incorporated a fluorine atom, often at the 2'- or 4'- position. As used herein, the term “peptide nucleic acid (PNA),” generally refers to a nucleotide analog wherein the backbone of the analog, for example a sugar backbone in DNA, is a pseudopeptide. A PNA backbone can comprise, for example, a sequence of repeated N-(2-amino-ethyl)-glycine units. A peptide nucleic acid analog can react as DNA would react in a given environment and can additionally bind complementary nucleic acid sequences and 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 and in addition, confer specificity and binding to complementary DNA under varying salt conditions. 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.

[0064] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to generally refer to 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. Some non-limiting examples of 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.

[0065] As used herein, the term “restriction endonuclease,” “restriction enzyme,” or grammatical equivalents thereof generally refers to an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. The restriction enzymes can originate in bacterial host defense and they can 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.

[0066] As used herein, the term “restriction endonuclease recognition sequence” generally refers to a location on a nucleic acid molecule (e.g., DNA molecule) containing specific sequences of nucleotides, which are recognized by various restriction enzymes. These sequences can comprise from 4-8 base pairs to 12-40 base pairs in length. These sites can be palindromic sequences.

[0067] As used herein, the term “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 can use a short, preexisting RNA or DNA strand, called a primer, to begin chain growth; and some DNA polymerases can utilize any free 3’ hydroxyl in a DNA duplex for extension. 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.

[0068] As used herein, the term “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 (Bst polymerase), exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, and OmniTaq 2 LA DNA polymerase.

[0069] As used herein, the terms “amplify,” “amplifies,” “amplified,” “amplification,” and “amplicon” 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. In some cases, 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.

[0070] The terms “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. [0071] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, generally refer to a sequence that is fully complementary to and hybridizable to the given sequence. In some cases, 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.

Overview

[0072] The present disclosure provides compositions, methods, systems, and kits for analyzing samples (e.g., biological samples). The samples can comprise target nucleic acid molecules. The target nucleic acid molecules can be amplified in the presence of reference nucleic acid molecules (e.g., a house keeping genes or a different target nucleic acid molecule) using various nucleic acid amplification methods. Relative quantitation, in the form of time to result value or Ct/Cq value, can be determined as readout for the target nucleic acid molecules. For example, the relative quantitation can be a relative value of the time to result value of the target amplification compared to the time to result value of the reference amplification. With the relative quantitation, one can quickly determine the abundance of a target nucleic acid molecule in a sample. The relative quantitation value can change depending on the abundance of the target nucleic acid molecule. Such methods or related compositions, systems, and kits, can be used in point-of-care tests since they can offer rapid testing, higher efficiency than exiting methods, less optimization for users, and/or, in some cases, less reactions for users (e.g., a single reaction). For example, the relative quantitation value can be used to quickly determine if a sample has a target pathogen nucleic acid or not.

[0073] The amplification methods described here can be isothermal amplifications. In such cases, a target nucleic acid molecule can compete with the reference nucleic acid molecule or a different nucleic acid molecule (e.g., or two or more different nucleic acid molecules) for reagents in the same reaction mixture. The amplification methods described herein can be polymerase chain reactions (PCRs). In such cases, it may be beneficial to deoptimize the PCR reactions such that the PCR efficiency is not optimal or less than 90%. [0074] The compositions, methods, systems, and kits for analyzing samples may not require well volume calibration, the need for standard curve, or the need for separate endogenous controls for comparison or quantitation.

Methods of Analyzing Samples

[0075] The present disclosure provides methods of analyzing samples. In some embodiments, a sample has or is suspected of having a target nucleic acid molecule. In some embodiments, the method comprises providing a reaction mixture. The reaction mixture may comprise a reference nucleic acid. The reaction mixture may comprise reagents (e.g., dNTPs, primers, guide molecules, salt (Mg, Na, etc.), and/or pH buffer) for performing a nucleic acid amplification reaction. In some embodiments of a method provided herein, a reaction mixture is subjected to conditions sufficient to perform a nucleic acid amplification reaction (e.g., according to a method disclosed herein). In some embodiments, a method of analyzing a sample does not comprise calibrating a volume of a reaction mixture.

[0076] In some embodiments, the nucleic acid amplification has a reaction launch time. The reaction launch time may be a time period from subjecting a reaction mixture to conditions sufficient to perform nucleic acid amplification reaction to initiation of the nucleic acid amplification reaction by a polymerase of a target nucleic acid molecule. The reaction launch time may be measured by a fluorescence detection method. A fluorescence detection method may comprise a molecular beacon. The reaction launch time may be measured by an electrochemical method. An electrochemical method may comprise detecting an electrochemical signal of the nucleic acid amplification reaction. The reaction launch time may vary depending on the abundance of a target nucleic acid molecule in the amplification reaction relative to a reference nucleic acid molecule (e.g., relative to an abundance of the reference nucleic acid molecule). The reaction launch time of a target nucleic acid molecule may vary depending on the abundance (e.g., concentration, molarity, or concentration relative to abundance of reaction reagents) of the target nucleic acid molecule relative to a reference nucleic acid molecule (e.g., an abundance, concentration, or molarity of the reference nucleic acid molecule). A reaction launch time may depend on a rate limiting step. A rate limiting step may be a cutting step (e.g., a cutting at a low frequency).

[0077] In some embodiments, the nucleic acid amplification reaction has a reaction end time. The amplification reaction can be stopped at an arbitrary time point. In certain embodiments, the end of the amplification reaction is an arbitrary time point. For example, the amplificant reaction can be ended after about 30 minutes from the start of the reaction. The amplification reaction can be ended arbitrarily at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 17 minutes, at least about 20 minutes, at least about 22 minutes, at least about 25 minutes, at least about 27 minutes, or at least about 30 minutes from the start of the reaction. In some embodiments, the amplification reaction can be ended arbitrarily at most about 30 minutes, of at most about 27 minutes, of at most about 25 minutes, of at most about 22 minutes, of at most about 20 minutes, of at most about 17 minutes, of at most about 15 minutes, at most about 12 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6.5 minutes, at most about 6 minutes, at most about 5.5 minutes, at most about 5 minutes, at most about 4.5 minutes, at most about 4 minutes, at most about 3.5 minutes, at most about 3 minutes, at most about 2.5 minutes, at most about 2 minutes, at most about 1.5 minutes, at most about 1 minute, at most about 30 seconds, or at most about 15 seconds from the start of the reaction.

[0078] In some embodiments of a method provided herein, a first signal may be detected. In some embodiments, a first and a second signal are detected. The first signal may be a signal of a target nucleic acid molecule. The signal of the first target nucleic acid molecule may be detected to obtain a first time to response. In some embodiments, a second signal may be a signal of a reference nucleic acid molecule. The second signal may be detected to obtain a second time to response. The signal may be a fluorescent, chemiluminescent, or colorimetric signal. The signal may be impedance-, conductance-, or voltage-based. The first time to response may be indicative of the presence of a target nucleic acid in a sample. The first time to response may be indicative of the absence of a target nucleic acid in a sample. The second time to response may be indicative of the presence of a target nucleic acid in a sample. The second time to response may be indicative of the absence of a target nucleic acid in a sample. The first time to response may be indicative of the quantity of a target nucleic acid in a sample. The second time to response may be indicative of the quantity of a target nucleic acid in a sample. The first time to response value of the target nucleic acid molecule may vary depending on the abundance of the target nucleic acid molecule relative to the abundance reference nucleic acid molecule in the reaction. The second time to response value of the reference nucleic acid molecule may vary depending on the abundance of the target nucleic acid molecule relative to the abundance reference nucleic acid molecule in the reaction. The first time to response value of the target nucleic acid molecule may be substantially independent of the abundance of the target nucleic acid molecule relative to the abundance reference nucleic acid molecule in the reaction. The second time to response value of the reference nucleic acid molecule may be substantially independent of the abundance of the target nucleic acid molecule relative to the abundance reference nucleic acid molecule in the reaction. The first time to response value of the target nucleic acid and the second time to response value of the reference nucleic acid may be affected by competition for reagents in a nucleic acid amplification reaction. [0079] In some embodiments, a time to response value is a cycle threshold (Ct or Cq). In some embodiments, a time to response is a time to result. For isothermal amplification, the time to response value can be a time to result value, and it may be referred to as a Ct value in some cases, and in such cases, the time to response value may be the shortest time (e.g., from the start of an amplification reaction) in which an instrument is capable of capturing a reading of a state of the reaction. In some embodiments, a time to response value is a time to the maximum value of the second derivative of the readout of the instrument (Cp). For isothermal amplification, the time to response value can be a time to result value, and it may be referred to as a Cp value in some cases, and in such cases, the time to response value may be the shortest time (e.g., from the start of an amplification reaction) in which an instrument is capable of capturing a metric of a reading of a state of the reaction, for example, the time to the maximum of the second derivative of the reading. The reading may be based on fluorescent, chemiluminescent, or colorimetric readouts from the reaction. The reading may be impedance-, conductance-, or voltage-based. In some embodiments, for PCRs, a Ct is a doubling of the abundance of the target nucleic acid.

[0080] In some embodiments, a reaction launch rate of the target nucleic acid molecule can vary depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule. A reaction launch rate can be referred to as copies of products per minute.

[0081] In some embodiments, a relative time to response value is determined. The relative time to response value may comprise a first time to response value and a second time to response value. The relative time to response value may comprise a first time to response value relative to a second time to response value. The relative time to response value may be indicative of a presence of a target nucleic acid in a sample. The relative time to response value may be indicative of an absence of a target nucleic acid in a sample. The relative time to response value may be indicative of a quantity of a target nucleic acid in a sample.

[0082] The method of analyzing a sample having or suspected of having a target nucleic acid molecule can comprise providing a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction. The method can further comprise subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction. The nucleic acid amplification reaction can be performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents. During the nucleic acid amplification reaction, a reaction launch time of the target nucleic acid molecule can vary depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule. In some cases, a reaction launch rate of the target nucleic acid molecule can vary depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule. The reaction launch rate can be defined as the copies of products (e.g., the products that can be extended in a nucleic acid amplification reaction) generated in a unit time (e.g., minute). For example, if the nucleic acid amplification is an isothermal amplification provided herein, the products generated during the reaction launch period can be the targets after cutting by a restriction enzyme that have an extendable 3 ’ end. The method can further comprise detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value. The method can further comprise determining a relative time to response value of the first time to response value and the second time to response value. The relative time to response value can be indicative of a presence or absence of the target nucleic acid molecule in the sample.

[0083] The method of analyzing a sample having or suspected of having a target nucleic acid molecule can comprise providing a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction. The method can further comprise subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction. The nucleic acid amplification reaction can be performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents. The method can further comprise detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value. The method can further comprise determining a relative time to response value of the first time to response value and the second time to response value. The relative time to response value can be indicative of a presence or absence of the target nucleic acid molecule in the sample. The second time to response value of the reference nucleic acid molecule can vary depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule.

[0084] In some embodiments, a first time to response value of a target nucleic acid molecule whose concentration is greater than 1 copy of the target nucleic acid per reaction is at least 2-fold less than a first time to response value of the target nucleic acid molecule if its concentration is less than 1 copy/reaction. In some cases, the target nucleic acid molecule is from a bacterium. The concentration of the bacterium in the reaction can be at least 0.01 colony -forming unit (CFU)/ reaction. The concentration of the bacterium in the reaction can be at least 0.1 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 1 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 10 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 100 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 10 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 100 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 1,000 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 10,000 CFU/ reaction. The concentration of the bacterium in the reaction can be at least 100,000 CFU/ reaction. In some cases, the target nucleic acid molecule is from a virus. The concentration of the virus in the reaction can be at least 0.01 infectious unit (IFU)/reaction. The concentration of the virus in the reaction can be at least 0.1 IFU/reaction. The concentration of the virus in the reaction can be at least 1 IFU/reaction. The concentration of the virus in the reaction can be at least 10 IFU/reaction. The concentration of the virus in the reaction can be at least 100 IFU/reaction. The concentration of the virus in the reaction can be at least 10 IFU/reaction. The concentration of the virus in the reaction can be at least 100 IFU/reaction. The concentration of the virus in the reaction can be at least 1,000 IFU/reaction. The concentration of the virus in the reaction can be at least 10,000 IFU/reaction. The concentration of the virus in the reaction can be at least 100,000 IFU/reaction. In some cases, the target nucleic acid molecule is isolated from a bacterium or a virus. The concentration of the isolated nucleic acid (e.g., the target nucleic acid) in the reaction can be 1 copy/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 2 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 5 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 10 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 50 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 100 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 20 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 200 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 1,000 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 10,000 copies/reaction. The concentration of the isolated nucleic acid in the reaction can be at least 100,000 copies/reaction. [0085] In some embodiment of a method disclosed herein, a nucleic acid amplification is performed under conditions such that a target nucleic acid molecule and a reference nucleic acid molecule compete for reagents. The conditions under which the nucleic acid amplification is performed may be suboptimal conditions for the nucleic acid amplification reaction. The suboptimal conditions may comprise less than 100% amplification efficiency of a target nucleic acid. The efficiency of an amplification (usually for PCRs) can be defined as the fraction of target molecules that are copied in one amplification cycle. For example, a properly designed assay shall, in the absence of interfering substances in the sample matrix, amplify target DNA with at least 90% efficiency. For isothermal amplifications, the time required to accumulate an arbitrary but fixed threshold number of copies of a target gene in a reaction vessel can be measured. Under saturating binding conditions, the time required to reach the threshold number can depend on (a) the number of copies present when the reaction is initiated, and (b) the rate of increase per unit time (which is referred to as reaction efficiency or amplification efficiency).

[0086] In some embodiments of a method disclosed herein, a target molecule and a reference molecule compete for one or more reagents. In some cases the reagent can be a sample preparation reagent or from sample preparation. In some cases, the reagent can be a reaction reagent or from an amplification reaction. Changing the compositions or concentrations of one or more of the reaction reagents in a sample can change the relative time to response value. For example, in some cases, a concentration of a salt in the final amplification reaction can be changed such that the relative time to response value is affected. In certain embodiments, the concentration of a salt, a surfactant, a polyol, a polymer, a sugar, a polyamine, or any combinations thereof can be changed such that the relative time to response value is affected. In some cases, the concentration of a polyol can be changed. In some embodiments, changing the composition or concentration of one or more reaction reagents can increase the relative time to response value. In some embodiments, changing the composition or concentration of one or more reaction reagents can decrease the relative time to response value.

[0087] Reagents of systems and methods provided herein can comprise a salt, a surfactant, a polyol, a polymer, a sugar, a polyamine, or any combination thereof. In some embodiments, a reagent comprises a salt. A salt can comprises a carbonate salt, a bicarbonate salt, a sulfate salt, a guanidine salt, a chloride salt, a lithium salt, or any combinations thereof. In certain embodiments, a carbonate salt comprises ammonium carbonate, magnesium carbonate, or any combination thereof. In certain embodiments, a bicarbonate salt comprises sodium bicarbonate. In certain embodiments, a sulfate salt comprises sodium sulfate, magnesium sulfate, ammonium sulfate, or any combination thereof. In certain embodiments, a guanidine salt comprises guanidine hydrochloride, guanidine thiocyanate, guanidine sulfate, guanidine carbonate, or any combination thereof. In certain embodiments, a chloride salt comprises sodium chloride, potassium chloride, magnesium chloride, or any combination thereof. In certain embodiments, a lithium salt comprises lithium chloride. In some embodiments, a reagent comprises a surfactant. A surfactant can comprise a cationic surfactant, an anionic surfactant, and nonionic surfactant, an amphoteric surfactant, a sulfate, a sulfonate, a carboxylate, a poloxamer, a zwitterionic surfactant, a Gemini surfactant, a polymeric surfactant, a co-block polymer, or any combination thereof. In certain embodiments, a reagent comprises a sugar. A sugar can be a non-reducing sugar. Examples of nonreducing sugars include, but are not limited to trehalose, sucrose, raffinose, genianose, verbascose, or any combination thereof. In certain embodiments, a reagent comprises a polyol. Examples of polyols include, but are not limited to mannitol, erythritol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol or any combination thereof. In certain embodiments, a reagent comprises a polymer. In certain specific embodiments, the polymer is dextran, ficoll, chitosan, alginate, or combinations thereof. A reagent in a nucleic acid amplification reaction provided herein can be a polyamine. In certain embodiments, a polyamine is linear. In certain embodiments, a polyamine is branched. Examples of polyamines include but are not limited to spermine, spermidine, (bis)aminopropylspermidine, Tetrakis(3-aminopropyl)-l,4-butanediamine, or any combination thereof.

[0088] In some cases, the conditions under which a PCR is performed may be suboptimal conditions for the PCR reaction. The suboptimal condition may comprise less than 98% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 96% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 95% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 94% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 92% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 90% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 88% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 86% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 85% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 84% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 82% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 80% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 75% amplification efficiency of a target nucleic acid. The suboptimal condition may comprise less than 70% amplification efficiency of a target nucleic acid. The suboptimal conditions may comprise less than 100% amplification efficiency of a reference nucleic acid (e.g., the number of molecules of the reference nucleic acid less than doubles each cycle). The suboptimal condition may comprise less than 98% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 96% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 95% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 94% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 92% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 90% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 88% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 86% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 85% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 84% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 82% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 80% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 75% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 70% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 84% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 82% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 80% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 77% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 75% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 72% amplification efficiency of a reference nucleic acid. The suboptimal condition may comprise less than 70% amplification efficiency of a reference nucleic acid.

[0089] The nucleic acid amplification reaction can be a real-time nucleic acid amplification reaction. The nucleic acid amplification reaction can be an isothermal amplification. The isothermal amplification reaction can comprise subjecting the reaction mixture to a constant temperature. The reaction mixture of the isothermal amplification reaction can further comprise a guide polynucleotide comprising a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and a target binding region configured to hybridize to a target sequence. The enzyme can exhibit at least two differential enzymatic activity rates. The at least two differential enzymatic activity rates can comprise two differential endonuclease activity rates when cutting two different cutting sites. One of the two differential endonuclease activity rates can comprise cutting the target sequence of the target nucleic acid molecule with low frequency. The cutting at the low frequency can be a rate limiting step for determining the reaction launch time. The nucleic acid amplification reaction can be polymerase chain reaction (PCR).

[0090] In some embodiments, a method of analyzing a sample disclosed herein comprises comparing a relative time to response value (e.g., a Ct value) with a standard value. A standard value may comprise a standard relative time to response value (e.g., a standard Ct value). A standard time to response value may be a time to response value associated (1) with amplifying a known concentration of a target nucleic acid molecule and with (2) a time to response value associated with amplifying a reference nucleic acid molecule in a single reaction (e.g., in the same reaction). A method disclosed herein may comprise a standard value. A standard value may comprise two or more standard relative time to response values. In some embodiments, two or more standard relative response time values may be generated by using two or more different known concentrations of a target nucleic acid molecule. Generating a standard value may comprise providing a plurality of reaction mixtures, each comprising a reference nucleic acid molecule at a first concentration and a target nucleic acid molecule at a second concentration, wherein the first concentration is constant across the plurality of reaction mixtures, and wherein the second concentration varies across the plurality of reaction mixtures. Generating a standard value may comprise subjecting a plurality of reaction mixtures to the nucleic acid amplification reaction. Generating a standard value may comprise subjecting a plurality of reaction mixtures to the nucleic acid amplification reaction. Generating a standard value may comprise detecting signals for a plurality of reaction mixtures to obtain a standard relative time to response value of a time to response value of a target nucleic acid molecule and a time to response value of a reference nucleic acid molecule for each reaction mixture of a plurality of reaction mixtures, thereby generating the standard value. Generating a standard value may comprise (i) providing a plurality of reaction mixtures, each comprising a reference nucleic acid molecule at a first concentration and a target nucleic acid molecule at a second concentration, where the first concentration is constant across the plurality of reaction mixtures, and where the second concentration varies across the plurality of reaction mixtures. Generating a standard value may further comprise (ii) subjecting a plurality of reaction mixtures to the nucleic acid amplification reaction. Generating a standard value may further comprise (iii) detecting signals for a plurality of reaction mixtures to obtain a standard relative time to response value of a time to response value of a target nucleic acid molecule and a time to response value of a reference nucleic acid molecule for each reaction mixture of a plurality of reaction mixtures, thereby generating the standard value. In some embodiments, a time to response value (e.g., a time to response value used to generate a standard value) of a reference nucleic acid molecule varies depending on a second concentration of a target nucleic acid molecule. In some embodiments, a standard value is generated prior to obtaining a relative time to response value. In some embodiments, a standard value is generated concurrently with obtaining a relative time to response value. In some embodiments, a standard value is generated subsequent to obtaining a relative time to response value. In some embodiments, a standard value may not be generated. For example, the standard curve may not be generated concurrently with the relative time to response value.

[0091] The methods described herein can be used to analyze or quantify multiple different targets. In some embodiments, a target nucleic acid molecule comprises two or more different target nucleic acid molecules in a reaction mixture. In some embodiments, a target nucleic acid molecule comprises three or more different target nucleic acid molecules in a reaction mixture. In some embodiments, a target nucleic acid molecule comprises four or more different target nucleic acid molecules in a reaction mixture. In some embodiments, a target nucleic acid molecule comprises five or more different target nucleic acid molecules in a reaction mixture. In some embodiments, a target nucleic acid molecule comprises seven or more different target nucleic acid molecules in a reaction mixture. In some embodiments, a target nucleic acid molecule comprises ten or more different target nucleic acid molecules in a reaction mixture. In some embodiments, a target nucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more different target nucleic acid molecules in a reaction mixture (e.g., a single reaction mixture).

[0092] In some embodiments, a method disclosed herein comprises calculating a relative time to response value of a first value of a target nucleic acid molecule and a reference value of a reference nucleic acid molecule for each target nucleic acid molecule in a reaction mixture (e.g., each of the two or more different target nucleic acid molecules in a reaction mixture). A ratio of a first value of a target nucleic acid molecule and a reference value of a reference nucleic acid molecule may be calculated to obtain a relative time to response value for each target nucleic acid molecule in a reaction mixture.

[0093] A method disclosed herein may comprise a plurality of reaction mixtures. The plurality of reaction mixtures may comprise a target nucleic acid at a concentration from about 0.01 to about 100,000 relative fluorescence unit (RFU)/reaction. The plurality of reaction mixtures may comprise a target nucleic acid at a concentration of at least about 0.01, at least about 0.1, at least about 1, at least about 10, at least about 20, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 1,000, at least about 10,000, or at least about 100,000 RFU/reaction. The plurality of reaction mixtures may comprise a target nucleic acid at a concentration from about 1 to about IxlO¹² copies/ml of sample. The plurality of reaction mixtures may comprise a target nucleic acid at a concentration of at least about 1, at least about 5, at least about 10, at least about 50, at least about 100, at least about 250, at least about 500, at least about 750, at least about 1,000, at least about 2,500, at least about 5,000, at least about 7,500, at least about 10,000, at least about 25,000, at least about 50,000, at least about 75,000, at least about 100,000, at least about 250,000, at least about 500,000, at least about 750,000, at least about 1,000,000, at least about 2,500,000, at least about 5,000,000, at least about 7,500,000, at least about 10,000,000, at least about 25,000,000, at least about 50,000,000, at least about 75,000,000, at least about 100,000,000, at least about 250,000,000, at least about 500,000,000, at least about 750,000,000, at least about 1,000,000,000, at least about 2,500,000,000, at least about 5,000,000,000, at least about, at least about 7,500,000,000, at least about 10,000,000,000, at least about 2,500,000,000, at least about 50,000,000,000, at least about 75,000,000,000, at least about 100,000,000,000, at least about 250,000,000,000, at least about 500,000,000,000, at least about 750,000,000,000, or at least about 1,000,000,000,000 copies of the target nucleic acid/ ml of sample. The concentration of a target nucleic acid molecule (e.g., a second concentration) may comprise a series dilution of the target nucleic acid molecule. The plurality of reaction mixtures may comprise a target nucleic acid at a concentration of at least about 0.01, at least about 0.1, at least about 0.5, at least about 1, 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 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 1,000, at least about 10,000, or more picogram (pg)/reaction.

[0094] The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 1 pg/ reaction to about 10,000 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of at least about 0.01, at least about 0.1, at least about 0.5, at least about 1, 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 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 1,000, at least about 10,000, or more pg/reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 10 pg/ reaction to about 1,000 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 50 pg/ reaction to about 750 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 75 pg/ reaction to about 700 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 80 pg/ reaction to about 600 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 90 pg/ reaction to about 500 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 100 pg/ reaction to about 400 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 125 pg/ reaction to about 375 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 150 pg/ reaction to about 350 pg/ reaction. The plurality of reaction mixtures may comprise a reference nucleic acid molecule at a concentration of about 175 pg/ reaction to about 325 pg/ reaction. The plurality of reaction mixture may comprise the reference nucleic acid molecule at a concentration of about 200 pg/ reaction to about 300 pg/ reaction. The plurality of reaction mixture may comprise the reference nucleic acid molecule at a concentration of about 225 pg/ reaction to about 275 pg/ reaction. The plurality of reaction mixture may comprise the reference nucleic acid molecule at a concentration of about 230 pg/ reaction to about 270 pg/ reaction. The plurality of reaction mixture may comprise the reference nucleic acid molecule at a concentration of about 240 pg/ reaction to about 260 pg/ reaction. The plurality of reaction mixtures may comprise the reference nucleic acid molecule at a concentration of about 250 pg/ reaction.

[0095] In some embodiments, a method of analyzing a sample disclosed herein comprises (a) providing a reaction mixture comprising the sample and a reference nucleic acid molecule; (b) subjecting the reaction mixture to a nucleic acid amplification reaction; (c) detecting a first signal of the target nucleic acid molecule to obtain a first time to response value, and a reference signal of the reference nucleic acid molecule to obtain a reference time to response value; and (d) determining a relative time to response value of the first time to response value and the reference time to response value, wherein the relative time to response value indicates the presence or absence of the target nucleic acid molecule in the sample. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 30 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 25 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 20 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 17 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 15 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 12 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 10 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 9 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 8 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 7 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 6 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 5 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 4 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 3 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 2 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 1 minutes or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 30 seconds or less. In some embodiments, the duration of analyzing the sample from (a) to (d) is equal to, at most, or about 15 seconds or less.

[0096] In some embodiments of a method disclosed herein, the method comprises determining a relative time to response value, and determining, based on the relative time to response value, whether a target nucleic acid is present in a sample. Based on the relative time to response value, it may be determined that the target nucleic acid molecule is present in the sample. Based on the relative time to response value, it may be determined that the target nucleic acid molecule is not present in the sample. In some embodiments of a method disclosed herein, the method comprises determining a relative time to response value, and determining, based on the relative time to response value, the amount (e.g., quantity, molarity, weight, abundance, or concentration) a target nucleic acid is present in a sample.

[0097] In some embodiments, a method of analyzing a sample disclosed herein comprises (a) providing a plurality of reaction mixtures comprising a first reaction mixture and a second reaction mixture, wherein the first reaction mixture comprises the target nucleic acid molecule at a first concentration and a reference nucleic acid molecule, and wherein the second reaction mixture comprises the target nucleic acid at a second concentration and the reference nucleic acid molecule, wherein the first concentration and the second concentration are different; (b) subjecting the plurality of reaction mixtures to a nucleic acid amplification reaction; (c) detecting from the first reaction mixture (i) a first signal of the target nucleic acid molecule to obtain a first value, and (ii) a first reference signal of the reference nucleic acid molecule to obtain a first reference value; (d) detecting from the second reaction mixture (i) a second signal of the target nucleic acid molecule to obtain a second value, and (ii) a second reference signal of the reference nucleic acid molecule to obtain a second reference value; (e) determining a first relative value based on the first value and the first reference value and a second relative value based on the second value and the second reference value to obtain a standard value; (f) subjecting the sample to the nucleic acid amplification reaction in the presence of the reference nucleic acid molecule; (g) detecting a signal of the target nucleic acid molecule to obtain a value, and a reference signal of the reference nucleic acid molecule to obtain a reference value, and determining a relative value of the value and the reference value; and (h) using the relative value, the first relative value and the second relative value to determine the presence or absence of the target nucleic acid molecule in the sample. In some embodiments, obtaining a standard value in (a)-(d) is not performed concurrently with (e)- (g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 7 days or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 14 days or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 21 days or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 1 month or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 2 months or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 5 months or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 10 months or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 1 year or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 2 years or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 3 years or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 5 years or more prior to (e)-(g). In some embodiments, obtaining a standard value in (a)-(d) is performed at least 10 years or more prior to (e)-(g). The plurality of reaction mixtures may comprise a third reaction mixture comprising the target nucleic acid molecule at a third concentration different from the first concentration and the second concentration and a reference nucleic acid molecule.

[0098] In some embodiments of a method disclosed herein, a different sample is analyzed using the same standard value. In some embodiments of a method disclosed herein, one or more samples are analyzed using the same standard value. In some embodiments, a standard value is not generated each time a sample is analyzed.

[0099] In some embodiments, a concentration of a reference nucleic acid molecule is constant in a first reaction mixture and a second reaction mixture (e.g., the concentration of a reference nucleic acid molecule is substantially the same in a first reaction mixture and a second reaction mixture). [0100] In some embodiments, a target nucleic acid molecule and a reference nucleic acid molecule compete for reaction reagents of a nucleic acid amplification reaction. A reaction launch time of a target nucleic acid may vary depending on an abundance (e.g., quantity, count, concentration, weight, or molarity) of a target nucleic acid molecule relative to a reference nucleic acid molecule (e.g., an abundance of a reference nucleic acid molecule).

[0101] In some embodiments of a method disclosed herein, using a relative value, a first relative value, and a second relative value comprises comparing a relative value to a first relative value and a second relative value (e.g., where the relative value, the first relative value, and the second relative value are described in (g)).

[0102] The present disclosure also provides corresponding systems for implementing the methods described herein. For example, the system can comprise an analytic unit for nucleic acid amplification. Detailed disclosures of such systems have been described in U.S. Patent No. 10,457,983 and U.S. Application No. 16/899,810, each of which is incorporated herein by reference in its entirety. In some cases, a system for analyzing a sample having or suspected of having a target nucleic acid molecule can comprise: an analytic unit configured to receive a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit. The method can comprise (a) subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents, and wherein during the nucleic acid amplification reaction a reaction launch time of the target nucleic acid molecule varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule; (b) detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value; and (c) determining a relative time to response value of the first time to response value and the second time to response value, wherein the relative time to response value is indicative of a presence or absence of the target nucleic acid molecule in the sample.

[0103] In some cases, a system of analyzing a sample having or suspected of having a target nucleic acid molecule can comprise an analytic unit configured to receive a reaction mixture comprising the sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit. The method can comprise (a) subjecting the reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, wherein the nucleic acid amplification reaction is performed under conditions such that the target nucleic acid molecule and the reference nucleic acid molecule compete for the reagents; (b) detecting (1) a first signal of the target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of the reference nucleic acid molecule to obtain a second time to response value; and (c) determining a relative time to response value of the first time to response value and the second time to response value, wherein the relative time to response value is indicative of a presence or absence of the target nucleic acid molecule in the sample, wherein the second time to response value of the reference nucleic acid molecule varies depending on an abundance of the target nucleic acid molecule relative to the reference nucleic acid molecule.

[0104] In some cases, a system of analyzing a sample having or suspected of having a target nucleic acid molecule can comprise: an analytic unit configured to receive a plurality of reaction mixtures comprising a first reaction mixture and a second reaction mixture, wherein the first reaction mixture comprises the target nucleic acid molecule at a first concentration and a reference nucleic acid molecule, and wherein the second reaction mixture comprises the target nucleic acid at a second concentration and the reference nucleic acid molecule, wherein the first concentration and the second concentration are different; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit. The method can comprise (a) subjecting the plurality of reaction mixtures to a nucleic acid amplification reaction; and (b) detecting from the first reaction mixture (i) a first signal of the target nucleic acid molecule to obtain a first value, and (ii) a first reference signal of the reference nucleic acid molecule to obtain a first reference value; (c) detecting from the second reaction mixture (i) a second signal of the target nucleic acid molecule to obtain a second value, and (ii) a second reference signal of the reference nucleic acid molecule to obtain a second reference value; (d) determining a first relative value based on the first value and the first reference value and a second relative value based on the second value and the second reference value to obtain a standard value; (e) subjecting the sample to the nucleic acid amplification reaction in the presence of the reference nucleic acid molecule; (f) detecting a signal of the target nucleic acid molecule to obtain a value, and a reference signal of the reference nucleic acid molecule to obtain a reference value, and determining a relative value of the value and the reference value; and (g) using the relative value, the first relative value and the second relative value to determine the presence or absence of the target nucleic acid molecule in the sample.

[0105] In some cases, a system of analyzing a sample having or suspected of having a target nucleic acid molecule can comprise: an analytic unit configured to receive a reaction mixture comprising the sample and a reference nucleic acid molecule; and one or more computer processors operatively coupled to the analytic unit, wherein the one or more computer processors are individually or collectively programmed to direct a method in the analytic unit, wherein the method comprises: (a) subjecting the reaction mixture to a nucleic acid amplification reaction; (b) detecting a first signal of the target nucleic acid molecule to obtain a first time to response value, and a reference signal of the reference nucleic acid molecule to obtain a reference time to response value; and (c) determining a relative time to response value of the first time to response value and the reference time to response value, wherein the relative time to response value indicates the presence or absence of the target nucleic acid molecule in the sample; wherein a duration of analyzing the sample from (a) to (d) is equal to, at most, or about 30 min or less.

Sample Preparations

[0106] The target nucleic acid molecules used in the nucleic acid amplification reactions can be processed first. For example, target nucleic acid molecule can be extracted or isolated from a sample. Such step may also be referred to as sample preparation.

[0107] In some embodiments, sample preparation can comprise extracting nucleic acids from a sample. In some embodiments, sample preparation can comprise extracting nucleic acids from a sample prior to subjecting the sample to a nucleic acid amplification reaction. In some embodiments, sample preparation can comprise extracting nucleic acids from a sample by heating the sample. For example, a target nucleic acid (e.g., target RNA, target DNA) may be extracted or released from a biological sample during heating phases of nucleic acid amplification. Alternatively or in addition to the heating, a target nucleic acid (e.g., target RNA, target DNA) may be extracted or released from a biological sample using a cartridge system wherein a sample can be mixed with a lysis buffer and then drawn through a filter thereby capturing the target nucleic acid in the filter. In some cases, a cartridge system can also comprise washing steps to remove contaminants. An elution buffer can be added to the cartridge to remove the target nucleic acid from the filter for further processing or analysis. The cartridge system can be an automated cartridge system. In some cases, the cartridge system can be the Ml Sample Prep® Cartridge Kit (SKU:3000536, Biomeme, Inc.). In some cases, the sample preparation method described herein can use the cartridge system for automated sample processing. Details of the sample preparation cartridge and related methods is described in the U.S. Application No. 16/817,733, the entire content of which is incorporated herein by reference. It is to be understood that the sample described herein can be processed by various other methods or any commercially available nucleic acid extraction kits or methods.

[0108] In some embodiments, the present disclosure provides a method for processing a sample. In some embodiments, the method comprises activating a system comprising a pump. Fluid from a sample chamber may be transferred to a waste chamber using a second pump, or to one or more assay tubes using a third pump for analysis. Generally, a conduit may pass through an assay tube cap, thereby providing a fluid connection between the assay tube and a conduit (e.g., a conduit extending from a sample chamber). As an alternative, a single pump and one or more valves may be used to draw fluid from the sample chamber into the waste chamber or the one or more assay tubes. In some embodiments, the method comprises activating a system comprising at least two multi-directional pumps in fluid communication with a fluid flow path for processing the sample, which fluid flow path does not include any valves. In some embodiments, the method further comprises subjecting fluid in the fluid flow path to flow along a first direction upon application of a first pressure drop by a first multi-directional pump of the at least two multi-directional pumps and a second pressure drop by a second multi-directional pump of the at least two multi-directional pumps. In some embodiments, the method further comprises subjecting fluid in the fluid flow path to flow along a second direction different than the first direction upon application of a third pressure drop by the first multi-directional pump and a fourth pressure drop by the second multi-directional pump. In some embodiments, the first pressure drop is different than the second pressure drop. In some embodiments, the third pressure drop is different than the fourth pressure drop. In some embodiments, the first pressure drop is different than the third pressure drop, or the second pressure drop is different than the fourth pressure drop. In some embodiments, the first pressure drop is different than the third pressure drop, and the second pressure drop is different than the fourth pressure drop.

[0109] FIG. 4 shows an example process flow. In a first operation 401, a valve is opened and lysis buffer is pumped from a reagent chamber into the sample chamber. In a second operation 402, a sample to be analyzed is added into the sample chamber now containing the lysis buffer. Filling the sample chamber with a buffer (e.g., a lysis buffer) prior to adding the sample may prevent loss of target nucleic acids within the sample (e.g., due to adhesion along the wall of the sample chamber). In a third operation 403, the lysis buffer and the sample are mixed in sample chamber. The mixing can be performed in a variety of ways. In an example, bubbles can be generated by positive pressure into the sample chamber from a pump (e.g., first pump, second pump, or third pump). Although any pump of the device may be used to generate bubbles within the sample chamber, the pump may be used to avoid situations in which reversing the flow of the second pump (e.g., the waste pump), for example, may increase the risk of contamination of the sample in the sample chamber with waste from the waste chamber. Other techniques may also be used to mix lysis buffer and sample in the sample chamber, such as agitating the chamber 101 or the entire device.

[0110] In a subsequent operation 404 the mixture of sample and lysis buffer is drawn through a filter by the second pump, thereby capturing targets (e.g., nucleic acids) in the filter and transferring waste to a waste chamber. In a subsequent operation 405, one or more wash buffers, and/or drying buffers, are serially pumped into sample chamber, and mixed with the targets captured in the filter. Subsequently, in operation 406, the mixture of buffer and target is drawn through the filter by pump, thereby capturing targets (e.g., nucleic acids) in the filter and transferring waste to a waste chamber. In some cases, in operation 407, following washing of the targets captured in the filter with a drying buffer (e.g., a volatile chemical such as acetone), the sample chamber may be heated (e.g., using a heating pad disposed along an outer surface of the sample chamber) to remove residual drying buffer (e.g., through vaporization). This may reduce contamination of the target by the drying agent. In a subsequent operation 408, elution buffer is pumped into sample chamber, thereby extracting a target (e.g., nucleic acids) from the filter into the elution buffer. In another operation 409, bubbles can be generated by positive pressure into the sample chamber from a pump to distribute the elution buffer throughout the sample chamber and enhance extraction of the target from the filter. In yet another operation 410, the mixture of elution buffer and target is pumped by the third pump from the sample chamber to one or more assay tubes for further processing and/or analysis.

[OHl] The present disclosure provides sample preparation cartridges. Generally, sample preparation cartridges can comprise (i) one or more wells, each well containing a reagent necessary for processing the sample, (ii) a sample chamber for reacting the buffers with a sample, (iii) a chamber for depositing waste from the sample chamber, and (iv) one or more assay tubes for collecting a processed sample and performing an assay. Generally, the chambers and assay tubes can be connected by conduits (e.g., connections capable of transferring fluid from one chamber to another). Any of these conduits can comprise openings for connecting with a pump or valve to regulate flow of a liquid (e.g., a buffer or a sample) along the conduit.

[0112] The present disclosure 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, amplification reaction buffers, or any combination thereof 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.

[0113] 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.

[0114] The 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.

[0115] 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) can comprise enzymes, primers and/or probes (e.g., guide oligonucleotides), reverse transcriptase primers, molecular beacons, dNTPs, or any combination thereof. The sample processing buffer (e.g., Sample Direct) 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, 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). In some embodiments, 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. In some cases, 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.

[0116] 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 shelf-stable formulation; (ii) screening and optimizing assays in a representative sample matrix (e.g., matrixbased 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.

[0117] Sample preparation 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.

[0118] In some aspects, the present disclosure provides compositions for sample processing for a nucleic acid amplification method. In some embodiments, the composition comprises a detergent, a solubilizer, and a cyclodextrin. Without wishing to be bound by theory, 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. In some embodiments, the detergent is sodium dodecyl sulfate (SDS). In some embodiments, the detergent comprises sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the detergent is an ionic detergent. In some embodiments, the detergent is a non-ionic detergent. In some embodiments, 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). In some embodiments, the lysis buffer is capable of handling challenging solid and liquid sample types.

[0119] In some embodiments, the detergent is present at a final concentration when mixed with the sample to be 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.

[0120] In some embodiments, the lysis buffer comprises reagents including, but not limited to, egtazic acid (EGTA), ethylenedi aminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)aminomethane (e.g., Tris), sodium acetate, or any combination thereof. In some embodiments, the lysis buffer comprises SDS, egtazic acid (EGTA), ethylenedi aminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), tri s(hydroxymethyl)aminom ethane (e.g., Tris), and/or sodium acetate. In some embodiments, 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 tetrahydropyran (THP). The lysis buffer can comprise any combination of reagents provided herein.

[0121] The lysis buffer can comprise sodium acetate. In some embodiments, the concentration (e.g., final concentration) of the sodium acetate in the lysis buffer in the presence of a sample (e.g., blood and/or swab-based sample) can be at least about ImM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 80 mM, or greater than about 80 mM. In some embodiments, the concentration (e.g., final concentration) of the sodium acetate in the lysis buffer in the presence of a sample (e.g., blood and/or swab-based sample) can be at most about 80 mM, at most about 70 mM, at most about 60 mM, at most about 50 mM, at most about 40 mM, at most about 30 mM, at most about 20 mM, at most about 10 mM, at most about 5 mM, or less than about 5 mM. In some embodiments, the concentration (e.g., final concentration) of the sodium acetate in the lysis buffer in the presence of a sample (e.g., blood and/or swab-based sample) can be from about 5 mM to about 70 mM. In some embodiments, the concentration (e.g., final concentration) of the sodium acetate in the lysis buffer in the presence of a sample (e.g., blood and/or swab-based sample) can be from about 5 mM to about 10 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 25 mM, about 5 mM to about 30 mM, about 5 mM to about 35 mM, about 5 mM to about 40 mM, about 5 mM to about 45 mM, about 5 mM to about 50 mM, about 5 mM to about 60 mM, about 5 mM to about 70 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, about 10 mM to about 25 mM, about 10 mM to about 30 mM, about 10 mM to about 35 mM, about 10 mM to about 40 mM, about 10 mM to about 45 mM, about 10 mM to about 50 mM, about 10 mM to about 60 mM, about 10 mM to about 70 mM, about 15 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 30 mM, about 15 mM to about 35 mM, about 15 mM to about 40 mM, about 15 mM to about 45 mM, about 15 mM to about 50 mM, about 15 mM to about 60 mM, about 15 mM to about 70 mM, about 20 mM to about 25 mM, about 20 mM to about 30 mM, about 20 mM to about 35 mM, about 20 mM to about 40 mM, about 20 mM to about 45 mM, about 20 mM to about 50 mM, about 20 mM to about 60 mM, about 20 mM to about 70 mM, about 25 mM to about 30 mM, about 25 mM to about 35 mM, about 25 mM to about 40 mM, about 25 mM to about 45 mM, about 25 mM to about 50 mM, about 25 mM to about 60 mM, about 25 mM to about 70 mM, about 30 mM to about 35 mM, about 30 mM to about 40 mM, about 30 mM to about 45 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 35 mM to about 40 mM, about 35 mM to about 45 mM, about 35 mM to about 50 mM, about 35 mM to about 60 mM, about 35 mM to about 70 mM, about 40 mM to about 45 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM, about 45 mM to about 50 mM, about 45 mM to about 60 mM, about 45 mM to about 70 mM, about 50 mM to about 60 mM, about 50 mM to about 70 mM, or about 60 mM to about 70 mM.

[0122] In some embodiments, the lysis buffer comprises a pH buffer. The pH buffer may have a pH of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12. The pH buffer may have a pH of at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1. The pH buffer may have a pH from about about 4 to about 10. The pH buffer may have a pH from about about 4 to about 4.5, 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 7.5, about 4 to about 8, about 4 to about 8.5, about 4 to about 9, about 4 to about 10, about 4.5 to about 5, about 4.5 to about 5.5, about 4.5 to about 6, about 4.5 to about 6.5, about 4.5 to about 7, about 4.5 to about 7.5, about 4.5 to about 8, about 4.5 to about 8.5, about 4.5 to about 9, about 4.5 to about 10, about 5 to about 5.5, about 5 to about 6, about 5 to about 6.5, about 5 to about 7, about 5 to about 7.5, about 5 to about 8, about 5 to about 8.5, about 5 to about 9, about 5 to about 10, about 5.5 to about 6, about 5.5 to about 6.5, about 5.5 to about 7, about 5.5 to about 7.5, about 5.5 to about 8, about 5.5 to about 8.5, about 5.5 to about 9, about 5.5 to about 10, about 6 to about 6.5, about 6 to about 7, about 6 to about 7.5, about 6 to about 8, about 6 to about 8.5, about 6 to about 9, about 6 to about 10, about 6.5 to about 7, about 6.5 to about 7.5, about 6.5 to about 8, about 6.5 to about 8.5, about 6.5 to about 9, about 6.5 to about 10, about 7 to about 7.5, about 7 to about 8, about 7 to about 8.5, about 7 to about 9, about 7 to about 10, about 7.5 to about 8, about 7.5 to about 8.5, about 7.5 to about 9, about 7.5 to about 10, about 8 to about 8.5, about 8 to about 9, about 8 to about 10, about 8.5 to about 9, about 8.5 to about 10, or about 9 to about 10. [0123] In some embodiments, the lysis buffer comprises a chelating agent. In some embodiments, the lysis buffer comprises 1, 2, 3, 4, or more chelating agents. In some embodiments, the chelating agent comprises 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). In some embodiments, the lysis buffer comprises a reducing agent. In some embodiments, the lysis buffer comprises 1, 2, 3, 4, 5, or more reducing agents. In some embodiments, 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).

[0124] In some embodiments, a concentration (e.g., final concentration) of a reagent of the lysis buffer described herein can be from about 0.1 mM to 100 mM. In some embodiments, a concentration (e.g., final concentration) of a reagent of the lysis buffer described herein can be at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or greater than about 100 mM. In some embodiments, a concentration (e.g., final concentration) of a reagent of the lysis buffer described herein can be at most about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less than about 0.1 mM.

[0125] In some embodiments, the lysis buffer has a pH value sufficient to lyse a desired cell. In some embodiments, 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. In some embodiments, the lysis buffer has a pH value of at most about 9, at most about 8, at most about 7, at most about 6.5, at most about 6, at most about 5.5, at most about 5, at most about 4.5, at most about 4, at most about 3, at most about 2, or at most about 1.

[0126] In some embodiments, the recovery buffer does not comprise a component in the lysis buffer. For example, the recovery buffer may not comprise a detergent or a reducing agent. In some cases, the recovery buffer may not comprise one or more agent selected from the group consisting of an egtazic acid (EGTA), an ethylenedi aminetetraacetic 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), and tetrahydropyran (THP). [0127] In some embodiments, 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 recovery buffer can comprise a cyclodextrin. The cyclodextrin can comprise (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, 3A-amino-3A-deoxy-(2AS,3AS)-y-cyclodextrin hydrate, or any combination thereof. The cyclodextrin can comprise an anionic cyclodextrin. The anionic cyclodextrin may comprise 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, carboxymethyl-y-cyclodextrin, (2-carboxyl)-y-cyclodextrin, sulfobutylated-a- cyclodextrin, succinyl-(2-hydroxypropyl)-P cyclodextrin, succinyl-(2-hydroxypropyl)-y cyclodextrin, sulfobutylated-y cyclodextrin, methyl-P-cyclodextrin, or any combination thereof. In some embodiments, the cyclodextrin in the recovery buffer can comprise two or more different cyclodextrin species described herein. For example, the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) P-cyclodextrin and (2-hydroxypropyl) y-cyclodextrin. For another example, the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) a-cyclodextrin and methyl-P-cyclodextrin. In some cases, the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) P-cyclodextrin and methyl-P-cyclodextrin. In some cases, altering the molar substitution ratio of a particular modified cyclodextrin species (e.g., (2-hydroxypropyl) P- cyclodextrin, methyl-P-cyclodextrin, or combinations thereof, or variants thereof) may improve reaction performance such as shortening time to result values, Ct values, or Cq values.

[0128] In some embodiments, the lysis buffer and the recovery buffer are in the same mixture. In some embodiments, the mixing of the lysis buffer and the recovery buffer is performed by hand. In some embodiments, the mixing of the lysis buffer and the recovery buffer is performed by a vortex. In some embodiments, the mixing of the lysis buffer and the recovery buffer is performed by an automated instrument, a consumable, or a microfluidic system. In some embodiments, the mixing of the lysis buffer and the recovery buffer is performed until the lysis buffer and the recovery buffer are mixed to homogeneity.

[0129] A composition (e.g., reaction mixture) described herein may 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. In some embodiments, 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. In some embodiments, 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, l,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid (BAPTA), or tetrahydropyran (THP), or any combination thereof.

[0130] A composition (e.g., reaction mixture) described herein may comprise a composition for sample amplification (e.g., sample amplification buffer). The composition for sample amplification may comprise a nonionic surfactant, a cyclodextrin, a sucrose/epichlorohydrin polymer, or any combination thereof. The composition may be configured to increase a rate of amplification. The amplification may be a nucleic acid amplification (e.g., a PCR or an isothermal nucleic amplification). The composition for sample amplification may be configured to stabilize one or more enzyme (e.g., a thermostable enzyme). The enzyme can be stabilized during an amplification (e.g., a nucleic acid amplification). The enzyme can be a polymerase, an endonuclease, or a reverse transcriptase, or any combination thereof. In some embodiments, the reverse transcriptase can be an avian myeloblastosis virus (AMV) reverse transcriptase or a murine leukemia virus (MMLV) reverse transcriptase. The nonionic surfactant of the composition for sample amplification may be nonoxynol-9.

[0131] In some embodiments, prior to analyzing a sample in a nucleic acid amplification, a sample may be processed with a sample processing buffer. The sample processing buffer can comprise one or more of the reagents described herein. The sample processing buffer can comprise one or more of buffers (e.g., lysis buffer, recovery buffer, etc.) described herein. For example, the sample processing buffer can comprise a lysis buffer described herein. As another example, the sample processing buffer can comprise a recovery buffer described herein.

[0132] The concentration of Tris in a recovery buffer described herein may be greater than a concentration of Tris after mixed with a sample. For example, a concentration of Tris in a recovery buffer described herein may be at least about 100 mM, at least about 200 mM, at least about 300 mM, at least about 400 mM, at least about 500 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, at least about 1000 mM, at least about 1500 mM, at least about 2000 mM, or greater than about 2000 mM. A concentration of Tris in a recovery buffer described herein may be at most about 2000 mM, at most about 1500 mM, at most about 1000 mM, at most about 900 mM, at most about 800 mM, at most about 700 mM, at most about 600 mM, at most about 500 mM, at most about 400 mM, at most about 300 mM, at most about 200 mM, at most about 100 mM, or less than about 100 mM.

[0133] In some aspects, provided herein are compositions for sample processing. The composition may comprise a detergent, a solubilizer, a cyclodextrin, or any combinations thereof. The composition may be configured to stabilize an enzyme during a nucleic acid amplification. The nucleic acid amplification can comprise any amplifications methods described herein. In some embodiments, the composition can be configured to reduce and/or eliminate activity of a degrading nuclease. A degrading nuclease may be a ribonuclease. The detergent may be part of a lysis buffer. The lysis buffer may have a pH. In some embodiments, the lysis buffer may have a pH of at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10.0, at least about 11.0, or greater than about 11.0. In some embodiments, the lysis buffer may have a pH of at most about 11.0, at most about 10.0, at most about 9.0, at most about 8.0, at most about 7.0, at most about 6.0, at most about 5.0, at most about 4.0, at most about 3.0, at most about 2.0, or less than about 2.0. In some embodiments, the lysis buffer may have a pH of less than about 7.0, less than about 6.0, or less than about 5.0. In some embodiments, the pH of the lysis buffer may be about 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

[0134] In some embodiments, a pH of the lysis buffer may be from about 1.0 to about 12.0. In some embodiments, a pH of the lysis buffer may be from at most about 12.0. In some embodiments, a pH of the lysis buffer may be from about 1.0 to about 2.0, about 1.0 to about 3.0, about 1.0 to about 4.0, about 1.0 to about 5.0, about 1.0 to about 6.0, about 1.0 to about 7.0, about 1.0 to about 8.0, about 1.0 to about 9.0, about 1.0 to about 10.0, about 1.0 to about 11.0, about 1.0 to about 12.0, about 2.0 to about 3.0, about 2.0 to about 4.0, about 2.0 to about 5.0, about 2.0 to about 6.0, about 2.0 to about 7.0, about 2.0 to about 8.0, about 2.0 to about 9.0, about 2.0 to about 10.0, about 2.0 to about 11.0, about 2.0 to about 12.0, about 3.0 to about 4.0, about 3.0 to about 5.0, about 3.0 to about 6.0, about 3.0 to about 7.0, about 3.0 to about 8.0, about 3.0 to about 9.0, about 3.0 to about 10.0, about 3.0 to about 11.0, about 3.0 to about 12.0, about 4.0 to about 5.0, about 4.0 to about 6.0, about 4.0 to about 7.0, about 4.0 to about 8.0, about 4.0 to about 9.0, about 4.0 to about 10.0, about 4.0 to about 11.0, about 4.0 to about 12.0, about 5.0 to about 6.0, about 5.0 to about 7.0, about 5.0 to about 8.0, about 5.0 to about 9.0, about 5.0 to about 10.0, about 5.0 to about 11.0, about 5.0 to about 12.0, about 6.0 to about 7.0, about 6.0 to about 8.0, about 6.0 to about 9.0, about 6.0 to about 10.0, about 6.0 to about 11.0, about 6.0 to about 12.0, about 7.0 to about 8.0, about

7.0 to about 9.0, about 7.0 to about 10.0, about 7.0 to about 11.0, about 7.0 to about 12.0, about

8.0 to about 9.0, about 8.0 to about 10.0, about 8.0 to about 11.0, about 8.0 to about 12.0, about

9.0 to about 10.0, about 9.0 to about 11.0, about 9.0 to about 12.0, about 10.0 to about 11.0, about

10.0 to about 12.0, or about 11.0 to about 12.0.

[0135] In some embodiments, a pH of the lysis buffer may be from about 5.0 to about 6.0. In some embodiments, a pH of the lysis buffer may be from about 5.0 to about 5.1, about 5.0 to about 5.2, about 5.0 to about 5.3, about 5.0 to about 5.4, about 5.0 to about 5.5, about 5.0 to about 5.6, about 5.0 to about 5.7, about 5.0 to about 5.8, about 5.0 to about 5.9, about 5.0 to about 6.0, about 5.1 to about 5.2, about 5.1 to about 5.3, about 5.1 to about 5.4, about 5.1 to about 5.5, about 5.1 to about 5.6, about 5.1 to about 5.7, about 5.1 to about 5.8, about 5.1 to about 5.9, about 5.1 to about 6.0, about 5.2 to about 5.3, about 5.2 to about 5.4, about 5.2 to about 5.5, about 5.2 to about 5.6, about 5.2 to about 5.7, about 5.2 to about 5.8, about 5.2 to about 5.9, about 5.2 to about 6.0, about 5.3 to about 5.4, about 5.3 to about 5.5, about 5.3 to about 5.6, about 5.3 to about 5.7, about 5.3 to about 5.8, about 5.3 to about 5.9, about 5.3 to about 6.0, about 5.4 to about 5.5, about 5.4 to about 5.6, about 5.4 to about 5.7, about 5.4 to about 5.8, about 5.4 to about 5.9, about 5.4 to about 6.0, about 5.5 to about 5.6, about 5.5 to about 5.7, about 5.5 to about 5.8, about 5.5 to about 5.9, about 5.5 to about 6.0, about 5.6 to about 5.7, about 5.6 to about 5.8, about 5.6 to about 5.9, about 5.6 to about 6.0, about 5.7 to about 5.8, about 5.7 to about 5.9, about 5.7 to about 6.0, about 5.8 to about 5.9, about 5.8 to about 6.0, or about 5.9 to about 6.0.

[0136] As an example, provided herein are compositions 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, wherein the composition is configured to reduce and/or eliminate activity of a degrading nuclease, and wherein the detergent is part of a lysis buffer, and wherein the lysis buffer has a pH of less than 8.0.

[0137] In some embodiments, the lysis buffer can comprise sodium acetate. The sodium acetate may comprise a pH. The pH of the sodium acetate may be at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10.0, at least about 11.0, or greater than about 11.0. The pH of the sodium acetate may be at most about 11.0, at most about 10.0, at most about 9.0, at most about 8.0, at most about 7.0, at most about 6.0, at most about 5.0, at most about 4.0, at most about 3.0, at most about 2.0, or less than about 2.0. In some embodiments, the sodium acetate may have a pH of less than about 7.0, less than about 6.0, or less than about 5.0. In some embodiments, the pH of the sodium acetate may be about 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, a pH of the sodium acetate may be from about 5.0 to about 6.0. In some embodiments, a pH of the sodium acetate may be from about 5.0 to about 5.1, about 5.0 to about 5.2, about 5.0 to about 5.3, about 5.0 to about 5.4, about 5.0 to about 5.5, about 5.0 to about 5.6, about 5.0 to about 5.7, about 5.0 to about 5.8, about 5.0 to about 5.9, about 5.0 to about 6.0, about 5.1 to about 5.2, about 5.1 to about 5.3, about 5.1 to about 5.4, about 5.1 to about 5.5, about 5.1 to about 5.6, about 5.1 to about 5.7, about 5.1 to about 5.8, about 5.1 to about 5.9, about 5.1 to about 6.0, about 5.2 to about 5.3, about 5.2 to about 5.4, about 5.2 to about 5.5, about 5.2 to about 5.6, about 5.2 to about 5.7, about 5.2 to about 5.8, about 5.2 to about 5.9, about 5.2 to about 6.0, about 5.3 to about 5.4, about 5.3 to about 5.5, about 5.3 to about 5.6, about 5.3 to about 5.7, about 5.3 to about 5.8, about 5.3 to about 5.9, about 5.3 to about 6.0, about 5.4 to about 5.5, about 5.4 to about 5.6, about 5.4 to about 5.7, about 5.4 to about

5.8, about 5.4 to about 5.9, about 5.4 to about 6.0, about 5.5 to about 5.6, about 5.5 to about 5.7, about 5.5 to about 5.8, about 5.5 to about 5.9, about 5.5 to about 6.0, about 5.6 to about 5.7, about

5.6 to about 5.8, about 5.6 to about 5.9, about 5.6 to about 6.0, about 5.7 to about 5.8, about 5.7 to about 5.9, about 5.7 to about 6.0, about 5.8 to about 5.9, about 5.8 to about 6.0, or about 5.9 to about 6.0.

[0138] The compositions provided herein may be configured to stabilize nucleic acids during a nucleic acid amplification. In some embodiments, the enzyme to be stabilized in the nucleic acid amplification may be a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

[0139] In some embodiments, the detergent is sodium dodecyl sulfate (SDS). In some embodiments, the detergent comprises sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof. In some embodiments, the detergent is an ionic detergent. In some embodiments, the detergent is a non-ionic detergent. In some embodiments, the solubilizer is a non-ionic surfactant. In some embodiments, 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. In some embodiments, the solubilizer is a Tergitol™ surfactant, a Triton™ surfactant, or a Igepal® surfactant. In some embodiments, the solubilizer is an alkoxylate or a cocamide. In some embodiments, the solubilizer is decyl glucoside, alkyl polyglycoside, lauryl glucoside, sorbitan tristearate, or a niosome.

[0140] The solubilizer and/or the cyclodextrin of the composition may be part of a recovery buffer. In some embodiments, the sample is contacted with the lysis buffer and the recovery buffer simultaneously. In some embodiments, the sample is contacted with the lysis buffer and the recovery buffer concurrently in the same mixture. In some embodiments, the sample is submerged in the lysis buffer. In some embodiments, mixing of the sample and lysis buffer is by a vortex and/or by hand. In some embodiments, the sample is not mixed with the lysis buffer. In some embodiments, the recovery buffer comprises a salt. In some embodiments, the recovery buffer does not comprise a salt. In some embodiments, the salt comprises a sodium 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 pH of the recovery buffer is at least about 3, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7, at least about 7.5, at least about 8, at least about 9, at least about 10, at least about 11, or at least about 12. In some embodiments, the pH of the recovery buffer is at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7.5, at most about 7, at most about 6.5, at most about 6, at most about 5.5, at most about 5, at most about 4.5, at most about 4, or at most about 3.

[0141] In some embodiments, 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.

[0142] The solubilizer and cyclodextrin of the composition may be configured to shorten a cycle threshold (Ct) value and/or a time to result value. In some cases, quantification cycle Cq value is also used and it can be used interchangeably with Ct value. In some embodiments, the solubilizer and cyclodextrin of the composition may be configured to shorten a cycle threshold (Ct) value and/or a time to result value of a nucleic acid amplification compared to a cycle threshold (Ct) value and/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. In some embodiments, 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 25, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1. In some embodiments, 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. [0143] In some embodiments, 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. The term “coefficient of variation” refers to a measure of precision of an amplification method. In some embodiments, 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%.

[0144] In some embodiments, 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. In some embodiments, 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.

[0145] In some embodiments, the detergent may be part of a lysis buffer, wherein the lysis buffer can comprise a chelating agent. The chelating agent can be deferiprone, ethylenediamine, 1,10- Phenanthroline, oxalic acid, pentetic acid, deferasirox, deferoxamine, deferoxamine mesylate, N,N,N',N'-tetrakis(2-pyridinylmethyl)-l,2-ethanediamine (TPEN), or any combination thereof. The composition can comprise a reducing agent. The reducing agent may be 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), tetrahydropyran (THP), or any combination thereof. The lysis buffer can comprise any compounds (e.g., components) as described herein. The composition can comprise a final concentration of EGTA, EDTA, TCEP, and/or Tris in a lysis buffer in the presence of a sample. The final concentration of EGTA, EDTA, TCEP, and/or Tris may be a concentration as described herein. In some embodiments, the composition of the present disclosure further comprises an agent capable of reducing a disulfide bond. In some embodiments, the agent capable of reducing said disulfide bond comprises dithiothreitol (DTT), hydroxylamine, hydroxylamine-HCl, 2-mercaptoethanol (BME), or TCEP. In some embodiments, the agent capable of reducing said disulfide bond comprises a compound in a monothiol class, a dithiol class, or a phosphine class.

[0146] The detergent may be present at a concentration (e.g., a final concentration) that can be sufficient for lysing cells. For example, when mixed with a sample described herein, the detergent of the sample processing buffer can lyse the cells. In some embodiments, 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 concentration of any agent described herein when mixed with a sample to be processed can be referred to as final concentration. In some embodiments, 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, at least about 2.0% w/v, at least about 3.0% w/v, at least about 4.0% w/v, at least about 5.0% w/v, at least about 6.0% w/v, at least about 7.0% w/v, at least about 8.0% w/v, at least about 9.0% w/v, or at least about 10.0% w/v.

[0147] In some embodiments, 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, at most about 0.35% w/v, at most about 0.3% w/v, at most about 0.25% w/v, at most about 0.2% w/v, at most about 0.15% w/v, at most about 0.1% w/v, or at most about 0.05% w/v.

[0148] 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 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 0.2% w/v to about 0.3% w/v, about 0.2% w/v to about 0.4% w/v, about 0.2% w/v to about 0.5% w/v, about 0.2% w/v to about 0.6% w/v, about 0.2% w/v to about 0.7% w/v, about 0.2% w/v to about 0.8% w/v, about 0.2% w/v to about 0.9% w/v, about 0.2% w/v to about 1% w/v, about 0.2% w/v to about 1.5% w/v, about 0.2% w/v to about 2% w/v, about 0.3% w/v to about 0.4% w/v, about 0.3% w/v to about 0.5% w/v, about 0.3% w/v to about 0.6% w/v, about 0.3% w/v to about 0.7% w/v, about 0.3% w/v to about 0.8% w/v, about 0.3% w/v to about 0.9% w/v, about 0.3% w/v to about 1% w/v, about 0.3% w/v to about 1.5% w/v, about 0.3% w/v to about 2% w/v, about 0.4% w/v to about 0.5% w/v, about 0.4% w/v to about 0.6% w/v, about 0.4% w/v to about 0.7% w/v, about 0.4% w/v to about 0.8% w/v, about 0.4% w/v to about 0.9% w/v, about 0.4% w/v to about 1% w/v, about 0.4% w/v to about 1.5% w/v, about 0.4% w/v to about 2% w/v, about 0.5% w/v to about 0.6% w/v, about 0.5% w/v to about 0.7% w/v, about 0.5% w/v to about 0.8% w/v, about 0.5% w/v to about 0.9% w/v, about 0.5% w/v to about 1% w/v, about 0.5% w/v to about 1.5% w/v, about 0.5% w/v to about 2% w/v, about 0.6% w/v to about 0.7% w/v, about 0.6% w/v to about 0.8% w/v, about 0.6% w/v to about 0.9% w/v, about 0.6% w/v to about 1% w/v, about 0.6% w/v to about 1.5% w/v, about 0.6% w/v to about 2% w/v, about 0.7% w/v to about 0.8% w/v, about 0.7% w/v to about 0.9% w/v, about 0.7% w/v to about 1% w/v, about 0.7% w/v to about 1.5% w/v, about 0.7% w/v to about 2% w/v, about 0.8% w/v to about 0.9% w/v, about 0.8% w/v to about 1% w/v, about 0.8% w/v to about 1.5% w/v, about 0.8% w/v to about 2% w/v, about 0.9% w/v to about 1% w/v, about 0.9% w/v to about 1.5% w/v, about 0.9% w/v to about 2% w/v, about 1% w/v to about 1.5% w/v, about 1% w/v to about 2% w/v, or about 1.5% w/v to about 2% w/v. [0149] The cyclodextrin may be present at a concentration (e.g., a final concentration) that can be sufficient for isolating the detergent in the composition. For example, when mixed with a sample described herein, the cyclodextrin of the sample processing buffer can isolate the detergent (e.g., a portion of the detergent) in the composition. The final concentration of the detergent, solubilizer, and/or cyclodextrin can comprise a final concentration as described herein. In some embodiments, the composition comprises a cyclodextrin. The cyclodextrin is configured to form a complex with the detergent of the present application. Without wishing to be bound by theory, 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. As a complexing agent, the cyclodextrin can increase the aqueous solubility of poorly soluble drugs and increase bioavailability and stability in solution. In some embodiments, 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, 3A- amino-3A-deoxy-(2AS,3AS)-y-cyclodextrin hydrate, or any combination thereof. The cyclodextrin can comprise an anionic cyclodextrin. The anionic cyclodextrin may comprise 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, carboxymethyl-y-cyclodextrin, (2-carboxyl)-y-cyclodextrin, sulfobutylated-a- cyclodextrin, succinyl-(2-hydroxypropyl)-P cyclodextrin, succinyl-(2-hydroxypropyl)-y cyclodextrin, sulfobutylated-y cyclodextrin, methyl-P-cyclodextrin, or any combination thereof. In some embodiments, the cyclodextrin in the recovery buffer can comprise two or more different cyclodextrin species described herein. For example, the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) P-cyclodextrin and (2-hydroxypropyl) y-cyclodextrin. For another example, the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) a-cyclodextrin and methyl-P-cyclodextrin. In some cases, the cyclodextrin in the recovery buffer can comprise (2-hydroxypropyl) P-cyclodextrin and methyl-P-cyclodextrin. In some cases, altering the molar substitution ratio of a particular modified cyclodextrin species (e.g., (2-hydroxypropyl) P- cyclodextrin, methyl-P-cyclodextrin, etc.) may improve reaction performance such as shortening time to result values, Ct values, or Cq values.

[0150] In some embodiments, 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. 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 0.05 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 5.0 mM, at least about 10.0 mM, at least about 15.0 mM, at least about 20.0 mM, at least about 25.0 mM, at least about 30.0 mM, at least about

35.0 mM, at least about 40.0 mM, at least about 50.0 mM, at least about 55.0 mM, at least about

60.0 mM, at least about 65.0 mM, at least about 70.0 mM, at least about 75.0 mM, at least about

80.0 mM, at least about 85.0 mM, at least about 90.0 mM, at least about 95.0 mM, at least about

100.0 mM, at least about 125.0 mM, at least about 150.0 mM, 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.

[0151] 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 300.0 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 25.0 mM, at most about 20.0 mM, at most about 15.0 mM, at most about 10.0 mM, at most about 5.0 mM, at most about 1.0 mM, at most about 0.5 mM, at most about 0.1 mM, or at most about 0.05 mM. In some embodiments, 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.

[0152] In some embodiments, the cyclodextrin has a higher binding affinity toward the detergent than a binding affinity of the solubilizer towards the detergent. In some embodiments, the binding affinity of the cyclodextrin to the detergent can be an association constant. In some embodiments, the binding affinity of the cyclodextrin to the detergent has an association constant (K_a) of at least about 2.5xl0³ M’¹, at least about 3xl0³ M’¹, at least about 3.5xl0³ M’¹, at least about 4xl0³ M’¹, at least about 5xl0³ M’¹, at least about IxlO⁴ M’¹, at least about 2xl0⁴ M’¹, at least about 3xl0⁴ M’¹, at least about 4xl0⁴ M’¹, at least about 5xl0⁴ M’¹, at least about IxlO⁵ M’¹, at least about 5xl0⁵ M’ or at least about IxlO⁶ M'¹ to the detergent. In some embodiments, the binding affinity of the cyclodextrin to the detergent has an association constant (K_a) of at most about IxlO⁶ M’¹, at most about 5xl0⁵ M’¹, at most about IxlO⁵ M’¹, at most about 5xl0⁴ M’¹, at most about 4xl0⁴ M’¹, at most about 3xl0⁴ M’¹, at most about 2xl0⁴ M’¹, at most about IxlO⁴ M’¹, at most about 5xl0³ M’ at most about 4xl0³ M’¹, at most about 3xl0³ M’¹, or at most about 2.5xl0³ M'¹.

[0153] The solubilizer may mix with the detergent of the present composition. In some embodiments, the solubilizer is capable of forming micelles comprising the detergent of the present application.

[0154] In some embodiments, the solubilizer is polysorbate 80. In some embodiments, the concentration (e.g., final concentration) of the solubilizer in the mixture in the presence of the sample is at least about 0.05% v/v, at least about 0.1% v/v, at least about 0.5% v/v, at least about 1% v/v, at least about 5% v/v, at least about 10% v/v, at least about 15% v/v, at least about 20% v/v, at least about 22.5% v/v, at least about 25% v/v, at least about 27.5% v/v, at least about 30% v/v, at least about 32.5% v/v, at least about 35% v/v, at least about 37.5% v/v, at least about 40% v/v, at least about 42.5% v/v, at least about 45% v/v, at least about 47.5% v/v, at least about 50% v/v, at least about 52.5% v/v, at least about 55% v/v, at least about 57.5% v/v, at least about 60% v/v, at least about 70% v/v, or at least about 75% v/v. [0155] In some embodiments, the concentration (e.g., final concentration) of the solubilizer in the mixture in the presence of the sample is at most about 75% v/v, at most about 70% v/v, at most about 65% v/v, at most about 60% v/v, at most about 57.5% v/v, at most about 55% v/v, at most about 52.5% v/v, at most about 50% v/v, at most about 47.5% v/v, at most about 45% v/v, at most about 42.5% v/v, at most about 40% v/v, at most about 37.5% v/v, at most about 35% v/v, at most about 32.5% v/v, at most about 30% v/v, at most about 27.5% v/v, at most about 25% v/v, at most about 22.5% v/v, at most about 20% v/v, at most about 15% v/v, at most about 10% v/v, at most about 5% v/v, at most about 1% v/v, at most about 0.5% v/v, at most about 0.1% v/v, or at most about 0.05% v/v.

[0156] In some embodiments, the lysis buffer may be frozen to stabilize the solution. In some embodiments, the lysis buffer may be frozen at a temperature of between about -50°C to 0°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -50°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -40°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -30°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -25°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -20°C. In some embodiments, the lysis buffer may be frozen at a temperature of about -15°C. In some embodiments, 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.

[0157] After freezing the lysis buffer, the buffer may be thawed and mixed with the sample. In some embodiments, the efficiency of the thawed lysis buffer is tested and compared with the efficiency of an unfrozen lysis buffer. In some embodiments, the efficiency of the thawed lysis buffer is similar to the efficiency of the unfrozen lysis buffer. Without wishing to be bound by theory, addition of sodium acetate to the lysis buffer can improve a freeze-thaw stability of the sample. This improved stability may lead to better results in a nucleic acid amplification (e.g., time to detection, Ct value, Cq value, or any combination thereof).

[0158] The compositions described herein may further comprise a sample. The sample may be any sample described herein. For example, the sample may be a biological sample. The sample can comprise 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, or any combination thereof. The sample can comprise a target nucleic acid molecule subject to sample processing. [0159] The compositions described herein may comprise a reaction mixture for nucleic acid amplification. The reaction mixture may be a reaction mixture described herein. The reaction mixture can be lyophilized. In some embodiments, the reaction mixture may not be lyophilized. The reaction mixture can comprise a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), one or more primers, one or more probes, or any combinations thereof. In some embodiments, the thermostable enzyme comprises a Bacillus stearothermophilus polymerase, 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, a Thermus aquaticus e.g., Taq-polA), a Thermotoga maritima (e.g., Tma-polA), a Pfu-polB, a Pab-polB, an OmniTaq 2 LA DNA polymerase, or any mutants thereof. 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. In some embodiments, the thermostable enzyme comprises a DNA polymerase. In some embodiments, the thermostable enzyme comprises a RNA polymerase. In some embodiments, the composition is configured to stabilize enzymatic activity of the thermostable enzyme for use during a nucleic acid amplification.

[0160] In some embodiments, the dNTPs of the reaction mixture comprise dATP, dCTP, dGTP, dTTP, and/or dUTP. In some embodiments, 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 pM to about 1,250 pM, about 100 pM to about 1,500 pM, about 100 pM to about 2,000 pM, about 100 pM to about 4,000 pM, about 100 pM to about 5,000 pM, about 100 pM to about 7,500 pM, about 250 pM to about 500 pM, about 250 pM to about 750 pM, about 250 pM to about 1,000 pM, about 250 pM to about 1,250 pM, about 250 pM to about 1,500 pM, about 250 pM to about 2,000 pM, about 250 pM to about 4,000 pM, about 250 pM to about 5,000 pM, about 250 pM to about 7,500 pM, about 500 pM to about 750 pM, about 500 pM to about 1,000 pM, about 500 pM to about 1,250 pM, about 500 pM to about 1,500 pM, about 500 pM to about 2,000 pM, about 500 pM to about 4,000 pM, about 500 pM to about 5,000 pM, about 500 pM to about 7,500 pM, about 750 pM to about 1,000 pM, about 750 pM to about 1,250 pM, about 750 pM to about 1,500 pM, about 750 pM to about 2,000 pM, about 750 pM to about 4,000 pM, about 750 pM to about 5,000 pM, about 750 pM to about 7,500 pM, about 1,000 pM to about 1,250 pM, about 1,000 pM to about 1,500 pM, about 1,000 pM to about 2,000 pM, about 1,000 pM to about 4,000 pM, about 1,000 pM to about 5,000 pM, about 1,000 pM to about 7,500 pM, about 1,250 pM to about 1,500 pM, about 1,250 pM to about 2,000 pM, about 1,250 pM to about 4,000 pM, about 1,250 pM to about 5,000 pM, about 1,250 pM to about 7,500 pM, about 1,500 pM to about 2,000 pM, about 1,500 pM to about 4,000 pM, about 1,500 pM to about 5,000 pM, about 1,500 pM to about 7,500 pM, about 2,000 pM to about 4,000 pM, about 2,000 pM to about 5,000 pM, about 2,000 pM to about 7,500 pM, about 4,000 pM to about 5,000 pM, about 4,000 pM to about 7,500 pM, or about 5,000 pM to about 7,500 pM.

[0161] In some embodiments, the primer or probe can be a stretch of nucleotides that hybridizes with a target nucleic acid sequence. In some embodiments, 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. In some embodiments, 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.

Samples

[0162] Described herein are methods of analyzing a sample. A sample described herein can comprise a biological sample. A sample can comprise a single-stranded nucleic acid molecule. Alternatively, a sample can comprise a double-stranded nucleic acid molecule.

[0163] A sample can comprise a fluid sample. Non-limiting examples of fluid samples can include blood, plasma, urine, feces saliva, sweat, tears, pericardial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, gastric juice, respiratory secretion, semen, synovial fluid, or amniotic fluid.

[0164] In some embodiment, 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, and/or a lymph sample for nucleic acid amplification. In some embodiments, the swab sample comprises a vaginal swab, an oral swab, a nasopharyngeal swab, a nasal swab, and/or a rectal swab. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject has a disease, a condition, or an infection. In some embodiments, the sample comprises a purified sample. In some embodiments, the sample is a combination of two, three, four, five, or more types of samples. In some embodiments, the sample comprises one, two, three, four, five, six, seven, eight, nine, ten, or more target nucleic acid molecules.

[0165] In some embodiments of a method disclosed herein, a sample is obtained. A sample may be obtained invasively (e.g., tissue biopsy) or non-invasively (e.g., venipuncture). The sample may be an environmental sample. The sample may be a water sample (e.g., a water sample obtained from a lake, stream, river, estuary, bay, or ocean). The sample may be a soil sample. The sample may be a tissue or fluid sample from a subject, such as saliva, semen, blood (e.g., whole blood), serum, synovial fluid, tear, urine, or plasma. A sample may be a blood sample, a fecal sample, a urine sample, a tissue sample, a vaginal sample, an oral swab, a rectal swab, or a tissue biopsy. The sample may be a tissue sample, such as a skin sample or tumor sample. The sample may be obtained from a portion of an organ of a subject. The sample may be a cellular sample. The sample may be a cell-free sample (e.g., a plasma sample comprising cell-free analytes or nucleic acids). A sample may be a solid sample or a liquid sample. A sample may be a biological sample or a non- biological sample. A sample may comprise an in-vitro sample or an ex-vivo sample. Non-limiting examples of a sample include an amniotic fluid, bile, bacterial sample, breast milk, buffy coat, cells, cerebrospinal fluid, chromatin DNA, ejaculate, nucleic acids, plant-derived materials, RNA, saliva, semen, blood, serum, soil, synovial fluid, tears, tissue, urine, water, whole blood or plasma, and/or any combination and/or any fraction thereof. In one example, the sample may be a plasma sample that may comprise DNA. In another example, the sample may comprise a cell sample that may comprise cell-free DNA.

[0166] A sample may be from an animal (e.g., a human or non-human animal). A sample may be a mammalian sample. For example, a sample may be a human sample. Alternatively, a sample may be a non-human animal sample. Non-limiting examples of a non-human sample include a cat sample, a dog sample, a goat sample, a guinea pig sample, a hamster sample, a mouse sample, a pig sample, a non-human primate sample (e.g., a gorilla sample, an ape sample, an orangutan sample, a lemur sample, or a baboon sample), a rat sample, a sheep sample, a cow sample, and a zebrafish sample. In some embodiments, a sample may be from an animal (e.g., a human or non- human animal) having or suspected of having an infection of a pathogen comprising a target nucleic acid molecule. In some embodiments, a sample may be from an animal (e.g., a human or non-human animal) at risk of having an infection of a pathogen comprising a target nucleic acid molecule. In some embodiments, a sample may be from an animal (e.g., a human or non-human animal) diagnosed with having an infection of a pathogen comprising a target nucleic acid molecule.

[0167] The sample may comprise nucleic acids (e.g., circulating and/or cell-free DNA fragments). Nucleic acids may be derived from eukaryotic cells, prokaryotic cells, or non-cellular sources (e.g., viral particles). A nucleic acid may refer to a substance whose molecules consist of many nucleotides linked in a long chain. Non-limiting examples of the nucleic acid include an artificial nucleic acid analog (e.g., a peptide nucleic acid, a morpholino oligomer, a locked nucleic acid, a glycol nucleic acid, or a threose nucleic acid), chromatin, miRNA, cDNA, DNA, single stranded DNA, double stranded DNA, genomic DNA, plasmid DNA, or RNA. A nucleic acid may be double stranded or single stranded. A sample may comprise a nucleic acid that may be intracellular. Alternatively, a sample may comprise a nucleic acid that may be extracellular (e.g., cell-free). A sample may comprise a nucleic acid (e.g., chromatin) that may be fragmented.

[0168] A sample can be obtained from a virus, a bacterium, an archaea, or a eukarya. In some embodiments, a sample is obtained from a bacterium. A bacterium can be a spherical-shaped bacterium, a rod-shaped bacterium, a spiral-shaped bacterium, a comma-shaped bacterium, or a corkscrew-shaped bacterium. Non-limiting examples of bacteria are Streptococcus pneumoniae, Streptococcus pyogenes, Legionella pneumonia, Bordetella bronchiseptica, Enterobacter aerogenes, Pasteurella multocida, Proteus mirabilis, Staphylococcus aureus, Haemophilus influenzae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasmoides genitalium, Chlamydia pneumoniae and Chlamydia trachomatis. In some embodiments, a sample is obtained from a virus. A virus can be a double-stranded DNA virus, a single-stranded DNA virus, a doublestranded RNA virus, a single-stranded RNA virus, a positive sense single-stranded reverse transcriptase virus, or a double-stranded DNA reverse transcriptase virus. In some embodiments, a sample is obtained from a parasite. A parasite can be an endoparasite (e.g., a protozoan organism, a helminth, a fluke, or a roundworm) or an ectoparasite. Non-limiting examples of parasites are Trichomonas vaginalis, Giardia lamblia, Blastocystis, Toxoplasma gondii, Dientamoeba fragilis, and species of the genus Plasmodium (e.g., Plasmodium falciparum.

[0169] In some cases, a sample comprises a target gene. In some embodiments, a sample comprises a target nucleic acid sequence. The target nucleic acid sequence may comprise a nucleic acid sequence from a pathogen. The target nucleic acid sequence may comprise a bacterial RNA (e.g., RNA from a bacterial species associated with an infection). The target nucleic acid sequence may comprise bacterial DNA (e.g., DNA from a bacterial species associated with an infection). Nonlimiting example of bacterial species associated with an infection are Streptococcus pneumoniae, Streptococcus pyogenes, Legionella pneumonia, Bordetella bronchiseptica, Enterobacter aerogenes, Pasteurella multocida, Proteus mirabilis, Staphylococcus aureus, Haemophilus influenzae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasmoides genitalium, Chlamydia pneumoniae and Chlamydia trachomatis. In some embodiments, the target nucleic acid sequence comprises bacterial RNA or DNA from Neisseria gonorrhoeae. In some embodiments, the target nucleic acid sequence comprises bacterial RNA or DNA from Chlamydia trachomatis. In some embodiments, the target nucleic acid sequence comprises parasitic DNA. In some embodiments, the target nucleic acid sequence comprises parasitic RNA. In some embodiment, the target nucleic acid sequence comprises DNA or RNA from a parasitic species associated with an infection. Non-limiting examples of parasitic species associated with infections include Trichomonas vaginalis, Giardia lamblia, Blastocystis, Toxoplasma gondii, Dientamoeba fragilis, and species of the genus Plasmodium (e.g., Plasmodium falciparum). In some embodiments, the target nucleic acid sequence comprises parasitic RNA or DNA from Trichomonas vaginalis. In some embodiments, a sample is obtained from a virus. A sample may contain viral DNA. A sample may contain viral RNA. A sample is obtained from a viral species associated with an infection. In some embodiments, a target nucleic acid molecule comprises a mutation. In some embodiments, a target nucleic acid comprises a mutation associated with a disease or condition (e.g., cancer).

[0170] In some cases, the sample comprises a reference gene. In some embodiments, the sample comprises a human deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The reference gene may be a human gene. The reference gene may comprise human DNA or RNA. Non-limiting examples of human genes are Ribonuclease P/MRP subunit p30 (RPP30); Arachidonate 15- lipoxygenase (Alox- 15); Glycealdehyde-3 -phosphate dehydrogenase (GAPDH); Hypoxanthine phosphoribosyl transferase (HPRT); Mitogen- Activated Protein Kinase 1 (MAPK1); Peptidylprolyl Isomerase A (PPIA); Ribosomal protein, large, P0 (RplpO); Ribosomal protein 18S (18S); and TATA box binding protein (TBP). The reference gene may comprise human DNA or RNA comprising a sequence encoding RPP30. The reference gene may comprise human DNA or RNA comprising a sequence encoding Alox-15. The reference gene may comprise human DNA or RNA comprising a sequence encoding GAPDH. The reference gene may comprise human DNA or RNA comprising a sequence encoding HPRT. The reference gene may comprise human DNA or RNA comprising a sequence encoding MAPK1. The reference gene may comprise human DNA or RNA comprising a sequence encoding PPIA. The reference gene may comprise human DNA or RNA comprising a sequence encoding RplpO. The reference gene may comprise human DNA or RNA comprising a sequence encoding 18S. The reference gene may comprise human DNA or RNA comprising a sequence encoding TBP. The reference gene may comprise human DNA or RNA comprising a sequence encoding MCTP1. The reference gene may comprise human DNA or RNA comprising a sequence encoding IL1RN.

Methods of Nucleic Acid Amplifications

[0171] The methods of analyzing the samples described herein can comprise subjecting a reaction mixture to conditions sufficient to perform a nucleic acid amplification reaction. Various nucleic acid amplification methods can be used. For example, any type of nucleic acid amplification reaction may be used to amplify a target nucleic acid and generate an amplified product. Moreover, amplification of a nucleic acid may linear, exponential, or a combination thereof. Amplification may be emulsion based or may be non-emulsion based. Non-limiting examples of nucleic acid amplification methods include real-time nucleic acid amplification, isothermal amplification, reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, asymmetric amplification, rolling circle amplification, strand displacement amplification (SDA), and multiple displacement amplification (MDA). In some embodiments, the amplified product may be DNA. In cases where a target RNA is amplified, DNA can be obtained by reverse transcription of the RNA and subsequent amplification of the DNA can be used to generate an amplified DNA product. The amplified DNA product may be indicative of the presence of the target RNA in the biological sample. In cases where DNA is amplified, various DNA amplification methods may be employed. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR), variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR, touchdown PCR), and ligase chain reaction (LCR). In some cases, DNA amplification is linear. In some cases, DNA amplification is exponential. In some cases, DNA amplification is achieved with nested PCR, which can improve sensitivity of detecting amplified DNA products. In some cases, nucleic acid amplification is isothermal. Non-limiting examples of isothermal nucleic acid amplification methods include helicase-dependent amplification, restriction endonuclease amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, and nucleic acid sequence based amplification.

[0172] Nucleic acid amplification reactions may be conducted in assay tubes in parallel. Nucleic acid amplification reactions may be conducted, for example, by including reagents necessary for each nucleic acid amplification reaction in a reaction vessel to obtain a reaction mixture and subjecting the reaction mixture to conditions necessary for each nucleic amplification reaction. Reverse transcription amplification and DNA amplification may be performed sequentially, such as, for example, performing reverse transcription amplification on RNA to generate complementary DNA (cDNA), and subsequently subjecting the cDNA to DNA amplification (e.g., PCR) to amplify the cDNA.

Di fferential Targeted Endonuclease Cutting Technology (DTECT) Isothermal Ampli fications [0173] In some cases, the nucleic acid amplification method described herein can be a DTECT isothermal amplification. The isothermal amplification methods described herein can provide advantages over existing nucleic acid amplification methods. Non-limiting examples of isothermal nucleic acid amplification methods can include helicase-dependent amplification, restriction endonuclease amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, and nucleic acid sequence based amplification.

[0174] The methods described herein may take advantage of DNA polymerases with high stranddisplacement activity and specially designed primer sets to exponentially amplify a target sequence. The methods provided herein may provide a faster time to amplify a target nucleic acid molecule compared to a time with an existing nucleic acid amplification method. The nucleic acid target processed (e.g., 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. Different existing isothermal amplification methods can utilize different DNA polymerases. Loop- mediated isothermal amplification (LAMP) utilizes two sets of specially designed primers, termed inner and outer primers and may be performed under a constant temperature of 50-65°C (122- 149°F). A limitation of LAMP can be use of non-specific detection methods, which may result in detection of false positives. Helicase-dependent amplification (HDA) utilizes DNA helicase activity to separate complementary strands of double strand DNA molecules, and thus may avoid temperature cycling to produce single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. The rolling circle amplification (RCA) method utilizes the continuous amplification of a circular DNA template by a strand-displacing DNA polymerase. RCA functions at a constant temperature (e.g., between 37°C-42°C, [98.6-107.6°F]) to produce a long single-stranded DNA molecule with tandem repeats of the circular template. Limitations of RCA may include challenges in mass production of target molecules, purification, and storage. Multiple displacement amplification (MDA) may utilize random exonuclease-resistant primers as well as a q>29 DNA polymerase with strand-displacement activity to produce target DNA strands at a constant temperature, e.g., 30 °C (86°F). MDA may also be used for whole genome amplification. The recombinase polymerase amplification (RPA) method is a low temperature (e.g., 37°C [98.6°F]) isothermal amplification that couples isothermal recombinase-driven primer targeting of a target molecule with strand-displacement DNA activity. RPA utilizes nucleoprotein complexes formed by oligonucleotide primers and recombinase proteins to guide and facilitate binding to a target DNA strand. Nucleic acid sequence-based amplification (NASBA) is an isothermal, transcription-based amplification method designed for the amplification of singlestranded RNA or DNA sequence and performed at a constant temperature of 41 °C (105.8°F).

[0175] The method can comprise contacting the single-stranded 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, in (b) the cut exposes an extendable 3' end of the target sequence. In some embodiments, the method further comprises reverse-transcribing the single- stranded nucleic acid molecule from an RNA.

[0176] The guide polynucleotide provided herein can be a forward guide polynucleotide (e.g., Forward Guide Oligo) configured for processing the target nucleic acid molecule in a reaction. The reaction can further comprise a reverse guide polynucleotide (e.g., Reverse Guide Oligo) configured for processing the target nucleic acid molecule or a reverse complement of the target nucleic acid molecule in the reaction. [0177] The enzyme described herein can comprise a type Ils restriction enzyme. The type Ils restriction enzyme can comprise one or more enzymes selected from the group consisting of BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, N.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, Nt.BpulOI, and any combinations thereof. The type Ils restriction enzyme can comprise type Ils restriction endonucleases such as N.BstNBI, N.BspD6I, N.Bst9 I and Nt.BstNBI, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutl, or any combination thereof. Alternatively, the type Ils restriction enzyme can comprise BfuAI, BsmAI, BsrDI, or BtsIMutl. Additional examples of type Ils restriction enzymes can be found at www.neb.com/tools-and- resources/selection-charts/type-iis-restriction-enzymes, which is herein incorporated by reference. [0178] In some embodiments, the type Ils restriction enzyme comprises an engineered type Ils restriction enzyme that has a nuclease-inactivating mutation in one of its two subunits to create a restriction endonuclease from an enzyme that is not naturally a restriction endonuclease. In some embodiments, the type Ils restriction enzyme comprises an engineered type Ils restriction enzyme that has a mutation in one of its two subunits that create different rates of enzymatic activity of cutting one strand over the opposite strand. In some cases, the enzyme comprises two enzymes with different activities or activity rates. In some cases, the enzyme can comprise a subunit of a type Ils restriction enzyme. In some cases, the enzyme can comprise a subunit of a restriction endonuclease. In some cases, the enzyme can comprise an activity for introducing a cut on the target nucleic acid sequence. For example, the enzyme can be N.BspD6I. In some cases, the enzyme can comprise an activity for introducing a cut on the complementary strand of the target nucleic acid sequence. In some cases, the enzyme can comprise an activity for introducing a cut on the guide polynucleotide (e.g., the target binding region of the guide polynucleotide). For example, the enzyme can be Nt.BstNBI. The enzyme can exhibit two different activities. The enzyme can exhibit a high-frequency endonuclease activity. In some embodiments, the high-frequency endonuclease activity is from a large subunit of the enzyme. The enzyme can exhibit a low- frequency endonuclease activity. In some embodiments, the low-frequency endonuclease activity is from a small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two differential enzymatic activity rates. In some embodiments, the at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites. In some embodiments, one of the two differential endonuclease activity rates comprises cutting the target sequence of the single-stranded nucleic acid molecule with low frequency. In some embodiments, one of the two differential endonuclease activity rates comprises cutting the target binding region of the guide polynucleotide with high frequency. In some embodiments, the two differential endonuclease activity rates are asymmetric or non-equal. In some embodiments, the enzyme comprises two different active sites or endonuclease domains conferring at least two differential enzymatic activities. In some embodiments, the target sequence comprises a recognition site specifically recognized by the enzyme or a first activity of the at least two differential enzymatic activities of the enzyme to introduce a cut.

[0179] In some embodiments, the target binding region of the guide polynucleotide comprises a recognition site specifically recognized by the enzyme or a second activity of the at least two differential enzymatic activities of the enzyme to introduce a cut.

[0180] The method of processing the single-stranded nucleic acid molecule can further comprise introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence. Optimal temperatures for specific type Ils restriction enzymes can be found in e.g. the Rebase database (accessible at http://rebase.neb.com/rebase/rebase.html).

[0181] In some aspects, the present disclosure provides for a method of amplifying a singlestranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting the single-stranded 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 a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end non- extendable by a polymerase; (b) introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in 5' to 3' order. In some embodiments, the nontarget 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. In some embodiments, the guide polynucleotide is a first guide polynucleotide, and the guide complex comprises a second guide polynucleotide, wherein the second guide polynucleotide comprises (i) a non-target binding region that is complementary with the non-target binding region of the first guide polynucleotide and (ii) a target binding region configured to hybridize to the target sequence. In some cases, when the first guide polynucleotide of the guide complex is hybridized to the target polynucleotide sequence, the target binding region of the second guide polynucleotide of the guide complex is not hybridized to the target sequence. In some embodiments, the first guide polynucleotide and the second guide polynucleotide of the guide complex hybridize to form a dimer. In some embodiments, the first guide polynucleotide and the second guide polynucleotide of the guide complex hybridize at a common 5' region. In some embodiments, the first guide polynucleotide and the second guide polynucleotide hybridize via the non-target binding region of the first guide polynucleotide and the second guide polynucleotide to form the dimer having a double-stranded binding region. In some embodiments, the double-stranded binding region comprises the restriction endonuclease recognition sequence. In some embodiments, the type Ils restriction enzyme binds to the doublestranded binding region of the dimer. A forward guide polynucleotide (or complex) can comprise one or more guide polynucleotides including the first guide polynucleotide and the second guide polynucleotide described herein. The first guide polynucleotide and the second guide polynucleotide can be homodimer or heterodimer. For example, the non-target binding region at the 5’ end of the first guide polynucleotide and the non-target binding region at the 5’ end of the second guide polynucleotide can comprise the same sequence (e.g., a palindromic sequence), and the target binding region at the 3’ end of the first or the second guide polynucleotide can be different. In some embodiments, a target binding region can be configured to hybridize to a target sequence. Alternatively, a target binding region can be configured to hybridize to a different target sequence.

[0182] In some cases, a reverse guide polynucleotide (or complex) can comprise a plurality of guide polynucleotides including the first guide polynucleotide and the second guide polynucleotide. In some cases, a reverse guide polynucleotide and a forward guide polynucleotide can comprise a same sequence (e.g., a palindromic sequence) at the 5’ end such that the reverse guide polynucleotide and the forward guide polynucleotide can hybridize to form a heterodimer. The target binding region of the forward guide polynucleotide and the target binding region of the reverse guide polynucleotide can comprise different sequences.

[0183] In some aspects, the present disclosure provides for a method of amplifying a singlestranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting a guide complex with the single-stranded nucleic acid molecule, wherein the guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with the target sequence of the single- stranded nucleic acid molecule, and (i) a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region binds to an enzyme; and (b) cutting the target sequence using the enzyme to expose an extendable 3' end of the target sequence. In some cases, an extendable 3' end is a 3' hydroxyl group. In some embodiments, if a target molecule is an RNA, the method can further comprise reverse-transcribing, prior to contacting the target molecule with the guide complex, the single-stranded nucleic acid molecule from the RNA. For example, the target RNA molecule can be reverse transcribed using a reverse transcriptase to generate a DNA molecule, which can be subject to further processing using the methods described herein. The DNA molecule can be a single- stranded DNA molecule (ssDNA). In some cases, a reverse transcription reaction can be used to make a ssDNA target from an initial RNA target. In some cases, a reverse transcription reaction can comprise a reverse transcriptase and a reverse transcription primer. The reverse transcriptase can comprise avian myeloblastosis virus (AMV) reverse transcriptase (RT), Moloney murine leukemia virus RT (M-MLV RT), telomerase RT, or human immunodeficiency virus type 1 RT (HIV-1 RT).

[0184] In some cases, the method of amplifying the single-stranded nucleic acid molecule comprising the target sequence further comprises extending the extendable 3' end of the target sequence with a polymerase to generate an extension product, wherein the extension product displaces the second guide polynucleotide. In some cases, the polymerase extension creates a double-stranded product displacing the second guide polynucleotide. In some embodiments, the extending comprises incubation in the presence of a DNA polymerase such as strand-displacing DNA polymerase, including any of the strand-displacing polymerases described herein. The extending can also comprise incubation in the presence of factors alongside the polymerase sufficient to add nucleotides to the 3' end, including dNTPs, appropriate buffering agents, and cofactors (e.g. divalent cations). The dNTPs may be natural or unnatural dNTPs. The natural dNTPs can comprise dATP, dCTP, dGTP, dTTP, and/or dUTP. The unnatural dNTPs can be a- thiol dNTPs (e.g., S-dNTPs). S-dNTPS can comprise dATPaS, dCTPaS, dGTPaS, and/or dTTPaS. [0185] In some cases, the method of amplifying the single-stranded nucleic acid molecule comprising the target sequence further comprises cutting the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide. In some embodiments the cutting can comprise introducing a type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut the first guide polynucleotide within the target binding region. In some embodiments, the extendable 3' end comprises a 3' hydroxyl.

[0186] In some cases, the method of amplifying the single-stranded nucleic acid molecule comprising the target sequence further comprises extending the extendable 3 ' end of the first guide polynucleotide using a polymerase to generate a complementary molecule of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule. The polymerase can be strand-displacing DNA polymerase, including any of the stranddisplacing polymerases described herein. The extending can also comprise incubation in the presence of factors alongside the polymerase sufficient to add nucleotides to the 3' end, including dNTPs, appropriate buffering agents, and cofactors (e.g., divalent cations). The dNTPs may be natural or unnatural dNTPs. The natural dNTPs can comprise dATP, dCTP, dGTP, dTTP, and/or dUTP. The unnatural dNTPs can be a-thiol dNTPs (e.g., S-dNTPs). S-dNTPS can comprise dATPaS, dCTPaS, dGTPaS, and/or dTTPaS.

[0187] In some embodiments, the second guide polynucleotide in the method of amplifying a single-stranded nucleic acid molecule comprising a target sequence comprises, from 5' to 3' (i) a non-target binding region that hybridizes with the non-target binding region of the first guide polynucleotide and (ii) a target binding region configured to hybridize with the target sequence. In some embodiments, the method further comprises prior to (b), cutting the first guide polynucleotide within the target binding region using the enzyme, wherein the guide complex dissociates from the single-stranded nucleic acid molecule. In some embodiments, the method further comprises cutting the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide and extending the extendable 3' end of the first guide polynucleotide using a polymerase to generate a complementary molecule of the target sequence of the singlestranded nucleic acid molecule repeatedly to generate a plurality of complementary molecules of the target sequence of the single-stranded nucleic acid molecule. In some embodiments, an additional guide complex binds to the complementary molecule. In some embodiments, the method further comprises using the complementary molecule with the additional guide complex bound thereto as a starting template to generate copies of the target molecule. In some embodiments, the enzyme is a type Ils restriction enzyme. In some embodiments, the type Ils restriction enzyme comprises N.BstNBI, N.Bst91 and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutl, BfuAI, BsmAI, BsrDI, BtsIMutl, a functional fragment thereof, or a combination thereof. In some embodiments, the guide polynucleotide comprises a blocked 3' end non-extendable by a polymerase. The blocked 3' end can comprise essentially any 3' chemical structure that prevents extension of the guide polynucleotide by a DNA polymerase, including any structures with such activity described herein. In some embodiments, the blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, or a spacer. In some embodiments, the singlestranded nucleic acid molecule with the cut and the guide polynucleotide bound thereto is used as a starting template for an amplification. In some embodiments, the amplification is an isothermal amplification. In some embodiments, the enzyme comprises asymmetric propensity to cleave one strand of a DNA duplex. In some embodiments, the enzyme exhibits a high-frequency endonuclease activity. In some embodiments, the high-frequency endonuclease activity is from a large subunit of the enzyme. In some embodiments, the enzyme exhibits a low-frequency endonuclease activity. In some embodiments, the low-frequency endonuclease activity is from a small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two differential enzymatic activity rates. In some embodiments, the at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites. In some embodiments, one of the two differential endonuclease activity rates comprises cutting the target sequence of the single-stranded nucleic acid molecule with low frequency. Cutting at the low frequency may be a rate limiting step (e.g., for determining a reaction launch time, as disclosed herein). In some embodiments, one of the two differential endonuclease activity rates comprises cutting the target binding region of the guide polynucleotide by with high frequency. In some embodiments, the two differential endonuclease activity rates are asymmetric or not equal. In some embodiments, the enzyme comprises N.BstNBI, N.Bst9 I and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutl, BfuAI, BsmAI, BsrDI, BtsIMutl, or a combination thereof.

Amplification Reaction Temperature

[0188] In some embodiments, a temperature is changed over the course of the method. In some embodiments, a first activity rate of the at least two differential enzymatic activity rates is favored at a first temperature, and a second activity rate of the at least two differential enzymatic activity rates is favored at a second temperature different from the first temperature. In some embodiments, changing a temperature during the course of a method disclosed herein (e.g., during or after an amplification reaction) changes a relative time to response value. In some embodiments, a first temperature wherein a first enzymatic activity rate is favored can be about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about

32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about

47°C, about 48°C, about 49°C, or about 50°C. In some embodiments, a first temperature wherein a first enzymatic activity rate is favored is between about 15°C-50°C, between about 20°C-45°C, between about 30°C-45°C, between about 30°C-40°C, or between about 32°C-39°C. In some embodiments, a second temperature wherein a second enzymatic activity rate is favored can be 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. In some embodiments, a second temperature wherein a second enzymatic activity rate is favored is between about 45°C-80°C, between about 50°C-80°C, between about 50°C-70°C, between about 50°C- 60°C, between about 52°C-58°C.

[0189] In some embodiments, a temperature may be changed over the course of the method for a period of time. For example, a temperature can be changed during a nucleic acid amplification reaction or subsequent to a nucleic acid amplification reaction. The period of time at which a temperature is changed may benefit the enzymatic activity rate during the reaction, and thereby change a relative time to response value. In some embodiments, a target nucleic acid molecule can comprise a plurality of different target nucleic acid molecules in a reaction mixture. Changing a temperature (e.g., from a first temperature to a second temperature) during or subsequent to a nucleic acid amplification reaction comprising two or more different target nucleic acid molecules can change a relative time to response value between the two or more different target nucleic acid molecules. A temperature change can comprise a first temperature or a second temperature. A first temperature can be maintained for a first period of time, changed to a second temperature, and then maintained at the second temperature for a second period of time. For example, during a nucleic acid amplification, a temperature can be maintained at a first temperature, changed to a second temperature, and then maintained at the second temperature until the end of the reaction. The first temperature can be greater than the second temperature. The first temperature can be less than the second temperature. In some embodiments, after changing from the first temperature to the second temperature, a temperature can change back to the first temperature during the reaction. In some embodiments, a temperature can be maintained at a first temperature for a first period of time until completion of a nucleic acid amplification, and then changed to a second temperature different from the first temperature after completion of the amplification reaction.

[0190] In some embodiments, a first temperature change or a second temperature change may occur over a duration of time of at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, or at least about 15 minutes. In some embodiments, a first temperature change or a second temperature change may occur over a duration of time of at most about 15 minutes, at most about 12 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6.5 minutes, at most about 6 minutes, at most about 5.5 minutes, at most about 5 minutes, at most about 4.5 minutes, at most about 4 minutes, at most about 3.5 minutes, at most about 3 minutes, at most about 2.5 minutes, at most about 2 minutes, at most about 1.5 minutes, at most about 1 minute, at most about 30 seconds, or at most about 15 seconds.

[0191] In some embodiments, a first temperature change or a second temperature change may occur over a duration of time from about 1 minute to about 15 minutes. In some embodiments, the sample may be heated from a range from about 1 minute to about 2 minutes, about 1 minute to about 2.5 minutes, about 1 minute to about 3 minutes, about 1 minute to about 3.5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 6 minutes, about 1 minute to about 7 minutes, about 1 minute to about 7.5 minutes, about 1 minute to about 10 minutes, about 1 minute to about 15 minutes, about 2 minutes to about 2.5 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 3.5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 5 minutes, about 2 minutes to about 6 minutes, about 2 minutes to about 7 minutes, about 2 minutes to about 7.5 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 15 minutes, about 2.5 minutes to about 3 minutes, about 2.5 minutes to about 3.5 minutes, about 2.5 minutes to about 4 minutes, about 2.5 minutes to about 5 minutes, about 2.5 minutes to about 6 minutes, about 2.5 minutes to about 7 minutes, about 2.5 minutes to about 7.5 minutes, about 2.5 minutes to about 10 minutes, about 2.5 minutes to about 15 minutes, about 3 minutes to about 3.5 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 5 minutes, about 3 minutes to about 6 minutes, about 3 minutes to about 7 minutes, about 3 minutes to about 7.5 minutes, about 3 minutes to about 10 minutes, about 3 minutes to about 15 minutes, about 3.5 minutes to about 4 minutes, about 3.5 minutes to about 5 minutes, about 3.5 minutes to about 6 minutes, about 3.5 minutes to about 7 minutes, about 3.5 minutes to about 7.5 minutes, about 3.5 minutes to about 10 minutes, about 3.5 minutes to about 15 minutes, about 4 minutes to about 5 minutes, about 4 minutes to about 6 minutes, about 4 minutes to about 7 minutes, about 4 minutes to about 7.5 minutes, about 4 minutes to about 10 minutes, about 4 minutes to about 15 minutes, about 5 minutes to about 6 minutes, about 5 minutes to about 7 minutes, about 5 minutes to about 7.5 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 15 minutes, about 6 minutes to about 7 minutes, about 6 minutes to about 7.5 minutes, about 6 minutes to about 10 minutes, about 6 minutes to about 15 minutes, about 7 minutes to about 7.5 minutes, about 7 minutes to about 10 minutes, about 7 minutes to about 15 minutes, about 7.5 minutes to about 10 minutes, about 7.5 minutes to about 15 minutes, or about 10 minutes to about 15 minutes. [0192] In some embodiments, a first temperature or a second temperature can be maintained for a duration of time of at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 17 minutes, at least about 20 minutes, at least about 22 minutes, at least about 25 minutes, at least about 27 minutes, or at least about 30 minutes. In some embodiments, a first temperature or a second temperature can be maintained for a duration of time of at most about 30 minutes, of at most about 27 minutes, of at most about 25 minutes, of at most about 22 minutes, of at most about 20 minutes, of at most about 17 minutes, of at most about 15 minutes, at most about 12 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6.5 minutes, at most about 6 minutes, at most about 5.5 minutes, at most about 5 minutes, at most about 4.5 minutes, at most about 4 minutes, at most about 3.5 minutes, at most about 3 minutes, at most about 2.5 minutes, at most about 2 minutes, at most about 1.5 minutes, at most about 1 minute, at most about 30 seconds, or at most about 15 seconds.

Guide polynucleotides

[0193] In some embodiments, a concentration of the guide polynucleotide is at least about 0.1 pM, at least about 1 pM, or about 0.1 pM to about 4 pM. In some embodiments, a concentration of the guide polynucleotide is at least about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.5 pM, 2.0 pM, 2.5 pM, 3.0 pM, 3.5 pM, 4 pM or more. In some embodiments, the non-target binding region comprises a palindromic sequence. In some embodiments, the non-target binding region is self-complementary or forms a self-annealing dimer under reaction conditions. In some embodiments, the non-target binding region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. In some embodiments, the non-target binding region is at most about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or less nucleotides in length. In some embodiments, the single- stranded nucleic acid molecule is a single-stranded deoxyribonucleic acid (ssDNA) or a single-stranded ribonucleic acid (ssRNA). In some embodiments, the method further comprises reverse-transcribing the singlestranded nucleic acid molecule from an RNA. In some embodiments, the target binding region comprises at least one peptide nucleic acid (PNA) residue. In some embodiments, the polymerase has strand displacement activity.

Cycle thresholds [0194] A method of analyzing a sample described herein can comprise detecting a signal (e.g., a first signal of a target nucleic acid molecule or a second signal of a refence nucleic acid molecule) to obtain a response value (e.g., a first time to response value or a second time to response value). The time to response value can be reflected by cycle threshold (Ct or Cq value). A “cycle threshold” can comprise a number of cycles needed for a signal (e.g., fluorescent signal) to exceed a background threshold level. A lower cycle threshold value can indicate a greater amount of target nucleic acid in a sample. In some embodiments, a nucleic acid amplification using the methods described herein can result in a lower cycle threshold compared to loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), or other amplification methods. In some embodiments, the methods described herein may result in a faster amplification result compared to nucleic acid amplification protocols without the programmed restriction enzyme. A metric of speed of an amplification may be a cycle threshold. A cycle threshold for a sample processing method described herein may be at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least 18%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% less than a cycle threshold for LAMP. A cycle threshold for a sample processing method described herein may be at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, or at most about 2% less than a cycle threshold for LAMP. A cycle threshold for a sample processing method described herein may be from about 1% to about 50% less than a cycle threshold for LAMP. A cycle threshold for a sample processing method described herein may be from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% less than a cycle threshold for LAMP.

[0195] In some embodiments, a cycle threshold value for a sample processing method described herein 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 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40. In some embodiments, a cycle threshold value for a sample processing method described herein may be at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1.

Second derivative method

[0196] A method of analyzing a sample described herein can comprise calculating a parameter of a signal (e.g., a time from the start of the reaction to the maximum value of the second derivative of the signal) to obtain a response value (e.g., a first time to response value or a second time to response value). The response value can be reflected by the time to the maximum of the second derivative of the signal (Cp value or second derivative). A “second derivative” can comprise a time needed for a signal (e.g., fluorescent signal) to reach the maximum value of its second derivative. A lower second derivative value can indicate a greater amount of target nucleic acid in a sample. In some embodiments, a nucleic acid amplification using the methods described herein can result in a lower second derivative (shorter time for the signal to reach the maximum value of its second derivative or Cp value) value compared to loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), or other amplification methods. In some embodiments, the methods described herein may result in a faster amplification result compared to nucleic acid amplification protocols without the programmed restriction enzyme. A metric of speed of an amplification can be a Cp value. A Cp for a sample processing method described herein may be at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least 18%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% less than a Cp for LAMP. A Cp for a sample processing method described herein may be at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about

-n- 20%, at most about 18%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, or at most about 2% less than a Cp for LAMP. A Cp for a sample processing method described herein may be from about 1% to about 50% less than a Cp for LAMP. A Cp for a sample processing method described herein may be from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about

50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about

15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about

10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about

25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% less than a Cp for LAMP.

[0197] In some embodiments, a Cp value for a sample processing method described herein may be at least about 0 seconds, at least about 1 us, at least about 1 ms, at least about 0.1 s, at least about 1 s, at least about 10 s, at least about 20, at least about 30 s, at least about 40 s, at least about 50 s, 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 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15, minutes at least about 18 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, or at least about 40 minutes. In some embodiments, a Cp value for a sample processing method described herein may be at most about 40 minutes, at most about 35 minutes, at most about 30 minutes, at most about 25 minutes, at most about 20 minutes, at most about 18 minutes, at most about 15 minutes, at most about 12 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, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, or at most about 1 minute.

Detections

[0198] The amplification product 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. 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).

[0199] A wide range of 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-carboxyfluorescein; 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-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro- Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5- Tetrachloro-Fluorescein ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6- carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4- dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2- aminoethyl)amino)naphthalene-l -sulfonic acid); 1,5-IAEDANS (5-((((2- iodoacetyl)amino)ethyl)amino)naphthalene-l -sulfonic acid); DABCYL (4-((4- (dimethylamino)phenyl) azo)benzoic acid) Cy5 (Indodicarbocyanine-5) Cy3 (Indo- dicarbocyanine-3); BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-proprionic acid); Quasar-670 (Bioresearch Technologies); CalOrange (Bioresearch Technologies); and Rox as well as suitable derivatives thereof. 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, DABMI 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.

[0200] A wide range of 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-carboxyfluorescein; 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-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro- Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5- Tetrachloro-Fluorescein ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6- carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4- dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2- aminoethyl)amino)naphthalene-l -sulfonic acid); 1,5-IAEDANS (5-((((2- iodoacetyl)amino)ethyl)amino)naphthalene-l -sulfonic acid); DABCYL (4-((4- (dimethylamino)phenyl) azo)benzoic acid) Cy5 (Indodicarbocyanine-5) Cy3 (Indo- dicarbocyanine-3); BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-proprionic acid); Quasar-670 (Bioresearch Technologies); CalOrange (Bioresearch Technologies); and Rox as well as suitable derivatives thereof. 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, DABMI 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

[0201] In some aspects, the present disclosure provides for a kit comprising any of the guide complexes or any of the guide polynucleotides described herein. In some embodiments, the kit comprises a reference nucleic acid molecule. The reference nucleic acid molecule can comprise a human nucleic acid sequence (e.g., RPP30). In some embodiments, the kit comprises one or more reaction reagents for nucleic acid amplification. The one or more reaction reagents can comprise one or more of enzymes (e.g., restriction enzymes or polymerases), primers, dNTPs, appropriate buffering agents, and cofactors (e.g., divalent cations). The dNTPs may be natural or unnatural dNTPs. The natural dNTPs can comprise dATP, dCTP, dGTP, dTTP, and/or dUTP. The unnatural dNTPs can be a-thiol dNTPs (e.g., S-dNTPs). S-dNTPS can comprise dATPaS, dCTPaS, dGTPaS, and/or dTTPaS.

[0202] In some embodiments, the kit further comprises a probe or a dye for detecting an amplification product generated using the kit. In some embodiments, the kit further comprises an informational material describing an instruction of using the kit (e.g., according to a method disclosed herein). In some embodiments, the information comprises optimal reaction temperatures for amplification using the guide complexes or the guide polynucleotides, or optimal buffer conditions for the same. In some embodiments, the kit comprises a standard value (e.g., a standard value in a method disclosed herein). In some embodiments, the kit further comprises a type II restriction enzyme compatible with the guide polynucleotides or guide complexes as described herein. In some embodiments, the kit further comprises a strand-displacing polymerase. The kits can be compartmentalized for ease of use and can include one or more containers with reagents. In some embodiments, all of the kit components are packaged together. Alternatively, one or more individual components of the kit can be provided in a separate package from the other kits components. [0203] In some aspects, the present disclosure provides kits for sample preparation and processing. Such kits may permit the automated processing of biological samples in a lab-free environment. The kit may comprise a device of a system. Devices and systems of the present disclosure may be portable, allowing users to employ such devices in remote locations, for example.

Multiplex

[0204] The methods, systems, or kits provided herein can be used to process or analyze one sample or one target nucleic acid molecule or target sequence. Alternatively, the methods, systems or kits provided herein can be used to process or analyze two or more different samples, or two or more different target nucleic acid molecules or target sequences in a same reaction mixture (e.g., a single reaction). Methods, systems, or kits provided herein can be used to process or analyze duplex reaction mixtures (e.g., processed to detect two different target nucleic acid molecules in a reaction mixture). Methods, systems, or kits provided herein can be used to process or analyze triplex reaction mixtures (e.g., processed to detect three different target nucleic acid molecules in a reaction mixture). Methods, systems, or kits provided herein can be used to process or analyze tetraplex reaction mixtures (e.g., processed to detect four different target nucleic acid molecules in a reaction mixture). Methods, systems, or kits provided herein can be used to process or analyze multiplex reaction mixtures (e.g., processed to detect two or more different target nucleic acid molecules in a reaction mixture). Methods, systems, or kits provided herein can be used to detect one or more target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect two or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect three or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect four or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect five or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect seven or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect nine or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect ten or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect twelve or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect fifteen or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect seventeen or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect twenty or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect 25 or more different target nucleic acid molecules in a reaction mixture. Methods, systems, or kits provided herein can be used to detect 50 or more different target nucleic acid molecules in a reaction mixture. For example, the methods, systems or kits provided herein can be used to process or analyze 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more different target nucleic acid sequences in a same reaction mixture. In some cases, a multiplexed reaction may contain more targets than the labels (e.g., fluorescent labels, colors, or dyes) used to detect them. For example, targets that increase or decrease in response to a condition (healthy vs. diseased) can be combined and be detected using the same label. In some cases, a multiplexed reaction may contain more targets than the color channels that an analytic device can detect.

[0205] In some embodiments, the reaction mixture is lyophilized. In some embodiments, the reaction mixture is not lyophilized.

[0206] In some aspects, the present disclosure provides for a method or a system of multiplexing the processing of more than one nucleic acid molecules, each nucleic acid molecule comprising a different target sequence. The method or system can comprise, for each nucleic acid molecule comprising a different target sequence, a nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide. The guide polynucleotide can comprise: (i) a nontarget binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end non-extendable by a polymerase. The enzyme can bind to the restriction endonuclease recognition sequence of the non-target binding region. In some aspects, a multiplexed processing of one or more nucleic acid molecules comprises using two or more different sets of primers or guide complexes, each targeting a different target. In some aspects, multiplexed processing of one or more nucleic acid molecules comprises a reaction mixture comprising two more different detection probes or fluorophores, each targeting a different target sequence. Each of the two or more different detection probes can be linked to a different fluorophore for multiplexed detection.

[0207] In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least 10, or more pluralities of single-stranded nucleic acid molecules can be analyzed in the same reaction. In some embodiments, each plurality of the multiplexed nucleic acid molecules is derived from a different sample. Computer Systems

[0208] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 3 shows a computer system 301 that can be programmed or otherwise configured to analyze target nuclei acid molecules, or to perform the methods of analyzing a sample described herein. For example, the computer system can be programmed or otherwise configured to subject a reaction mixture to conditions sufficient to perform the nucleic acid amplification reaction, detect signals from the nucleic acid amplification reaction, and/or determine the relative time to response value of the target nucleic acid molecule and the reference nucleic acid molecule as described herein. The computer system 301 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0209] The computer system 301 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 305, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 301 also includes memory or memory location 310 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 315 (e.g., hard disk), communication interface 320 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 325, such as cache, other memory, data storage and/or electronic display adapters. The memory 310, storage unit 315, interface 320 and peripheral devices 325 are in communication with the CPU 305 through a communication bus (solid lines), such as a motherboard. The storage unit 315 can be a data storage unit (or data repository) for storing data. The computer system 301 can be operatively coupled to a computer network (“network”) 330 with the aid of the communication interface 320. The network 330 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 330 in some cases is a telecommunication and/or data network. The network 330 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 330, in some cases with the aid of the computer system 301, can implement a peer-to-peer network, which may enable devices coupled to the computer system 301 to behave as a client or a server.

[0210] The CPU 305 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 310. The instructions can be directed to the CPU 305, which can subsequently program or otherwise configure the CPU 305 to implement methods of the present disclosure. Examples of operations performed by the CPU 305 can include fetch, decode, execute, and writeback. [0211] The CPU 305 can be part of a circuit, such as an integrated circuit. One or more other components of the system 301 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0212] The storage unit 315 can store files, such as drivers, libraries and saved programs. The storage unit 315 can store user data, e.g., user preferences and user programs. The computer system 301 in some cases can include one or more additional data storage units that are external to the computer system 301, such as located on a remote server that is in communication with the computer system 301 through an intranet or the Internet.

[0213] The computer system 301 can communicate with one or more remote computer systems through the network 330. For instance, the computer system 301 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 301 via the network 330.

[0214] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 301, such as, for example, on the memory 310 or electronic storage unit 315. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 305. In some cases, the code can be retrieved from the storage unit 315 and stored on the memory 310 for ready access by the processor 305. In some situations, the electronic storage unit 315 can be precluded, and machine-executable instructions are stored on memory 310.

[0215] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[0216] Aspects of the systems and methods provided herein, such as the computer system 301, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machineexecutable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0217] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0218] The computer system 301 can include or be in communication with an electronic display 335 that comprises a user interface (UI) 340 for providing, for example, analysis of amplification data. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface. [0219] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 305. The algorithm can, for example, analyze single- stranded nucleic acid molecule processing data.

EXAMPLES

Example 1: Triplexed Isothermal Amplification

[0220] In these experiments an isothermal strand displacement amplification was run on samples comprising Nc/.s.sc/va gonorrhoeas , Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). Ml Sample Prep® was performed using 400 uL of frozen sample culture, resuspended in approximately 800 uL of total culture. The target quantitation was based on an Ml Sample Prep® with an assumed 100% recovery from sample culture.

[0221] The readout of this experiment was the relative quantitation in the cycle threshold (Ct; Time to Result; time to response) value between two or more targets in an amplification reactions. The amplification reactions were optimized so that they predictably amplified the targets in a healthy sample state. The targets were optimized at the same time to exhibit competitive amplification once the healthy sample state was disturbed. This reaction optimization relied on knowledge of changes that occurred with targets in sample states other than the healthy state.

[0222] In a first experiment, an isothermal strand displacement amplification was run on samples comprising Neisseria gonorrhoeas (ATCC 19424 culture (Im Quest BioSciences Inc. (ATCC))), Chlamydia trachomatis (ATCC vr885 Serovar D culture (ImQuest BioSciences Inc. (ATCC))), and Ribonuclease P/MRP subunit p30 (RPP30). C. trachomatis RNA in varying titrations was amplified in 250 picogram/ reaction of purified human RNA (RPP30). The titrations of C. trachomatis RNA used in this experiment were (in IFU/Rxn): 29375; 2937.5; 293.75; 29.375; 2.9375; 0.29375; 0.029375; and 0 (no target). The Time to Result (time to response of C. trachomatis RNA or RPP30 RNA amplification product; Ct value; time to response) was considered. The results of this experiment are depicted in FIG. 1A and the data are provided in

Table 1

[0223] Table 1 : Time to Result in isothermal amplification of C. trachomatis RNA titrations in 250 pg/reaction of RPP30 RNA

[0224] In this reaction, the Time to Result (detection or time to response value) of C. trachomatis RNA was negatively correlated with the concentration of C. trachomatis RNA present in the reaction (R² = 0.9769), while the Time to Result of RPP30 was stable across different concentration of C. trachomatis RNA present in the reaction. The abundance of C. trachomatis RNA could be determined by considering the Time to Result value for C. trachomatis (e.g., relative to the Time to Result value of RPP30).

[0225] In a second experiment, an isothermal strand displacement amplification was run on samples comprising Neisseria gonorrhoeae, Chlamydia trachomatis, and Ribonuclease P/MR.P subunit p30 (RPP30). N. gonorrhoeae RNA in varying titrations was amplified in 250 picogram/ reaction of purified human RNA (RPP30). The titrations of N. gonorrhoeae RNA used in this experiment were (in IFU/Rxn): 17750; 1775; 177.5; 17.75; 1.775; 0.1775; 0.01775; and 0 (no target). The Time to Result (detection of N. gonorrhoeae RNA or RPP30 RNA amplification product) was considered. The results of this experiment are depicted in FIG. IB and the data are provided in Table 2.

[0226] Table 2 : Time to Result in isothermal amplification of N gonorrhoeae RNA titrations in 250 pg/reaction of RPP30 RNA [0227] In this reaction, the Time to Result (detection) of N. gonorrhoeae RNA was negatively correlated with the concentration of N. gonorrhoeae RNA present in the reaction (R² = 0.8862), while the Time to Result of RPP30 was stable across different concentration of N. gonorrhoeae RNA present in the reaction. The abundance of N. gonorrhoeae RNA could be determined by considering the Time to Result value for A. gonorrhoeae (e.g., relative to the Time to Result value ofRPP30).

[0228] In a third experiment, an isothermal strand displacement amplification was run on samples comprising Ac /.s.sc/va gonorrhoeae , Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). A gonorrhoeae RNA and C. trachomatis RNA in varying titrations were amplified in 250 picogram/ reaction of purified human RNA (RPP30). The Time to Result (detection of A gonorrhoeae RNA, C. trachomatis RNA, or RPP30 RNA amplification product) was considered. The results of this experiment are depicted in FIG. 1C.

[0229] In this reaction, the Time to Result (detection) of A gonorrhoeae RNA and C. trachomatis RNA was negatively correlated with the concentration of A gonorrhoeae RNA and C. trachomatis RNA present in the reaction, while the Time to Result of RPP30 was stable across different concentration of A gonorrhoeae RNA and C. trachomatis RNA present in the reaction. The abundance of A gonorrhoeae RNA and C. trachomatis RNA could be determined by considering the Time to Result value for the A gonorrhoeae and C. trachomatis (e.g., relative to the Time to Result value of RPP30).

Example 2: Tetraplexed Isothermal Amplification

[0230] In these experiments an isothermal strand displacement amplification was run on samples comprising Trichomonas vaginalis (ATCC 30001 culture (ImQuest BioSciences Inc. (ATCC))), Neisseria gonorrhoeae (ATCC 19424 culture (ImQuest BioSciences Inc. (ATCC))), Chlamydia trachomatis (ATCC vr885 Serovar D culture (ImQuest BioSciences Inc. (ATCC))), and Ribonuclease P/MRP subunit p30 (RPP30). Ml Sample Prep® was performed using 400 uL of frozen sample culture, resuspended in approximately 800 uL of total culture. The target quantitation was based on an Ml Sample Prep® with an assumed 100% recovery from sample culture.

[0231] The readout of this experiment was the relative quantitation in the cycle threshold (Ct; Time to Result; time to response) value between two or more targets in an amplification reactions. The amplification reactions were optimized so that they predictably amplified the targets in a healthy sample state. The targets were optimized at the same time to exhibit competitive amplification once the healthy sample state was disturbed. This reaction optimization relied on knowledge of changes that occurred with targets in sample states other than the healthy state.

[0232] In a first experiment, an isothermal strand displacement amplification was run on samples comprising Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). Ml purified T. vaginalis RNA in varying titrations was amplified in 250 picogram/ reaction of purified human RNA (RPP30). The titrations of T. vaginalis RNA used in this experiment were (in cells/Rxn): 74250; 7425; 742.5; 74.25; 7.425; 0.7425; 0.07425; and 0 (no target). The RFU for T. vaginalis and for RPP30 as a function of amplification cycle are shown in FIG. 2A and FIG. 2B, respectively. The Time to Result (time to detection of T. vaginalis,' time to response; Ct value) of T. vaginalis RNA is provided in FIG. 2C, and the data are provided in Table 3.

[0233] Table 3: Time to Result in isothermal amplification of T. vaginalis RNA titrations in 250 pg/reaction of RPP30 RNA

[0234] In this reaction, the Time to Result (detection or time to response value) of T. vaginalis RNA was negatively correlated with the concentration of T. vaginalis RNA present in the reaction (R² = 0.9475). The abundance of T. vaginalis RNA could be determined by considering the Time to Result value for T. vaginalis (e.g., relative to the Time to Result value of RPP30).

[0235] In a second experiment, an isothermal strand displacement amplification was run on samples comprising Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). Ml purified T. vaginalis, N. gonorrhoeae, and C. trachomatis RNA in varying titrations were amplified in 250 picogram/ reaction of purified human RNA (RPP30). The titrations of T. vaginalis, N. gonorrhoeae, and C. trachomatis RNA used in this experiment were (in cells/IFU/CFU per reaction): 10,000; 1,000; 1000 and 0 (no target cells). The RFU for T. vaginalis, N. gonorrhoeae, and C trachomatis as a function of time for concentrations of 10,000; 1,000; 100; and 0 (no target cells) cells/IFU/CFU per reaction are shown in FIG. 2D, FIG. 2E, FIG. 2F, and FIG. 2G, respectively

[0236] These data show the dynamics of the competitive isothermal amplification reactions comprising Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Ribonuclease P/MRP subunit p30 (RPP30). At lower concentrations of Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis present in the reaction, there was a higher RFU at each time for RPP30 than when higher concentrations of T. vaginalis, N. gonorrhoeae, and C. trachomatis were present (compare, e.g., FIG. 2D to FIG. 2F). Additionally, at lower concentrations of T. vaginalis, N. gonorrhoeae, and C. trachomatis, the time to launch of RPP30 was shorter than when higher concentrations were present (compare, e.g., FIG. 2D to FIG. 2F). Finally, no T. vaginalis, N. gonorrhoeae, and C. trachomatis were detected in the reaction when there were no T. vaginalis, N. gonorrhoeae, and C. trachomatis cells present in the reaction (FIG. 2G).

Example 3: Isothermal Amplification with Different Lysis Buffer Compositions

[0237] This experiment used lysis buffer with either Tris or sodium acetate and evaluated the effects on the resulting amplification of target products. Reagents and concentrations for the amplification reaction are shown in Table 4.

[0238] Table 4: Experimental reagents.

[0239] “BST” denotes the polymerase, “Nt.BstNBI” denotes the endonuclease, and AMV reverse transcriptase (avian myeloblastosis virus reverse transcriptase) was the reverse transcriptase. The terms “IL1RN” and “MCTP1” denote amplification targets. The concentrations of each component in the lysis buffer can vary. The lysis buffer was prepared with either sodium acetate or Tris. When prepared with sodium acetate, the surfactant was present in the presence of the sample at a concentration of about 0.2% to 0.8%; EGTA was present in the presence of the sample at a concentration of about 1 mM to 6 mM; and sodium acetate was present in the presence of the sample at a concentration of about 10 mM to 75 mM. The sodium acetate had a pH of about 4 to 7. As an example, the lysis buffer used can comprise 0.5% surfactant, 4 mM EGTA, and 50 mM sodium acetate final concentration for each component in the presence of the sample. When prepared with sodium acetate, the surfactant was present in the presence of the sample at a concentration of about 0.2% to 0.8%; EGTA was present in the presence of the sample at a concentration of about 1 mM to 6 mM; and Tris was present in the presence of the sample at a concentration of about 10 mM to 75 mM. The Tris had a pH of about 6 to 9. As an example, the lysis buffer used can comprise 0.5% surfactant, 4 mM EGTA, and 50 mM Tris final concentration for each component in the presence of the sample.

[0240] 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 100 mM to 150 mM, EDTA was present in the presence of the sample at a concentration of about 2 mM to 8 mM; polysorbate 80 was present in the presence of the sample at a concentration of about 0.5% v/v to 5.0% v/v; magnesium sulfate (MgSCU) was present in the presence of the sample at a concentration of about 30 mM to 100 mM; sodium sulfate (NaSCU) was present in the presence of the sample at a concentration of about 30 mM to 100 mM; ammonium sulfate (NH4SO4) was present in the presence of the sample at a concentration of about 30 mM to 100 mM; and Tris acid was present in the presence of the sample at a concentration of about 200 mM to 300 mM. As an example, the recovery buffer used can comprise 120 mM cyclodextrin, 4 mM EDTA, 4.8% v/v polysorbate 80, 60 mM MgSO4, 60 mM NaSCU, 60 mM NH4SO4, and 240 mM of Tris acid final concentration for each component in the presence of the sample.

[0241] The experiment was conducted at timepoints TO and T2H. TO corresponds to “time zero”, which refers to the sample assayed after being lysed. T2H corresponds to a timepoint at which the sample is lysed, then assayed after 2 hour room temperature (~22°C) incubation. The number of dropouts in the amplification reaction were compared between samples prepared with lysis buffer comprising sodium acetate or lysis buffer comprising Tris (FIG. 5A). Samples prepared with lysis buffer comprising sodium acetate showed fewer dropouts compared to those samples prepared with lysis buffer comprising Tris. The coefficient of variation was also measured in the amplification reaction between samples prepared with lysis buffer comprising sodium acetate or lysis buffer comprising Tris (FIG. 5B). The coefficient of variation (CV) informs about the relative variability or dispersion of a dataset by comparing the standard deviation to the mean. A lower CV can indicate less variability or dispersion around the mean. As shown in FIG. 5B, samples prepared with lysis buffer comprising sodium acetate showed lower CV values compared to those samples prepared with lysis buffer comprising Tris.

[0242] Time to detection, measured in minutes, was also evaluated and compared between samples prepared with lysis buffer comprising sodium acetate or lysis buffer comprising Tris (FIGs. 6A-6C). For target IL1RN, samples prepared with lysis buffer comprising either sodium acetate or Tris showed similar times to detection in the amplification reaction (FIG. 6A). For the blood sample of patient 2 (P2_Blood), there was no detection at timepoint T2H with samples prepared using lysis buffer comprising Tris. For target MCTP1, samples prepared with lysis buffer comprising either sodium acetate or Tris showed similar times to detection in the amplification reaction (FIG. 6B) For the blood sample of patient 2 (P2_Blood), there was no detection at timepoint T2H with samples prepared using lysis buffer comprising Tris. A summary of the patient data in FIG. 6A is shown in FIG. 6C, with all patients combined for each timepoint.

Example 4: Analysis of Freeze-Thaw Stability in Samples Prepared with Different Lysis Buffer Compositions

[0243] This experiment used lysis buffer with either Tris or sodium acetate and evaluated the effects on the resulting amplification of target products. Reagents and concentrations for the amplification reaction are shown in Table 5.

[0244] Table 5: Experimental reagents. [0245] “BST” denotes the polymerase, “Nt.BstNBI” denotes the endonuclease, and AMV reverse transcriptase (avian myeloblastosis virus reverse transcriptase) was the reverse transcriptase. The terms “IL1RN” and “MCTP1” denote amplification targets. The concentrations of each component in the lysis buffer can vary. The lysis buffer was prepared with either sodium acetate or Tris. When prepared with sodium acetate, the surfactant was present in the presence of the sample at a concentration of about 0.2% to 0.8%; EGTA was present in the presence of the sample at a concentration of about 1 mM to 6 mM; and sodium acetate was present in the presence of the sample at a concentration of about 10 mM to 75 mM. The sodium acetate had a pH of about 4 to 7. As an example, the lysis buffer used can comprise 0.5% surfactant, 4 mM EGTA, and 50 mM sodium acetate final concentration for each component in the presence of the sample. When prepared with sodium acetate, the surfactant was present in the presence of the sample at a concentration of about 0.2% to 0.8%; EGTA was present in the presence of the sample at a concentration of about 1 mM to 6 mM; and Tris was present in the presence of the sample at a concentration of about 10 mM to 75 mM. The Tris had a pH of about 6 to 9. As an example, the lysis buffer used can comprise 0.5% surfactant, 4 mM EGTA, and 50 mM Tris final concentration for each component in the presence of the sample.

[0246] 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 100 mM to 150 mM, EDTA was present in the presence of the sample at a concentration of about 2 mM to 8 mM; polysorbate 80 was present in the presence of the sample at a concentration of about 0.5% v/v to 5.0% v/v; magnesium sulfate (MgSCU) was present in the presence of the sample at a concentration of about 30 mM to 100 mM; sodium sulfate (NaSCU) was present in the presence of the sample at a concentration of about 30 mM to 100 mM; ammonium sulfate (NH4SO4) was present in the presence of the sample at a concentration of about 30 mM to 100 mM; and Tris acid was present in the presence of the sample at a concentration of about 200 mM to 300 mM. As an example, the recovery buffer used can comprise 120 mM cyclodextrin, 4 mM EDTA, 4.8% v/v polysorbate 80, 60 mM MgSO4, 60 mM NaSCU, 60 mM NH4SO4, and 240 mM of Tris acid final concentration for each component in the presence of the sample.

[0247] Four timepoints were assessed in the amplification reaction: TO, T2H, and then each timepoint with a freeze-thaw period (T0_F/Txl and T2H_F/Txl, respectively). TO corresponded to “time zero”, which refers to the sample assayed after being lysed. T0_F/Txl corresponded to a timepoint at which the sample is frozen (e.g., frozen at -80°C) immediately after lysis, thawed, then assayed. T2H corresponded to a timepoint at which the sample is lysed, then assayed after 2 hour room temperature (~22°C) incubation. T2H_F/Txl corresponded to a timepoint at which the sample is lysed, incubated at room temperature for 2 hours, frozen at -80°C, thawed, then assayed. Time to detection was measured in minutes for samples prepared with lysis buffer comprising sodium acetate or lysis buffer comprising Tris. The two targets were IL1RN and MCTP1. As shown in FIGs. 7A-7B, for both targets, samples prepared with lysis buffer comprising sodium acetate showed improved freeze-thaw stability over those samples prepared with lysis buffer comprising Tris.

[0248] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of analyzing a sample having or suspected of having a target nucleic acid molecule, the method comprising:

(a) providing a reaction mixture comprising said sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction;

(b) subjecting said reaction mixture to conditions sufficient to perform said nucleic acid amplification reaction, wherein said nucleic acid amplification reaction is performed under conditions such that said target nucleic acid molecule and said reference nucleic acid molecule compete for said reagents, and wherein during said nucleic acid amplification reaction a reaction launch time of said target nucleic acid molecule varies depending on an abundance of said target nucleic acid molecule relative to said reference nucleic acid molecule;

(c) detecting (1) a first signal of said target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of said reference nucleic acid molecule to obtain a second time to response value; and

(d) determining a relative time to response value of said first time to response value and said second time to response value, wherein said relative time to response value is indicative of a presence or absence of said target nucleic acid molecule in said sample.

2. A method of analyzing a sample having or suspected of having a target nucleic acid molecule, the method comprising:

(b) subjecting said reaction mixture to conditions sufficient to perform said nucleic acid amplification reaction, wherein said nucleic acid amplification reaction is performed under conditions such that said target nucleic acid molecule and said reference nucleic acid molecule compete for said reagents;

(c) detecting (1) a first signal of said target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of said reference nucleic acid molecule to obtain a second time to response value; and (d) determining a relative time to response value of said first time to response value and said second time to response value, wherein said relative time to response value is indicative of a presence or absence of said target nucleic acid molecule in said sample, wherein said second time to response value of said reference nucleic acid molecule varies depending on an abundance of said target nucleic acid molecule relative to said reference nucleic acid molecule.

3. The method of claim 1 or 2, further comprising, subsequent to (d), determining based on said relative time to response value whether said target nucleic acid is present in the sample.

4. The method of any one of claims 1-3, further comprising, subsequent to (d), determining based on said relative time to response value an amount of said target nucleic acid molecule in said sample.

5. The method of any one of claims 1-4, wherein said conditions comprise a sub-optimal condition for said nucleic acid amplification reaction, and wherein said sub-optimal condition comprises less than 100%, less than 95%, less than 90%, less than 85%, or less than 80% amplification efficiency of said target nucleic acid and/or said reference nucleic acid molecule.

6. The method of any one of claims 1 and 3-5, wherein said reaction launch time is a time period from subjecting said reaction mixture to conditions sufficient to perform said nucleic acid amplification reaction to initiation of said nucleic acid amplification by a polymerase of said target nucleic acid molecule.

7. The method of claim 6, wherein said reaction launch time is measured by a fluorescence detection method or an electrochemical method.

8. The method of claim 7, wherein said fluorescence detection method comprises molecular beacon.

9. The method of claim 7, wherein said electrochemical method comprises detecting an electrochemical signal of said nucleic acid amplification reaction.

10. The method of any one of claims 1-9, wherein changing compositions or concentrations of one or more reagents of said reagents changes said relative time to response value.

11. The method of claim 10, wherein said one or more reagents comprises a salt, a surfactant, a polyol, a polymer, a sugar, a polyamine, or any combinations thereof.

12. The method of claim 11, wherein said salt comprises a carbonate salt, a bicarbonate salt, a sulfate salt, a guanidine salt, a chloride salt, a lithium salt, or any combinations thereof.

13. The method of claim 12, wherein said carbonate salt comprises ammonium carbonate, magnesium carbonate, or any combination thereof.

14. The method of claim 12, wherein said bicarbonate salt comprises sodium bicarbonate.

15. The method of claim 12, wherein said sulfate salt comprises sodium sulfate, magnesium sulfate, ammonium sulfate, or any combination thereof.

16. The method of claim 12, wherein said guanidine salt comprises guanidine hydrochloride, guanidine thiocyanate, guanidine sulfate, guanidine carbonate, or any combination thereof.

17. The method of claim 12, wherein said chloride salt comprises sodium chloride, potassium chloride, magnesium chloride, or any combination thereof.

18. The method of claim 12, wherein said lithium salt comprises lithium chloride.

19. The method of claim 11, wherein said surfactant comprises a cationic surfactant, an anionic surfactant, and nonionic surfactant, an amphoteric surfactant, a sulfate, a sulfonate, a carboxylate, a poloxamer, a zwitterionic surfactant, a Gemini surfactant, a polymeric surfactant, a co-block polymer surfactant, or any combination thereof.

20. The method of claim 11, wherein said sugar is a non-reducing sugar.

21. The method of claim 20, wherein said non-reducing sugar is trehalose, sucrose, raffinose, or any combination thereof.

22. The method of claim 11, wherein said polyol is mannitol.

23. The method of claim 11, wherein said polymer is dextran, ficoll, or any combination thereof.

24. The method of claim 11, wherein said polyamine is a linear polyamine, a branched polyamine, or any combination thereof.

25. The method of claim 11, wherein said polyamine is spermine, spermidine, (bis)aminopropylspermidine, Tetrakis(3-aminopropyl)-l,4-butanediamine, or any combination thereof.

26. The method of any one of claims 1-25, further comprising, prior to (a), processing said sample with a sample processing buffer.

27. The method of claim 26, wherein said sample processing buffer comprises a lysis buffer.

28. The method of claim 27, wherein said lysis buffer comprises sodium acetate, 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), tetrahydropyran (THP), or any combination thereof.

29. The method of claim 27 or 28, wherein said lysis buffer comprises sodium acetate.

30. The method of any one of claims 27-29, wherein said sodium acetate is present in said lysis buffer mixed with said sample at a final concentration of about 20 mM to 80 mM.

31. The method of of any one of claims 27-30, wherein said sodium acetate is present at a pH from about 3 to 6.

32. The method of any one of claims 29-31, wherein said sodium acetate is configured to improve a freeze-thaw stability of the sample in said nucleic acid amplification reaction.

33. The method of any one of claims 27-32, wherein said lysis buffer further comprises a chelating agent.

34. The method of claim 33, wherein said 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).

35. The method of any one of claims 27-34, wherein said lysis buffer further comprises a reducing agent.

36. The method of claim 35, wherein said 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'-tetraacetic acid (BAPTA), or tetrahydropyran (THP).

37. The method of any one one of claims 26-36, wherein said sample processing buffer comprises a recovery buffer.

38. The method of claim 37, wherein said recovery buffer comprises a cyclodextrin, an ethylenediaminetetraacetic acid (EDTA), a solubilizer, a Tris, magnesium sulfate, ammonium sulfate, sodium sulfate, or any combination thereof.

39. The method of claim 38, wherein said solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

40. The method of claim 38 or 39, wherein said 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,

3 A-amino-3 A-deoxy-(2AS,3 AS)-y-cyclodextrin hydrate, an anionic cyclodextrin, or any combination thereof.

41. The method of claim 10 or 11, further comprising changing compositions or concentrations of said one or more reagents of said reagents.

42. The method of any one of claims 10-41, wherein said target nucleic acid molecule comprises two or more different target nucleic acid molecules in said reaction mixture, and wherein changing said compositions or concentrations of said one or more reagents of said reagents changes a relative time to response value between two or more different target nucleic acid molecules.

43. The method of any one of claims 1-9, wherein said first time to response value of said target nucleic acid molecule at a concentration of higher than 1 copy/reaction is at least 2- fold less than said first time to response value of said target nucleic acid molecule at a concentration of less than 1 copy/reaction.

44. The method of any one of claims 1-43, wherein said time to response value is a Ct value.

45. The method of any one of claims 1-44, wherein said nucleic acid amplification reaction is a real-time nucleic acid amplification reaction.

46. The method of any one of claims 1-45, wherein said nucleic acid amplification reaction is an isothermal amplification.

47. The method of claim 46, wherein said isothermal amplification reaction comprises subjecting said reaction mixture to a constant temperature.

48. The method of claim 46 or 47, wherein said reaction mixture of said isothermal amplification reaction further comprises a guide polynucleotide comprising a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and a target binding region configured to hybridize to a target sequence.

49. The method of claim 48, wherein said enzyme exhibits at least two differential enzymatic activity rates.

50. The method of claim 49, wherein said at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites.

51. The method of claim 50, wherein one of said two differential endonuclease activity rates comprises cutting said target sequence of said target nucleic acid molecule with low frequency.

52. The method of claim 51, wherein said cutting at said low frequency is a rate limiting step for determining said reaction launch time.

53. The method of any one of claims 1-52, further comprising changing a temperature during said nucleic acid amplification or subsequent to said nucleic acid amplification.

54. The method of claim 53, wherein changing said temperature during said nucleic acid amplification comprises maintaining said temperature at a first temperature for a first period of time and changing said temperature to a second temperature different from said first temperature for a second period of time during said nucleic acid amplification.

55. The method of claim 54, further comprising changing said temperature back to said first temperature during said nucleic acid amplification.

56. The method of claim 53, wherein changing said temperature subsequent to said nucleic acid amplification comprises maintaining said temperature at a first temperature for a first period of time until completion of said nucleic acid amplification, and changing said temperature to a second temperature different from said first temperature.

57. The method of any one of claims 53-56, wherein changing a temperature during said nucleic acid amplification or subsequent to said nucleic acid amplification changes said relative time to response value.

58. The method of any one of claims 53-57, wherein said target nucleic acid molecule comprises two or more different target nucleic acid molecules in said reaction mixture, and wherein changing a temperature during said nucleic acid amplification or subsequent to said nucleic acid amplification changes a relative time to response value between two or more different target nucleic acid molecules.

59. The method of any one of claims 1-45, wherein said nucleic acid amplification reaction is polymerase chain reaction (PCR).

60. The method of any one of claims 1-59, further comprising in (d) comparing said relative time to response value with a standard value, wherein said standard value comprises a standard relative time to response value of a time to response value associated with amplifying a known concentration of said target nucleic acid molecule and a time to response value associated with amplifying said reference nucleic acid molecule in a single reaction.

61. The method of claim 60, further comprising generating said standard value.

62. The method of claim 60 or 61, wherein said standard value comprises two or more standard relative time to response values, each being a standard relative time to response value of a time to response value associated with amplifying a known concentration of said target nucleic acid molecule and a time to response value associated with amplifying said reference nucleic acid molecule in a single reaction, and wherein the two or more standard relative time to response values are generated using two or more different known concentrations of said target nucleic acid molecule.

63. The method of claim 61, wherein generating said standard value comprises:

(i) providing a plurality of reaction mixtures, each comprising a reference nucleic acid molecule at a first concentration and a target nucleic acid molecule at a second concentration, wherein said first concentration is constant across the plurality of reaction mixtures, and wherein said second concentration varies across the plurality of reaction mixtures;

(ii) subjecting said plurality of reaction mixtures to said nucleic acid amplification reaction;

(iii) detecting signals for said plurality of reaction mixtures to obtain a standard relative time to response value of a time to response value of said target nucleic acid molecule and a time to response value of said reference nucleic acid molecule for each reaction mixture of said plurality of reaction mixtures, thereby generating said standard value.

64. The method of claim 63, wherein said time to response value of said reference nucleic acid molecule varies depending on said second concentration of said target nucleic acid molecule.

65. The method of claim 63 or 64, wherein a reaction launch time of said reference nucleic acid molecule varies depending on said second concentration of said target nucleic acid molecule.

66. The method of claim 61, wherein said standard value is not generated concurrently with said relative time to response value.

67. The method of claim 61, wherein said standard value is generated prior to, concurrently with, or subsequent to obtaining said relative time to response value.

68. The method of any one of claims 1-67, wherein said target nucleic acid molecule comprises two or more different target nucleic acid molecules in said reaction mixture.

69. The method of claim 68, wherein said two or more different target nucleic acid molecules in said reaction mixture is detected by a same signal.

70. The method of claim 68, further comprising, for each target nucleic acid molecule, calculating a relative time to response value of a first value of said target nucleic acid molecule and a reference value of said reference nucleic acid molecule.

71. The method of claim 68, further comprising, for each target nucleic acid molecule, calculating a ratio of a first value of said target nucleic acid molecule and a reference value of said reference nucleic acid molecule to obtain a relative time to response value.

72. The method of any one of claims 1-71, wherein said reference nucleic acid molecule is an additional target nucleic acid molecule different from said target nucleic acid molecule.

73. The method of any one of claims 63-72, wherein the plurality of reaction mixtures comprises said target nucleic acid at a concentration from about 0.01 to about 100,000 RFU/reaction.

74. The method of any one of claims 63-73, wherein said second concentration comprises a series dilution of said target nucleic acid molecule.

75. The method of any one of claims 63-74, wherein the plurality of reaction mixtures further comprises said refence nucleic acid molecule at a concentration of about 50 pg/reaction to about 500 pg/ reaction.

76. The method of any one of claims 63-74, wherein said plurality of reaction mixtures further comprises said refence nucleic acid molecule at a concentration of about 100 pg/reaction to about 400 pg/ reaction.

77. The method of any one of claims 63-74, wherein said plurality of reaction mixtures further comprises said refence nucleic acid molecule at a concentration of about 200 pg/reaction to about 300 pg/ reaction.

78. The method of any one of claims 63-74, wherein said plurality of reaction mixtures further comprises said reference nucleic acid molecule at a concentration of about 250 pg/reaction.

79. The method of any one of claims 63-78, wherein said reference nucleic acid molecule comprises a human deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

80. The method of claim 79, wherein said human DNA or RNA comprises a sequence encoding ribonuclease P/MRP subunit p30 (RPP30).

81. The method of any one of claims 1-80, wherein said target nucleic acid molecule comprises a nucleic acid sequence from a pathogen.

82. The method of claim 81, wherein said target nucleic acid molecule comprises a bacterial DNA or RNA.

83. The method of claim 82, wherein said bacterial DNA or RNA is from a bacterial species associated with an infection.

84. The method of claim 83, wherein said bacterial species is Neisseria gonorrhoeae.

85. The method of claim 83, wherein said bacterial species is Chlamydia trachomatis.

86. The method of claim 81, wherein said target nucleic acid molecule comprises a parasitic DNA or RNA.

87. The method of claim 86, wherein said parasitic DNA or RNA is from a parasitic species associated with an infection.

88. The method of claim 87, wherein said parasitic species is Trichomonas vaginalis.

89. The method of claim 81, wherein said target nucleic acid molecule comprises a viral DNA or RNA.

90. The method of claim 82, wherein said viral DNA or RNA is from a viral species associated with an infection.

91. The method of any one of claims 1-80, wherein said target nucleic acid molecule comprises a mutation associated with a disease or a condition.

92. The method of claim 91, wherein said disease or said condition comprises a cancer.

93. The method of any one of claims 1-92, further comprising obtaining said sample.

94. The method of any one of claims 1-93, wherein said sample is from a human or a nonhuman animal.

95. The method of any one of claims 1-93, wherein said sample comprises a blood sample, a fecal sample, a urine sample, a tissue sample, a vaginal swab, an oral swab, or a rectal swab.

96. The method of any one of claims 1-93, wherein said human or said non-human animal has, is diagnosed with having, is suspected of having, or is at risk of having an infection of a pathogen comprising said target nucleic acid molecule.

97. A method of analyzing a sample having or suspected of having a target nucleic acid molecule, said method comprising:

(a) providing a plurality of reaction mixtures comprising a first reaction mixture and a second reaction mixture, wherein said first reaction mixture comprises said target nucleic acid molecule at a first concentration and a reference nucleic acid molecule, and wherein said second reaction mixture comprises said target nucleic acid at a second concentration and said reference nucleic acid molecule, wherein said first concentration and said second concentration are different;

(b) subjecting said plurality of reaction mixtures to a nucleic acid amplification reaction; and

(c) detecting from said first reaction mixture (i) a first signal of said target nucleic acid molecule to obtain a first value, and (ii) a first reference signal of said reference nucleic acid molecule to obtain a first reference value; (d) detecting from said second reaction mixture (i) a second signal of said target nucleic acid molecule to obtain a second value, and (ii) a second reference signal of said reference nucleic acid molecule to obtain a second reference value;

(e) determining a first relative value based on said first value and said first reference value and a second relative value based on said second value and said second reference value to obtain a standard value;

(f) subjecting said sample to said nucleic acid amplification reaction in the presence of said reference nucleic acid molecule;

(g) detecting a signal of said target nucleic acid molecule to obtain a value, and a reference signal of said reference nucleic acid molecule to obtain a reference value, and determining a relative value of said value and said reference value; and

(h) using said relative value, said first relative value and said second relative value to determine the presence or absence of said target nucleic acid molecule in said sample.

98. The method of claim 97, wherein said target nucleic acid molecule and said reference nucleic acid molecule compete for reaction reagents of said nucleic acid amplification reaction and a reaction launch time of said target nucleic acid varies depending on an abundance of said target nucleic acid molecule relative to said reference nucleic acid molecule.

99. The method of claim 97 or 98, wherein using said relative value, said first relative value and said second relative value in (g) comprises comparing said relative value to said first relative value and said second relative value.

100. The method of any one of claims 97-99, wherein obtaining said standard value in (a)-(d) is not performed concurrently with (e)-(g).

101. The method of any one of claims 97-99, wherein obtaining said standard value in (a)-(d) is performed at least 7 days, 14 days, 21 days, 1 month, 2 months, 5 months, 10 months, 1 year, 2 years or more prior to (e)-(g).

102. The method of any one of claims 97-101, further comprising analyzing a different sample using the same standard value.

103. The method of any one of claims 97-102, wherein said standard value is not generated each time a sample is analyzed.

104. The method of any one of claims 97-103, wherein a concentration of said reference nucleic acid molecule is constant in said first reaction mixture and said second reaction mixture.

105. The method of any one of claims 97-104, wherein said plurality of reaction mixtures comprises a third reaction mixture comprising said target nucleic acid molecule at a third concentration different from said first concentration and said second concentration and a reference nucleic acid molecule.

106. The method of any one of claims 97-105, wherein said plurality of reaction mixtures comprises the target nucleic acid molecule at a concentration from about 0.01 to about 100,000 RFU/reaction.

107. The method of any one of claims 97-105, wherein said plurality of reaction mixtures further comprise said refence nucleic acid molecule at a concentration of about 50 pg/reaction to about 500 pg/ reaction.

108. The method of any one of claims 97-105, wherein said plurality of reaction mixtures further comprises said refence nucleic acid molecule at a concentration of about 100 pg/reaction to about 400 pg/ reaction.

109. The method of any one of claims 97-105, wherein said plurality of reaction mixtures further comprises said refence nucleic acid molecule at a concentration of about 200 pg/reaction to about 300 pg/ reaction.

110. The method of any one of claims 97-105, wherein said plurality of reaction mixtures further comprises said reference nucleic acid molecule at a concentration of about 250 pg/reaction.

111. The method of any one of claims 97-110, wherein said reference nucleic acid molecule comprises a human deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

112. The method of claim 111, wherein said human DNA or RNA comprises a sequence encoding ribonuclease P/MRP subunit p30 (RPP30).

113. The method of any one of claims 97-112, wherein said target nucleic acid molecule comprises a nucleic acid sequence from a pathogen.

114. The method of claim 113, wherein said target nucleic acid molecule comprises a bacterial DNA or RNA.

115. The method of claim 114, wherein said bacterial DNA or RNA is from a bacterial species associated with an infection.

116. The method of claim 113, wherein said target nucleic acid molecule comprises a parasitic DNA or RNA.

117. The method of claim 116, wherein said parasitic DNA or RNA is from a parasitic species associated with an infection.

118. The method of claim 117, wherein said parasitic species is Trichomonas vaginalis.

119. The method of claim 113, wherein said target nucleic acid molecule comprises a viral DNA or RNA.

120. The method of claim 119, wherein said viral DNA or RNA is from a viral species associated with an infection.

121. The method of any one of claims 97-120, further comprising, prior to (e), obtaining said sample.

122. The method of any one of claims 97-121, wherein said sample is from a human or a nonhuman animal.

123. The method of any one of claims 97-122, wherein said sample comprises a blood sample, a fecal sample, a urine sample, a tissue sample, a vaginal swab, an oral swab, or a rectal swab.

124. The method of any one of claims 97-123, wherein said human or said non-human animal has, is diagnosed with having, is suspected of having, or is at risk of having an infection of a pathogen comprising said target nucleic acid molecule.

125. A method of analyzing a sample having or suspected of having a target nucleic acid molecule, said method comprising:

(a) providing a reaction mixture comprising said sample and a reference nucleic acid molecule;

(b) subjecting said reaction mixture to a nucleic acid amplification reaction;

(c) detecting a first signal of said target nucleic acid molecule to obtain a first time to response value, and a reference signal of said reference nucleic acid molecule to obtain a reference time to response value; and

(d) determining a relative time to response value of said first time to response value and said reference time to response value, wherein said relative time to response value indicates the presence or absence of said target nucleic acid molecule in said sample; wherein a duration of analyzing said sample from (a) to (d) is equal to, at most, or about 30 min or less.

126. The method of claim 125, wherein said target nucleic acid molecule and said reference nucleic acid molecule compete for reaction reagents of said nucleic acid amplification reaction and a reaction launch time of said target nucleic acid varies depending on an abundance of said target nucleic acid molecule relative to said reference nucleic acid molecule.

127. The method of claim 125 or 126, wherein said duration of the analyzing is equal to, at most, or about 15 min or less, 12 min or less, 10 min or less, 8 min or less, 7 min or less, 6 min or less, or 5 min or less.

128. The method of any one of claims 1-127, wherein said sample is processed to extract genetic materials from said sample prior to subjecting said sample to nucleic acid amplification reaction.

129. The method of claim 128, wherein said sample is processed by a sample extraction device or by heating.

130. The method of any one of claims 1-129, wherein said method does not comprise calibrating a volume of the reaction mixture.

131. A kit comprising a reference nucleic acid molecule in said method of any one of claims 1- 130, one or more reaction reagents for nucleic acid amplification, and an instruction for user to perform said method of any one of claims 1-130.

132. The kit of claim 131, further comprising said standard value in said method of any one of claims 60-130.

133. A system for analyzing a sample having or suspected of having a target nucleic acid molecule, the system comprising: an analytic unit configured to receive a reaction mixture comprising said sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; and one or more computer processors operatively coupled to said analytic unit, wherein said one or more computer processors are individually or collectively programmed to direct a method in said analytic unit, wherein said method comprises:

(a) subjecting said reaction mixture to conditions sufficient to perform said nucleic acid amplification reaction, wherein said nucleic acid amplification reaction is performed under conditions such that said target nucleic acid molecule and said reference nucleic acid molecule compete for said reagents, and wherein during said nucleic acid amplification reaction a reaction launch time of said target nucleic acid molecule varies depending on an abundance of said target nucleic acid molecule relative to said reference nucleic acid molecule;

(b) detecting (1) a first signal of said target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of said reference nucleic acid molecule to obtain a second time to response value; and (c) determining a relative time to response value of said first time to response value and said second time to response value, wherein said relative time to response value is indicative of a presence or absence of said target nucleic acid molecule in said sample.

134. A system of analyzing a sample having or suspected of having a target nucleic acid molecule, said system comprising: an analytic unit configured to receive a reaction mixture comprising said sample, a reference nucleic acid molecule and reagents for performing a nucleic acid amplification reaction; and one or more computer processors operatively coupled to said analytic unit, wherein said one or more computer processors are individually or collectively programmed to direct a method in said analytic unit, wherein said method comprises:

(a) subjecting said reaction mixture to conditions sufficient to perform said nucleic acid amplification reaction, wherein said nucleic acid amplification reaction is performed under conditions such that said target nucleic acid molecule and said reference nucleic acid molecule compete for said reagents;

(b) detecting (1) a first signal of said target nucleic acid molecule to obtain a first time to response value, and (2) a second signal of said reference nucleic acid molecule to obtain a second time to response value; and

(c) determining a relative time to response value of said first time to response value and said second time to response value, wherein said relative time to response value is indicative of a presence or absence of said target nucleic acid molecule in said sample, wherein said second time to response value of said reference nucleic acid molecule varies depending on an abundance of said target nucleic acid molecule relative to said reference nucleic acid molecule.

135. A system of analyzing a sample having or suspected of having a target nucleic acid molecule, said system comprising: an analytic unit configured to receive a plurality of reaction mixtures comprising a first reaction mixture and a second reaction mixture, wherein said first reaction mixture comprises said target nucleic acid molecule at a first concentration and a reference nucleic acid molecule, and wherein said second reaction mixture comprises said target nucleic acid at a second concentration and said reference nucleic acid molecule, wherein said first concentration and said second concentration are different; and one or more computer processors operatively coupled to said analytic unit, wherein said one or more computer processors are individually or collectively programmed to direct a method in said analytic unit, wherein said method comprises:

(a) subjecting said plurality of reaction mixtures to a nucleic acid amplification reaction; and

(b) detecting from said first reaction mixture (i) a first signal of said target nucleic acid molecule to obtain a first value, and (ii) a first reference signal of said reference nucleic acid molecule to obtain a first reference value;

(c) detecting from said second reaction mixture (i) a second signal of said target nucleic acid molecule to obtain a second value, and (ii) a second reference signal of said reference nucleic acid molecule to obtain a second reference value;

(d) determining a first relative value based on said first value and said first reference value and a second relative value based on said second value and said second reference value to obtain a standard value;

(e) subjecting said sample to said nucleic acid amplification reaction in the presence of said reference nucleic acid molecule;

(f) detecting a signal of said target nucleic acid molecule to obtain a value, and a reference signal of said reference nucleic acid molecule to obtain a reference value, and determining a relative value of said value and said reference value; and

(g) using said relative value, said first relative value and said second relative value to determine the presence or absence of said target nucleic acid molecule in said sample.

136. A system of analyzing a sample having or suspected of having a target nucleic acid molecule, said system comprising: an analytic unit configured to receive a reaction mixture comprising said sample and a reference nucleic acid molecule; and one or more computer processors operatively coupled to said analytic unit, wherein said one or more computer processors are individually or collectively programmed to direct a method in said analytic unit, wherein said method comprises:

(a) subjecting said reaction mixture to a nucleic acid amplification reaction;

(b) detecting a first signal of said target nucleic acid molecule to obtain a first time to response value, and a reference signal of said reference nucleic acid molecule to obtain a reference time to response value; and

(c) determining a relative time to response value of said first time to response value and said reference time to response value, wherein said relative time to response value indicates the presence or absence of said target nucleic acid molecule in said sample; wherein a duration of analyzing said sample from (a) to (d) is equal to, at most, or about 30 min or less.

137. A composition for sample processing comprising: a detergent, a solubilizer, and a cyclodextrin, wherein said composition is configured to stabilize an enzyme during a nucleic acid amplification, wherein said composition is configured to reduce and/or eliminate activity of a degrading nuclease, and wherein said detergent is part of a lysis buffer, and wherein said lysis buffer has a pH of less than 8.0.

138. The composition of claim 137, wherein the pH is less than 7.0, less than 6.0, or less than 5.5.

139. The composition of claim 137 or 138, wherein the pH is 5.0 to 6.0.

140. The composition of any one of claims 137-139, wherein the pH is 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

141. The composition of any one of claims 137-140, wherein the lysis buffer comprises sodium acetate.

142. The composition of claim 141, wherein the sodium acetate has a pH of less than 8.0, less than 7.0, less than 6.0, or less than 5.5, or wherein the sodium acetate has a pH 5.0 to 6.0, or wherein the sodium acetate has pH 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

143. The composition of any one of claims 137-142, wherein said composition is configured to stabilize nucleic acids during said nucleic acid amplification.

144. The composition of any one of claims 137-143, wherein said enzyme is a polymerase, an endonuclease, a reverse transcriptase, or any combination thereof.

145. The composition of any one of claims 137-144, wherein said detergent is sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate, or a functional variant thereof.

146. The composition of any one of claims 137-145, wherein said solubilizer is a non-ionic surfactant.

147. The composition of any one of claims 137-146, wherein said solubilizer is a polysorbate, octylphenoxypolyethoxyethanol, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, or a secondary alcohol ethoxylate.

-I l l-

148. The composition of any one of claims 137-147, wherein said solubilizer is polysorbate 80, polysorbate 20, polysorbate 40, polysorbate 60, or a functional variant thereof.

149. The composition of any one of claims 137-148, wherein said solubilizer and said cyclodextrin are part of a recovery buffer.

150. The composition of claim 149, wherein said lysis buffer and said recovery buffer are in a same mixture.

151. The composition of claim 149 or 150, wherein said recovery buffer comprises a salt.

152. The composition of claim 149 or 150, wherein said recovery buffer does not comprise a salt.

153. The composition of any one of claims 149-152, wherein said recovery buffer comprises a pH buffer.

154. The composition of any one of claims 149-152, wherein said recovery buffer does not comprise a pH buffer.

155. The composition of any one of claims 149-154, wherein said recovery buffer is lyophilized.

156. The composition of any one of claims 137-155, wherein said lysis buffer is lyophilized.

157. The composition of any one of claims 137-156, wherein said solubilizer and said cyclodextrin are configured to shorten a cycle threshold value or a time to result value in said 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.

158. The composition of claim 157, wherein said cycle threshold value is at most 40 or said time to result value is at most 15 minutes.

159. The composition of any one of claims 137-158, wherein said solubilizer and said cyclodextrin are configured to decrease a coefficient of variation.

160. The composition of any one of claims 137-159, wherein said solubilizer and said cyclodextrin are configured to lower a limit of detection.

161. The composition of any one of claims 137-160, wherein said degrading nuclease is a ribonuclease.

162. The composition of any one of claims 137-161, wherein said lysis buffer further comprises a chelating agent.

163. The composition of claim 162, wherein said 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).

164. The composition of any one of claims 137-163, wherein said lysis buffer further comprises a reducing agent.

165. The composition of claim 164, wherein said reducing agent is 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).

166. The composition of any one of claims 137-165, wherein said lysis buffer comprises an egtazic acid (EGTA), an ethylenediaminetetraacetic acid (EDTA), a tris(2- carboxyethyl)phosphine (TCEP), 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), tetrahydropyran (THP), or any combination thereof.

167. The composition of any one of claims 137-166, wherein said composition further comprises an agent capable of reducing a disulfide bond.

168. The composition of claim 167, wherein said agent capable of reducing said disulfide bond comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2- mercaptoehtanol (PME).

169. The composition of any one of claims 137-168, wherein said detergent is present in said composition mixed with a sample at a final concentration that is effective for lysing cells.

170. The composition of claim 169, wherein said final concentration of said detergent is about

O.1% to 10% w/v (g of solute / 100 mL of solution).

171. The composition of any one of claims 137-170, wherein said cyclodextrin is present in said composition mixed with a sample at a final concentration that is effective for isolating said detergent within said composition.

172. The composition of claim 171, wherein said final concentration of said cyclodextrin is about 0.1 mM to 70 mM.

173. The composition of any one of claims 137-172, wherein said detergent is configured to form a complex with said solubilizer and/or said cyclodextrin to stabilize said enzyme.

174. The composition of claim 173, wherein said cyclodextrin is configured to increase the efficiency of forming said complex.

175. The composition of any one of claims 137-174, wherein said cyclodextrin has a higher binding affinity toward said detergent than a binding affinity of said solubilizer.

176. The composition of any one of claims 137-175, wherein said 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.

177. The composition of any one of claims 137-176, wherein said solubilizer is present in said composition mixed with a sample at a final concentration of about 0.1% to 50% w/v.

178. The composition of claim 177, wherein said final concentration of said solubilizer is effective for forming micelles comprising said detergent.

179. The composition of any one of claims 137-178, wherein said composition further comprises a sample.

180. The composition of claim 179, wherein said sample is a biological sample.

181. The composition of claim 180, wherein said biological sample comprises a target nucleic acid molecule subject to sample processing.

182. The composition of any one of claims 137-181, wherein said composition further comprises a reaction mixture for nucleic acid amplification.

183. The composition of claim 182, wherein said reaction mixture is lyophilized.

184. The composition of claim 182 or 183, wherein said reaction mixture comprises a thermostable enzyme, deoxynucleoside triphosphates (dNTPs), a primer, or a probe.

185. The composition of claim 184, wherein said composition is configured to stabilize enzymatic activity of said thermostable enzyme for use during a nucleic acid amplification.

186. The composition of claim 184 or 185, wherein said 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.

187. The composition of any one of claims 184-186, wherein said dNTPs comprise dATP, dCTP, dGTP, dTTP, or dUTP.

188. The composition of any one of claims 184-187, wherein a concentration of said dNTPs in said reaction mixture is about 40 micromolar (pM) to 5000 pM.

189. The composition of any one of claims 184-188, wherein said primer is at least 4 nucleotides in length.