Isothermal amplification of nucleic acids

Abstract

The present disclosure relates to the amplification of nucleic acids in a sample using isothermal application methods, such as RPA. In particular, the present disclosure provides methods of amplifying at least one target nucleic acid in a sample wherein reagents used in the methods include trehalose and polyethylene glycol (PEG). The present disclosure also provides compositions, systems, and kits for performing such methods.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 530,676, filed August 3, 2023, and U.S. Provisional Application No. 63 / 562,603, filed March 7, 2024 (the contents of each of which are incorporated herein by reference in their entirety). Technical Field

[0003] The topics disclosed herein relate to isothermal amplification methods for target nucleic acids, as well as compositions, kits, and systems for performing such methods. Background Technology

[0004] The ability to amplify nucleic acids is central to modern biological and medical research. In particular, nucleic acid amplification is frequently used in nucleic acid-based diagnostic methods to detect infections (e.g., viral or bacterial), diseases, and / or gene mutations in a subject. For example, identifying bacterial or viral nucleic acids in a blood sample can be used to determine whether the blood is safe for donation. Other examples include identifying specific gene mutations for early cancer detection or to determine the optimal treatment for cancer.

[0005] Among various nucleic acid amplification techniques, polymerase chain reaction (PCR) is the most commonly used due to its sensitivity and efficiency in amplifying short nucleic acid sequences. While PCR is widely applicable, it also has limitations. In particular, PCR relies on multiple cycles of thermal desorption (denaturation) at high temperatures, followed by hybridization and extension at lower temperatures (“thermal cycling”), which is a time-consuming process and requires specialized equipment. Given the technical drawbacks of PCR and other amplification techniques that require thermal cycling, methods capable of isothermal amplification of nucleic acids have been developed. Summary of the Invention

[0006] The purposes and advantages of this disclosure will be set forth in and apparent from the following description, and will be learned by practicing the subject matter of this disclosure. Further advantages of the disclosed subject matter will be realized and obtained through the means particularly pointed out in this specification and its claims, as well as the accompanying drawings.

[0007] To achieve these and other advantages, and in accordance with the purposes of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes methods for amplifying target nucleic acids in a sample, such as isothermal amplification methods for amplifying target nucleic acids in a sample. In some embodiments, the isothermal amplification methods of this disclosure include using a composition comprising trehalose and PEG, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.79 or less. In some embodiments, the isothermal amplification methods of this disclosure include using a composition comprising trehalose and PEG, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.75 or less. In some embodiments, the isothermal amplification methods of this disclosure include using a composition comprising trehalose and PEG, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.70 or less. In some embodiments, the isothermal amplification methods of this disclosure include using a composition comprising trehalose and PEG, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.65 or less. In some embodiments, the isothermal amplification method of this disclosure includes using a composition comprising trehalose and PEG, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.60 or less. In some embodiments, the isothermal amplification method of this disclosure includes using a composition comprising trehalose and PEG, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.55 or less.

[0008] This disclosure provides an isothermal amplification method for amplifying a target nucleic acid, the method comprising: (i) preparing a reagent composition using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less; and (ii) contacting the target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the method may include: (i) providing a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), and wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less; and (ii) contacting the target nucleic acid with the reagent composition to amplify the target nucleic acid.

[0009] In some embodiments, the wt% ratio of trehalose to PEG in the composition is from about 0.25 to about 0.79. In some embodiments, the wt% ratio of trehalose to PEG in the composition is from about 0.25 to about 0.75. In some embodiments, the wt% ratio of trehalose to PEG in the composition is from about 0.30 to about 0.70. In some embodiments, the wt% ratio of trehalose to PEG in the composition is from about 0.30 to about 0.60. In some embodiments, the wt% ratio of trehalose to PEG in the composition is from about 0.30 to about 0.55. In some embodiments, PEG is present in the composition at a weight-volume ratio (w / v) of about 10% to about 30%. In some embodiments, PEG is present in the composition at a weight-volume ratio (w / v) of about 15% to about 20%. In some embodiments, the reagent composition contains PEG at a weight-volume ratio (w / v) of about 2% to about 8%. In some embodiments, trehalose is present in the composition at a weight-volume ratio (w / v) of about 5% to about 20%. In some embodiments, trehalose is present in the composition at an amount of about 5% to about 15% w / v. In some embodiments, trehalose is present in the composition at an amount of about 5% to about 12% w / v. In some embodiments, the reagent composition contains about 3% to about 12% w / v of trehalose. In some embodiments, the reagent composition contains about 3% to about 10% w / v of trehalose.

[0010] In some embodiments, the composition further comprises one or more of the following: ATP, dNTPs, creatine phosphate, and one or more salts. In some embodiments, the reagent composition further comprises one or more of the following: DNA polymerase, recombinase-loading protein, single-stranded binding protein, ATP, dNTPs or a mixture of dNTPs and ddNTPs, a reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probes, and reverse transcriptase.

[0011] In some embodiments, the isothermal amplification method of this disclosure includes an isothermal amplification method performed in the presence of trehalose and PEG at a weight percentage (wt%) of about 0.79 or less. In some embodiments, the method includes an isothermal amplification method performed in the presence of trehalose and PEG at a weight percentage (wt%) of about 0.75 or less.

[0012] This disclosure also provides an isothermal amplification method for amplifying a target nucleic acid, the method comprising contacting the target nucleic acid (or a sample containing the target nucleic acid) with a reagent composition comprising trehalose and polyethylene glycol (PEG) to amplify the target nucleic acid, wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.79 or less. In some embodiments, the wt% ratio of trehalose to PEG is about 0.75 or less. In some embodiments, the wt% ratio of trehalose to PEG is about 0.70 or less. In some embodiments, the wt% ratio of trehalose to PEG is about 0.30 to about 0.79. In some embodiments, the wt% ratio of trehalose to PEG is about 0.40 to about 0.79. In some embodiments, the wt% ratio of trehalose to PEG is about 0.45 to about 0.75. In some embodiments, the wt% ratio of trehalose to PEG is about 0.50 to about 0.70. In some embodiments, the wt% ratio of trehalose to PEG is from about 0.50 to about 0.60. In some embodiments, the wt% ratio of trehalose to PEG is from about 0.30 to about 0.50. In some embodiments, the wt% ratio of trehalose to PEG is from about 0.30 to about 0.45.

[0013] In some embodiments, PEG is present in the isothermal amplification method and / or reagent composition at a weight-to-volume ratio (w / v) of about 2% to about 9%, for example, about 2% to about 6%, about 2% to about 6% w / v, or about 6% to about 8% w / v. In some embodiments, PEG is present in the isothermal amplification method and / or reagent composition at a weight-to-volume ratio (w / v) of about 2% to about 8%. In some embodiments, PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons; for example, PEG has a molecular weight of about 35,000 Daltons.

[0014] In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 1% to about 12% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 2% to about 12% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 3% to about 12% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 5% to about 9% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 3% to about 5% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 2% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 3% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 4% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at an amount of about 6% w / v.

[0015] In some embodiments, the isothermal amplification method further includes contacting the target nucleic acid (or a sample containing the target nucleic acid) with one or more of the following: DNA polymerase, recombinase, recombinase-loading protein, single-strand binding protein, ATP, dNTPs or a mixture of dNTPs and ddNTPs, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe, reverse transcriptase, and activator.

[0016] In some embodiments, the reagent composition further includes one or more of the following: DNA polymerase, recombinase, recombinase-loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probes, and reverse transcriptase.

[0017] In some embodiments, the isothermal amplification method is selected from the group consisting of: rolling circle amplification (RCA), sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single-primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR). In some embodiments, the isothermal amplification method is RPA. In some embodiments, the isothermal amplification method is NEAR.

[0018] In some embodiments, the target nucleic acid is bacterial, eukaryotic, or viral nucleic acid. In some embodiments, the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, parvovirus B19, HAV, chlamydia, gonorrhea, WNV, Zika virus, dengue virus, chikungunya virus, influenza, babesi, malaria, Usutu virus, or HEV.

[0019] In some implementations, isothermal amplification methods, for example, amplify at least two or at least three target nucleic acids in a multiplex reaction.

[0020] In some embodiments, the target nucleic acid is present in and / or obtained from the sample. In some embodiments, the sample is a tissue sample. In some embodiments, the target nucleic acid is isolated from the tissue sample prior to amplification.

[0021] In some embodiments, the sample is a biological fluid. In some embodiments, the biological fluid is blood, such as whole blood, lysed whole blood, serum, or plasma. In some embodiments, the target nucleic acid is isolated from the biological fluid prior to amplification.

[0022] This disclosure also provides a composition for performing an isothermal amplification method. In some embodiments, the composition comprises trehalose and PEG in a weight percentage (wt%) ratio of about 0.79 or less. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.75 or less. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.70 or less. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.3 to about 0.79. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.4 to about 0.79. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.45 to about 0.75. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.50 to about 0.70. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.50 to about 0.60. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.30 to about 0.50. In some embodiments, the composition comprises trehalose and PEG in a wt% ratio of about 0.30 to about 0.45.

[0023] In some embodiments, the compositions of this disclosure comprise about 2% to about 9% by weight (w / v), for example, about 2% to about 6% w / v, about 2% to about 6% w / v, or about 6% to about 8% w / v of PEG. In some embodiments, the PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons, for example, the PEG has a molecular weight of about 35,000 Daltons.

[0024] In some embodiments, the composition of this disclosure contains about 1% to about 12% w / v trehalose. In some embodiments, the composition contains about 2% to about 12% w / v trehalose. In some embodiments, the composition contains about 4% to about 9% w / v trehalose. In some embodiments, the composition contains about 3% to about 5% w / v trehalose. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at about 2% w / v. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at about 3% w / v. In some embodiments, the composition contains about 4% w / v trehalose. In some embodiments, trehalose is present in the isothermal amplification method and / or reagent composition at about 6% w / v.

[0025] In some embodiments, the composition further comprises one or more of the following: ATP, dNTPs, or a mixture of dNTPs and ddNTPs. In some embodiments, the composition further comprises one or more of the following: ATP, dNTPs, or a mixture of dNTPs and ddNTPs, and a reducing agent. In some embodiments, the composition further comprises one or more salts and / or phosphocreatine. In some embodiments, the composition further comprises one or more of the following: DNA polymerase, recombinase, recombinase-loading protein, single-stranded binding protein, ATP, dNTPs, or a mixture of dNTPs and ddNTPs, a reducing agent, creatine kinase, nuclease, one or more primers, nucleic acid probes, and reverse transcriptase.

[0026] This disclosure provides a composition for an isothermal amplification method, the composition comprising or primarily consisting of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.79 or less. In some embodiments, the composition for an isothermal amplification method comprises or primarily consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.75 or less. In some embodiments, the composition for an isothermal amplification method comprises or primarily consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.70 or less. In some embodiments, the composition for the isothermal amplification method comprises or mainly consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.25 to about 0.79. In some embodiments, the composition for the isothermal amplification method comprises or mainly consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.30 to about 0.79. In some embodiments, the composition for the isothermal amplification method comprises or mainly consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.40 to about 0.79. In some embodiments, the composition for the isothermal amplification method comprises or mainly consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.45 to about 0.75. In some embodiments, the composition for the isothermal amplification method comprises or mainly consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.50 to about 0.70. In some embodiments, the composition for the isothermal amplification method comprises or mainly consists of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.50 to about 0.60. In some embodiments, the composition for the isothermal amplification method comprises or consists primarily of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a wt% ratio of about 0.30 to about 0.50.In some embodiments, the composition for the isothermal amplification method comprises or consists primarily of trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a wt% ratio of about 0.30 to about 0.45.

[0027] In some embodiments, the composition is used in an isothermal amplification method selected from the group consisting of: rolling circle amplification (RCA), sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single-primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR). In some embodiments, the isothermal amplification method is RPA. In some embodiments, the isothermal amplification method is NEAR.

[0028] In another aspect, this disclosure provides a system for performing the methods described herein. In some embodiments, the system is an automated system. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.79 or less. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.78 or less. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.75 or less. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.70 or less. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.30 to about 0.79. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.40 to about 0.79. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.45 to about 0.75. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.50 to about 0.70. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.50 to about 0.60. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.30 to about 0.50. In some embodiments, the system includes a container or reservoir comprising at least one composition containing trehalose and PEG in a wt% ratio of about 0.30 to about 0.45.

[0029] This disclosure also provides a kit for performing the methods described herein.

[0030] This disclosure provides kits comprising the compositions described herein. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.79 or less. For example, but not as a limitation, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.75 or less. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.3 to about 0.79. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.40 to about 0.79. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.45 to about 0.75. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.50 to about 0.70. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.50 to about 0.60. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.30 to about 0.50. In some embodiments, the kit comprises a composition containing trehalose and PEG in a weight percentage (wt%) ratio of about 0.30 to about 0.45.

[0031] Brief description of the attached figures

[0032] The following figures are included to illustrate certain aspects of this disclosure and should not be considered as the only embodiments. The disclosed subject matter can be modified, altered, combined, and equivalent in form and function without departing from the scope of this disclosure.

[0033] Figure 1 Exemplary results are provided associated with RPA amplification of HCV and HIV-2 target nucleic acids with the addition of 4% trehalose and polyethylene glycol (PEG).

[0034] Figure 2 Exemplary results are provided associated with RPA amplification of HCV and internal control (IC) target nucleic acids with 4% trehalose and PEG at near-limit of detection (LOD) levels.

[0035] Figure 3A and 3B Exemplary results are provided for RPA amplification of chikungunya (CHIKV) and dengue fever (DENV) with added trehalose and PEG.

[0036] Figure 4 Exemplary results are provided regarding the RPA amplification of HCV target nucleic acids as trehalose concentration increases.

[0037] Figures 5A-5C It provides information on how HBV (HBV) decreases with increasing PEG concentration. Figure 5A ), HCV ( Figure 5B ) and internal control (IC; Figure 5C Exemplary results of RPA amplification of the target.

[0038] Figure 6A and 6B Provides that as PEG concentration increases, DENV ( Figure 6A ) and elution buffer negative control (“EB only”) Figure 6B Exemplary results of RPA amplification. Detailed Implementation

[0039] This disclosure provides an improved method for amplifying and detecting nucleic acids in a sample using isothermal amplification methods (e.g., recombinase polymerase amplification (RPA)). This disclosure also provides compositions, systems, and kits for performing isothermal amplification reactions.

[0040] This invention is partly based on the observation that trehalose and polyethylene glycol (PEG), present in specific weight percentage (wt%) ratios and amounts in compositions used in RPA reactions, can significantly improve the amplification of target nucleic acids. As shown in Example 1, reagent compositions (e.g., reagent compositions containing non-protein components) containing trehalose and PEG at wt% ratios of about 0.3 to about 0.79 (e.g., about 0.5 to about 0.75) of target nucleic acids resulted in a significant increase in the amplification of target nucleic acids (including target nucleic acids of HBV, HCV, HIV, HEV, chikungunya, and dengue fever) and the multiplex amplification of target nucleic acids. Specifically, as... Figure 4-6A As shown, the amplification of target nucleic acids was significantly improved in RPA reactions with different trehalose and PEG ratios.

[0041] For clarity, but not as a limitation, the detailed description of the subject matter disclosed herein is divided into the following sections:

[0042] I. Definition;

[0043] II. Isothermal amplification method;

[0044] III. Composition;

[0045] IV. Instructions for use;

[0046] V. Reagent kit;

[0047] VI. System; and

[0048] VII. Exemplary Implementation.

[0049] I. Definition

[0050] In the context of this disclosure and in the specific context in which each term is used, the terms used in this specification generally have their common meaning in the art. Some terms are discussed below or in other parts of this specification to provide additional guidance to practitioners in describing the compositions and methods of this disclosure and how to prepare and use them.

[0051] For the purposes of this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any definition described below conflicts with any document incorporated herein by reference, the definition described below shall prevail.

[0052] As used herein, the terms "a" or "an" used in conjunction with "comprising" in the claims and / or specification may refer to "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more than one." For example, but not as a limitation, references to "an" or "the" "target nucleic acid" include both a single target nucleic acid and combinations and / or mixtures of two or more different target nucleic acids.

[0053] As used herein, the terms “about” or “approximately” mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, the range of error depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may, according to practice in the art, mean within three or more standard deviations. Alternatively, “about” may mean a range of up to 20% for a given value, preferably up to 10%, more preferably up to 5%, and even more preferably still up to 1%.

[0054] As used in this article, the term "amplified" refers to the process of preparing multiple copies of nucleic acid from a single or small number of nucleic acid molecules. The amplified nucleic acid may be called an amplicon.

[0055] The term "amplification process" generally refers to any process in which a portion of a nucleic acid is copied or replicated into at least one additional nucleic acid molecule. The amplification process produces amplified nucleic acid.

[0056] As used herein, the term "biological fluid" refers to any body fluid or body fluid derivative in which the analyte can be measured. Non-limiting examples of biological fluids include dermal fluid, tissue fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, tears, etc. In some embodiments, the biological fluid is blood.

[0057] As used herein, the terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)”, and variations thereof are intended as open-ended transitional phrases, terms, or words that do not exclude other actions or structures. This disclosure also contemplates other embodiments that “comprise the embodiments or elements presented herein,” “consist of the embodiments or elements presented herein,” and “consist primarily of the embodiments or elements presented herein,” whether or not explicitly listed.

[0058] The term "coupling" can refer to the connection or binding of two or more components through interaction, bonding, connection, force, or knot, so as to keep the two or more components together. In some embodiments, the term "coupling" covers direct or indirect binding, for example, the first component directly binding to the second component, or one or more intermediate molecules located between the first and second components. Exemplary bonds include covalent bonds, ionic bonds, van der Waals interactions, and other bonds that are recognizable by those skilled in the art.

[0059] As used herein, the term "detection" or "detection" refers to determining the presence of a target nucleic acid in a limited space (including, but not limited to, a sample, reaction mixture, molecular complex, and substrate). As used herein, "detection" or "detection" may include determining the chemical and / or biological properties of a target, including but not limited to its ability to interact with (especially bind to) other compounds, its ability to activate other compounds, and other properties that may be recognized by a person skilled in the art upon reading this disclosure. Detection may be quantitative or qualitative. A detection is "quantitative" when it refers to, concerns, or relates to a measurement of the quantity or amount of a target or signal (also referred to as quantification), and includes, but is not limited to, any analysis designed to determine the quantity or proportion of a target or signal. A detection is "qualitative" when it refers to, concerns, or relates to identifying the presence or absence of a target or signal, regardless of the quantity or amount of the target or signal (as long as it is present or absent).

[0060] As used herein, the term “expression” or “expresses” refers to transcription and translation occurring within a cell. The expression levels of genes and / or nucleic acids in a cell can be determined based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by genes and / or nucleic acids produced by the cell. For example, it is preferable to quantify mRNA transcribed from genes and / or nucleic acids using northern hybridization. (Sambrook et al., *Molecular Cloning: A Laboratory Manual*, pp. 7.3–7.57 (Cold Spring Harbor Laboratory Press, 1989)). Proteins encoded by genes and / or nucleic acids can be quantified by measuring the protein’s biological activity or by using assays unrelated to such activity, such as Western blotting or radioimmunoassay using antibodies that react with the protein. (Sambrook et al., *Molecular Cloning: A Laboratory Manual*, pp. 18.1–18.88 (Cold Spring Harbor Laboratory Press, 1989)).

[0061] As used in this article, the term "hybridization" refers to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide.

[0062] As used herein, “label” means an agent that allows for direct or indirect detection. Labels include, but are not limited to, fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels. Non-limiting examples of labels include green fluorescent protein (“GFP”), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005), which is incorporated herein by reference). 32 P, 14 C, 125 I, 3 H and 131I. Fluoresceins (such as rare earth chelates or luciferin yellow and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferases (such as firefly luciferase and bacterial luciferase) (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, and enzymes that generate detectable signals, such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase).

[0063] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” refer to any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base (particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group). Typically, nucleic acid molecules are described by their base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from the 5' end to the 3' end. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA; and ribonucleic acid (RNA), particularly messenger RNA. Nucleic acid molecules can be mRNA, synthetic forms of DNA or RNA, or mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, and single-stranded and double-stranded forms. Additionally, nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone bonds or chemically modified residues. In some embodiments, the nucleic acid is isolated. In some embodiments, the term "isolated nucleic acid" can refer to nucleic acid isolated from a subject or sample, for example, nucleic acid isolated from its original environment (e.g., the natural environment or host cell if recombinant expression is involved).

[0064] As used herein, the term "oligonucleotide" refers to a short nucleic acid sequence comprising about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values). As used herein, the terms "nucleic acid" and "polynucleotide" refer to nucleotides of any length in polymeric form, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. The terms include RNA or DNA analogs made from nucleotide analogs and modified polynucleotides (such as, for example, methylated and / or capped polynucleotides) as equivalents. Nucleic acids are typically linked together via phosphate bonds to form nucleic acid sequences or polynucleotides, although many other linkages (e.g., phosphate thioides, borosiphosphates, etc.) are known in the art.

[0065] Oligonucleotides can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Oligonucleotides can be DNA (genomic DNA and complementary DNA (cDNA)), RNA, or hybrids, wherein the nucleic acids may include combinations of deoxyribonucleotides and ribonucleotides, and base combinations comprising uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Oligonucleotides can be obtained through chemical synthesis or recombinant methods.

[0066] Any oligonucleotide described herein may be modified in any suitable manner to stabilize or enhance its binding affinity to its target. For example, the oligonucleotide sequence described herein may contain one or more modified oligonucleotide bases.

[0067] Any oligonucleotide sequence described herein may comprise, consist primarily of, or consist of complementary sequences to any sequence disclosed herein. As used herein, the term "complementary sequence" refers to a nucleic acid sequence that forms a stable duplex with the oligonucleotide described herein by means of the Watson-Crick base pairing rule and typically shares approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% greater identity with the disclosed oligonucleotide.

[0068] The oligonucleotides described herein can be prepared using any suitable method, and many such methods are known in the art (see, for example, Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supplement, Cold Spring Harbor Laboratory Press: New York, NY; MA Innis (ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, NY (1990); P. Tijssen, Hybridization with Nucleic Acid Probes - Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); MA Innis (ed.), PCR Strategies, Academic Press: New York, NY (1995); and FMAusubel (ed.), Short Protocols in Molecular Biology, John Wiley & Sons: Seculus, NJ (2002); Narang et al., Meth. Enzymol., 68: 90-98). (1979); Brown et al., Meth. Enzymol., 68: 109-151 (1979); and Belousov et al., Nucleic Acids Res., 25:3440-3444 (1997) (each of which is incorporated herein by reference in its entirety). Oligonucleotide pairs can also be designed using a variety of tools, such as the Primer-BLAST tool available from the National Center for Biotechnology Information (NCBI). Oligonucleotide synthesis can be performed on oligonucleotide synthesizers, such as those commercially available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, California), DuPont (Wilmington, Delaware), or Milligen (Bedford, Massachusetts). Alternatively, oligonucleotides can be customized and sourced from a variety of commercial sources well-known in the art, including, for example, Midland Certified Reagent Company (Midland, Texas), EurofinsScientific (Louisville, Kentucky), and BioSearch Technologies, Inc. (Novato, California).Oligonucleotides can be purified using any suitable method known in the art, such as, for example, natural acrylamide gel electrophoresis, anion-exchange HPLC (see, for example, Pearson et al., J. Chrom., 255: 137-149 (1983), which is incorporated herein by reference), and reversed-phase HPLC (see, for example, McFarland et al., Nucleic Acids Res., 7:1067-1080 (1979), which is incorporated herein by reference).

[0069] The oligonucleotide sequence can be validated using any suitable sequencing method known in the art, including but not limited to chemical degradation (see, e.g., Maxam et al., Methods of Enzymology, 65: 499-560 (1980), which is incorporated herein by reference), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Pieles et al., Nucleic Acids Res., 21: 3191-3196 (1993), which is incorporated herein by reference), and mass spectrometry analysis after combined digestion with alkaline phosphatase and exonuclease (Wu et al., Anal. Biochem., 290: 347-352 (2001), which is incorporated herein by reference).

[0070] The term "multiple" refers to a number greater than one. In some implementations, the term "multiple nucleic acids" refers to a number of nucleic acids greater than one. For example, but not as a limitation, multiple target nucleic acids include at least two target nucleic acids.

[0071] As used herein, the terms “primer,” “primer sequence,” “primer oligonucleotide,” and “amplification oligonucleotide” refer to an oligonucleotide that, when placed under suitable amplification conditions (e.g., buffers, salts, temperature, and pH) and in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., DNA-dependent or RNA-dependent polymerases), functions as the starting point for the synthesis of an elongation product, which is the complementary strand of a nucleic acid (all types of DNA or RNA). The amplification oligonucleotides of this disclosure can be of any suitable size and ideally comprise about 15 to 50 nucleotides (preferably about 20 to 40 nucleotides), consisting primarily of or composed of about 15 to 50 nucleotides (preferably about 20 to 40 nucleotides). The oligonucleotides of this disclosure may contain other nucleotides besides those described herein.

[0072] The terms “probe,” “probe sequence,” and “probe oligonucleotide” refer to oligonucleotides that, under suitable hybridization conditions, selectively hybridize with at least a portion of a target sequence (e.g., a portion of an amplified target sequence). Typically, probe sequences are identified as “complementary” (i.e., complementary to the coding strand or the positive (+) strand) or “anticomplementary” (i.e., complementary to the antisense strand (−) strand). The probes of this disclosure may have any suitable size and ideally comprise about 10-50 nucleotides (preferably about 12-35 nucleotides), consisting primarily of or composed of about 10-50 nucleotides (preferably about 12-35 nucleotides).

[0073] As used herein, the terms “set,” “primer set,” “probe set,” and “primer and probe set” refer to two or more oligonucleotides that together induce amplification of a target sequence or nucleic acid of interest (e.g., a target sequence in a pathogenic factor), and / or at least one probe capable of detecting the target sequence or nucleic acid. In some embodiments, the term “set” refers to a pair of oligonucleotides comprising a first oligonucleotide (referred to herein as a “forward primer”) that hybridizes to the 5’ end of the target sequence or nucleic acid to be amplified and a second oligonucleotide (referred herein as a “reverse primer”) that hybridizes to a complementary sequence of the target sequence or nucleic acid to be amplified.

[0074] As used herein, a “reference sequence” is a specific sequence used as the basis for sequence alignment. A reference sequence can be a subset or the entirety of a specific sequence; for example, a fragment or segment of a full-length protein. A reference sequence can be a sequence identifiable in databases such as GenBank and UniProt, as well as other sequences recognizable to those skilled in the art.

[0075] As used herein, “sequence identity” or “identity” in the context of two polynucleotide or polypeptide sequences refers to the same nucleotide bases or amino acid residues in two sequences when a maximum match is performed within a specified alignment window. When using sequence identity or similarity percentages to refer to proteins, it should be recognized that dissimilar residue positions are often due to conserved amino acid substitutions, where an amino acid residue is replaced by a residue with similar physicochemical properties and functional equivalence, and therefore without altering the functional characteristics of the molecule.

[0076] As used herein, “sequence identity percentage” or “identity percentage” refers to the value determined by comparing two optimally aligned sequences within an alignment window, where a portion of the polynucleotide sequence in the alignment window may contain additions or deletions (vacancies) relative to a reference sequence (which does not contain additions or deletions), used for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues appear in the two sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the alignment window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0077] As will be understood by those skilled in the art, certain well-known mathematical algorithms can be used to determine the percentage identity between any two sequences. Non-limiting examples of such mathematical algorithms include the algorithm of Myers and Miller; the local homology algorithm of Smith et al.; the homology alignment algorithm of Needleman and Wunsch; the similarity search method of Pearson and Lipman; and the algorithm of Karlin and Altschul (as modified by Karlin and Altschul). Computer implementations of suitable mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to, CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, FASTA, and other implementations recognizable by those skilled in the art. Sequence alignment algorithms have also been published in publications such as Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009); Durbin et al., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7): 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge, UK (1997). (Each of these articles is incorporated into this paper in its entirety through citation).

[0078] As used herein, the terms "subject" or "individual" refer to a vertebrate or invertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, livestock, locomotor animals, rodents, and pets. Non-limiting examples of non-human animal subjects include, but are not limited to, rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, apes, and monkeys. In some embodiments, the individual or subject is a human.

[0079] As used herein, the terms “target nucleic acid,” “target sequence,” or “target nucleic acid sequence” refer to the nucleic acid sequence of interest amplified using the methods of this disclosure.

[0080] II. Isothermal Amplification Method

[0081] This disclosure provides methods for amplifying target nucleic acids in samples. In some embodiments, the amplification method is an isothermal amplification method. Isothermal amplification methods include amplification methods that amplify target nucleic acids without temperature cycling or rapid heating and cooling.

[0082] Non-limiting examples of isothermal amplification methods include rolling circle amplification (RCA), sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single-primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR). Other non-limiting disclosures regarding isothermal amplification methods are provided in Oliveira et al., Frontiers in Sensors 2:752600 (2021) (the contents of which are incorporated herein by reference in their entirety).

[0083] In some implementations, the isothermal amplification method is RPA.

[0084] In some implementations, the isothermal amplification method is called NEAR.

[0085] This disclosure provides isothermal amplification methods in the presence of trehalose and PEG at specific weight percentage (wt%) ratios, and / or using compositions with trehalose and PEG at specific weight percentage (wt%) ratios. As shown in Example 1, for example in non-protein component (NPC) compositions, trehalose and PEG at wt% ratios of about 0.31, about 0.52, about 0.62, about 0.72, and about 0.73 result in significantly improved RPA methods compared to other ratios analyzed. In some embodiments, adding trehalose to NPC compositions containing PEG allows for increased crowding agent concentration without affecting the viscosity of the NPC composition. In some embodiments, increasing the viscosity of the NPC can render the composition unusable or difficult to use.

[0086] In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.25 to about 0.79 or about 0.3 to about 0.79 (e.g., about 0.4 to about 0.75), and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.25 to about 0.79 or about 0.3 to about 0.79 (e.g., about 0.4 to about 0.75). In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.25 to about 0.79 or about 0.3 to about 0.79 (e.g., about 0.4 to about 0.75). The percentage ratios are approximately 0.3 to approximately 0.7, approximately 0.3 to approximately 0.65, approximately 0.3 to approximately 0.6, approximately 0.3 to approximately 0.55, approximately 0.3 to approximately 0.45, approximately 0.3 to approximately 0.4, approximately 0.45 to approximately 0.75, approximately 0.5 to approximately 0.75, approximately 0.55 to approximately 0.75, approximately 0.6 to approximately 0.75, approximately 0.65 to approximately 0.75, approximately 0.7 to approximately 0.75, and approximately 0.4 to approximately 0. 7. Trehalose in the presence of PEG at concentrations of about 0.4 to about 0.65, about 0.4 to about 0.6, about 0.4 to about 0.55, about 0.4 to about 0.45, about 0.5 to about 0.7, about 0.45 to about 0.55, about 0.45 to about 0.65, about 0.55 to about 0.65, about 0.5 to about 0.55, about 0.6 to about 0.65, or about 0.7 to about 0.75, and / or using wt% trehalose. The percentage ratios are approximately 0.3 to approximately 0.7, approximately 0.3 to approximately 0.65, approximately 0.3 to approximately 0.6, approximately 0.3 to approximately 0.55, approximately 0.3 to approximately 0.45, approximately 0.3 to approximately 0.4, approximately 0.45 to approximately 0.75, approximately 0.5 to approximately 0.75, approximately 0.55 to approximately 0.75, approximately 0.6 to approximately 0.75, approximately 0.65 to approximately 0.75, approximately 0.7 to approximately 0.75, and approximately 0.4 to... The composition may be carried out in the presence of trehalose and PEG at a wt% ratio of about 0.7, about 0.4 to about 0.65, about 0.4 to about 0.6, about 0.4 to about 0.55, about 0.4 to about 0.45, about 0.5 to about 0.7, about 0.45 to about 0.55, about 0.45 to about 0.65, about 0.55 to about 0.65, about 0.5 to about 0.55, about 0.6 to about 0.65, or about 0.7 to about 0.75. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.5 to about 0.7, and / or using a composition comprising trehalose and PEG at a wt% ratio of about 0.5 to about 0.7. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.45 to about 0.75, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.45 to about 0.75. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.5 to about 0.75, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.5 to about 0.75.In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.45 to about 0.65, and / or using a composition containing trehalose and PEG in a wt% ratio of about 0.45 to about 0.65. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.5 to about 0.65, and / or using a composition containing trehalose and PEG in a wt% ratio of about 0.5 to about 0.55. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.55 to about 0.65, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.55 to about 0.65. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.6 to 0.65, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.6 to about 0.65. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.7 to about 0.75, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.7 to about 0.75. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.3 to about 0.65, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.3 to about 0.65. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.3 to about 0.60, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.60. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.3 to about 0.45, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.45.

[0087] In some embodiments, the isothermal amplification method uses a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.75.

[0088] In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in the following wt% ratios and / or using a composition containing trehalose and PEG in the following wt% ratios: about 0.79 or less, about 0.78 or less, about 0.77 or less, about 0.76 or less, about 0.75 or less, about 0.74 or less, about 0.73 or less, about 0.72 or less, about 0.71 or less, about 0.7 or less, about 0.69 or less, about 0.68 or less, about 0.67 or less, about 0.66 or less, about 0.65 or less, about 0.64 or less, about 0.63 or less, about 0.62 or less, about 0.61 or less, about 0.60 or less, about 0.59 or less, about 0.58 or less, about 0.57 or less, about 0.56 or less Less, about 0.55 or less, about 0.54 or less, about 0.53 or less, about 0.52 or less, about 0.51 or less, about 0.50 or less, about 0.49 or less, about 0.48 or less, about 0.47 or less, about 0.46 or less, about 0.45 or less, about 0.44 or less, about 0.43 or less, about 0.42 or less, about 0.41 or less, about 0.40 or less, about 0.39 or less, about 0.38 or less, about 0.37 or less, about 0.36 or less, about 0.35 or less, about 0.34 or less, about 0.33 or less, about 0.32 or less, about 0.31 or less, or about 0.3 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.79 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.79 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.75 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.75 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.7 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.7 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.65 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.65 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.60 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.60 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.55 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.55 or less.In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.50 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.50 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.45 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.45 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.40 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.40 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of about 0.35 or less, and / or using a composition containing trehalose and PEG at a wt% ratio of about 0.35 or less. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in a wt% ratio of about 0.30 or less, and / or using a composition containing trehalose and PEG in a wt% ratio of about 0.30 or less.

[0089] In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG in the following wt% ratios and / or using a composition containing trehalose and PEG in the following wt% ratios: not greater than about 0.79, not greater than about 0.78, not greater than about 0.77, not greater than about 0.76, not greater than about 0.75, not greater than about 0.74, not greater than about 0.73, not greater than about 0.72, not greater than about 0.71, not greater than about 0.7, not greater than about 0.69, not greater than about 0.68, not greater than about 0.67, not greater than about 0.66, not greater than about 0.65, not greater than about 0.64, not greater than about 0.63, not greater than about 0.62, not greater than about 0.61, not greater than about 0.60, not greater than about 0.59, not greater than about 0.58, not greater than about 0.57, not greater than about 0.56, not greater than about 0.55 Not greater than about 0.56, not greater than about 0.55, not greater than about 0.54, not greater than about 0.53, not greater than about 0.52, not greater than about 0.51, not greater than about 0.50, not greater than about 0.49, not greater than about 0.48, not greater than about 0.47, not greater than about 0.46, not greater than about 0.45, not greater than about 0.44, not greater than about 0.43, not greater than about 0.42, not greater than about 0.41, not greater than about 0.40, not greater than about 0.39, not greater than about 0.38, not greater than about 0.37, not greater than about 0.36, not greater than about 0.35, not greater than about 0.34, not greater than about 0.33, not greater than about 0.32, not greater than about 0.31 or not greater than about 0.30. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.79, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.79. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.75, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.75. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.73, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.73. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.72, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.72. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of no more than about 0.7, and / or using a composition containing trehalose and PEG at a wt% ratio of no more than about 0.7.In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.65, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.65. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.62, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.62. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.60, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.60. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.55, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.55. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.52, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.52. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.45, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.45. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.42, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.42. In some embodiments, the isothermal amplification method is performed in the presence of trehalose and PEG at a wt% ratio not greater than about 0.35, and / or using a composition containing trehalose and PEG at a wt% ratio not greater than about 0.35. In some embodiments, the isothermal amplification method is carried out in the presence of trehalose and PEG at a wt% ratio of no more than about 0.31, and / or using a composition containing trehalose and PEG at a wt% ratio of no more than about 0.31.

[0090] In some embodiments, the PEG used in the isothermal amplification method of this disclosure has a molecular weight between about 15,000 and about 50,000 Daltons. In some embodiments, the PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons. In some embodiments, the PEG has a molecular weight between about 30,000 Daltons and about 40,000 Daltons, for example, about 35,000 Daltons. In some embodiments, the PEG is PEG1450, PEG3000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000, PEG30000, PEG35000, or PEG40000. In some embodiments, the PEG is PEG35000.

[0091] In some embodiments, PEG is present in the isothermal amplification method of this disclosure, for example, in reagent mixtures and / or solutions, in an amount of about 2% to about 9% by weight (w / v). In some embodiments, PEG is present in the isothermal amplification method of this disclosure, for example, in reagent mixtures or solutions, in an amount of about 2% to about 9% by weight (w / v). In some embodiments, PEG is present in the isothermal amplification method of this disclosure, for example, in reagent mixtures or solutions, in an amount of about 2% to about 8.5% by weight (w / v).For example, but not as a limitation, PEG may be present in amounts of about 2.2% to about 8.5% w / v, about 2.4% to about 8.5% w / v, about 2.6% to about 8.5% w / v, about 2.8% to about 8.5% w / v, about 3% to about 8.5% w / v, about 3.2% to about 8.5% w / v, about 3.4% to about 8.5% w / v, about 3.6% to about 8.5% w / v, about 3.8% to about 8.5% w / v, about 4% to about 8.5% w / v, about 4.2% to about 8.5% w / v, about 4.4% to about 8.5% w / v, about 4.6% to about 8.5% w / v, about 4.8% to about 8.5% w / v, about 5% to about 8.5% w / v, about 5.2% to about 8.5% w / v, and about 5.4% to about 8.5%. w / v, about 5.6% to about 8.5% w / v, about 5.8% to about 8.5% w / v, about 6% to about 8.5% w / v, about 6.2% to about 8.5% w / v, about 6.4% to about 8.5% w / v, about 6.5% to about 8.5% w / v, about 6.6% to about 8.5% w / v, about 6.8% to about 8.5% w / v, about 7% to about 8.5% w / v, about 7.2% to about 8.5% w / v, about 7.4% to about 8.5% w / v, about 7.6% to about 8.5% w / v, about 7.8% to about 8.5% w / v, about 2% to about 7.8% w / v, about 2% to about 7.6% w / v, about 2% to about 7.4% w / v, about 2% to about 7.2% w / v, about 2% to about 7% w / v, about 2% to about 6.8% w / v, about 2% to about 6.6% w / v, about 2% to about 6.4% w / v, about 2% to about 6.2% w / v, about 2% to about 6% w / v, about 2% to about 5.8% w / v, about 2% to about 5.6% w / v, about 2% to about 5.4% w / v, about 2% to about 5.2% w / v, about 2% to about 5% w / v, about 2% to about 5% w / v, about 2% to about 4.8% w / v, about 2% to about 4.6% w / v, about 2% to about 4.4% w / v, about 2% to about 4.2% w / v, about 2% to about 4% w / v, about 2% to about 3.8% w / v, about 2% to about 3.6% w / v, about 2% to about 3.4% w / v, about 2% to about 3.2% w / v, about 2% to about 3% w / v, about 2% to about 2.8% It is present at approximately 2% to approximately 2.6% w / v, approximately 2% to approximately 2.4% w / v, approximately 2% to approximately 2.2% w / v, approximately 3% to approximately 5% w / v, approximately 3% to approximately 7% w / v, approximately 4% to approximately 6% w / v, approximately 4% to approximately 7% w / v, or approximately 4% to approximately 7% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of approximately 4% to approximately 9% w / v, for example, in a reagent mixture or solution.In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 4% to about 6% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 5% to about 7% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 6% to about 7% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 5% to about 8% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 5% to about 9% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 6% to about 8% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 7% to about 8% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 8% to about 9% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 5.5% w / v. In some embodiments, PEG is present in the isothermal amplification method at a concentration of about 6.3% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of about 6.5% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of about 7% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of about 7.4% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of about 7.7% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of 8.25% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of about 8.4% w / v. In some embodiments, PEG is present in the isothermal amplification method at an amount of about 8.5% w / v.

[0092] In some embodiments, trehalose is present in the isothermal amplification method of this disclosure at an amount of about 1% to about 12% w / v, for example, in a reagent mixture or solution. In some embodiments, trehalose is present in the isothermal amplification method of this disclosure at an amount of about 2% to about 12% w / v, for example, in a reagent mixture or solution. In some embodiments, trehalose is present in the isothermal amplification method of this disclosure at an amount of about 5% to about 12% w / v, for example, in a reagent mixture or solution. In some embodiments, trehalose is present in the isothermal amplification method of this disclosure at an amount of about 2% to about 9% w / v, for example, in a reagent mixture or solution. In some embodiments, trehalose is present in the isothermal amplification method of this disclosure at an amount of about 5% to about 9% w / v, for example, in a reagent mixture or solution. For example, but not by limitation, trehalose is expressed in amounts of about 1% to about 9% w / v, about 1.5% to about 9% w / v, about 1.8% to about 9% w / v, about 1.9% to about 9% w / v, 2.2% to about 9% w / v, about 2.4% to about 9% w / v, about 2.6% to about 9% w / v, about 2.8% to about 9% w / v, about 3% to about 9% w / v, about 3.2% to about 9% w / v, about 3.4% to about 9% w / v, about 3.6% to about 9% w / v, about 3.8% to about 9% w / v, about 4% to about 9% w / v, about 4.2% to about 9% w / v, about 4.4% to about 9% w / v, about 4.6% to about 9% w / v, about 4.8% to about 9% w / v, about 5% to about 9% w / v, and about 5.2% to about 9%. w / v, about 5.4% to about 9% w / v, about 5.6% to about 9% w / v, about 5.8% to about 9% w / v, about 6% to about 9% w / v, about 6.2% to about 9% w / v, about 6.4% to about 9% w / v, about 6.5% to about 9% w / v, about 6.6% to about 9% w / v, about 6.8% to about 9% w / v, about 7% to about 9% w / v, about 7.2% to about 9% w / v, about 7.4% to about 9% w / v, about 7.6% to about 9% w / v, about 7.8% to about 9% w / v, about 8% to about 9% w / v, about 8.2% to about 9% w / v, about 8.4% to about 9% w / v, about 8.6% to about 9% w / v, about 8.8% to about 9% w / v, 2% to about 8.8% w / v, about 2% to about 8.6% w / v, about 2% to about 8.4% w / v, about 2% to about 8.2% w / v, about 2.2% to about 8% w / v, about 2% to about 7.8% w / v, about 2% to about 7.6% w / v, about 2% to about 7.4% w / v, about 2% to about 7.2% w / v, about 2% to about 7% w / v, about 2% to about 6.8% w / v, about 2% to about 6.6% w / v, about 2% to about 6.4% w / v, about 2% to about 6.2% w / v, about 2% to about 6% w / v, about 2% to about 5.8% w / v, about 2% to about 5.6% w / v, about 2% to about 5.4% w / v, about 2% to about 5.2% w / v, about 2% to about 5% w / v, about 2% to about 4.8% w / v, about 2% to about 4.6% w / v, about 2% to about 4.4% w / v, about 2% to about 4.2% w / v, about 2% to about 4% w / v, about 2% to about 3.8% w / v, about 2% to about 3.6% w / v, about 2% to about 3.4% w / v, about 2% to about 3.2% w / v, about 2% to about 3% w / v, about 2% to about 2.8% w / v, about 2% to about 2.6% In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 12% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 7% to about 12% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 8% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 8% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 7% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 7% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 6% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 3% to about 5% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 4% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 8% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 9% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 11% w / v. In some embodiments, trehalose is present in the isothermal amplification method at a concentration of about 12% w / v.

[0093] A. RPA method

[0094] In some implementations, the isothermal amplification method is known as RPA. RPA relies on the properties of recombinases and their associated protein components to utilize single-stranded homologous nucleic acids to invade double-stranded nucleic acids, allowing sequence-specific initiation of the nucleic acid polymerase reaction.

[0095] RPA amplification utilizes an enzyme called a recombinase, which forms a complex with an oligonucleotide primer and pairs the primer with its homologous sequence in the double-stranded nucleic acid. A single-stranded nucleic acid binding (SSB) protein binds to the replaced nucleic acid strand and stabilizes the resulting loop. Nucleic acid amplification is then initiated from the primer, but only in the presence of the target sequence. Once initiated, the amplification reaction proceeds rapidly, thus starting with only a small number of copies of the target nucleic acid, and highly specific amplification reaches detectable levels within minutes.

[0096] In some embodiments, in the first step, a first single-stranded nucleic acid primer and a second single-stranded nucleic acid primer are contacted with a recombinase (e.g., UvsX), a recombinase loader (e.g., UvsY), and a single-stranded DNA-binding protein (e.g., gp32) to form a first nucleoprotein primer and a second nucleoprotein primer. The single-stranded nucleic acid primers are specifically targeted at and complementary to the target nucleic acid molecule. In the second step, the first nucleoprotein primer is contacted with the double-stranded target nucleic acid molecule to create a first D-loop structure in the first portion of the double-stranded target nucleic acid molecule (step 2a). Furthermore, the second nucleoprotein primer is contacted with the double-stranded target nucleic acid molecule to create a second D-loop structure in the second portion of the double-stranded target nucleic acid molecule (step 2b). The formation of the D-loop structure is such that the 3' ends of the first and second nucleic acid primers are oriented toward each other on the same double-stranded target nucleic acid molecule without completely denaturing the target nucleic acid molecule. It should be noted that steps 2a and 2b can be performed in any order or simultaneously.

[0097] In the D-loop structure, the primer hybridizes with one strand of the double-stranded target nucleic acid molecule to form a double-stranded structure. The second strand of the target nucleic acid molecule is then replaced by the primer. The structure resembles the capital letter D, where the straight portion of the D represents the double-stranded portion of the structure, and the curved portion of the D represents the second strand of the target nucleic acid molecule that has been replaced by a single strand.

[0098] In the third step, the 3' ends of the first and second nucleoprotein primers are extended with one or more polymerases and dNTPs capable of strand substitution synthesis to generate first and second double-stranded target nucleic acid molecules and first and second substitution strand nucleic acids. The first and second double-stranded target nucleic acid molecules can serve as target nucleic acid molecules in the second step of subsequent amplification rounds.

[0099] Repeat steps two and three until the desired level of target nucleic acid amplification is achieved.

[0100] During the amplification process described above, the first and second substitutional strand nucleic acids can hybridize with each other after step (c) to form a third double-stranded target nucleic acid molecule.

[0101] In some embodiments, this disclosure provides an RPA method comprising, for example, the ratios and amounts of trehalose and PEG disclosed herein in the above-disclosed ratios and amounts. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.25 to about 0.79, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.25 to about 0.79. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.79, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.79. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.5, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.5. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.4, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.4. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.4 to about 0.79, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.4 to about 0.79. For example, but not as a limitation, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.45 to about 0.75 (e.g., about 0.5 to about 0.7), and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.45 to about 0.75 (e.g., about 0.5 to about 0.7). In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.4 to about 0.45, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.4 to about 0.45. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.79 or less, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.79 or less.In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.75 or less (e.g., 0.65 or less or 0.55 or less), and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.75 or less (e.g., 0.65 or less or 0.55 or less). In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.65 or less, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.65 or less. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.55 or less, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.55 or less. In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising trehalose and PEG in a wt% ratio of about 0.55 or less, and / or using a composition comprising trehalose and PEG in a wt% ratio of about 0.55 or less.

[0102] In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising about 2% to about 9% w / v of PEG, and / or using a composition comprising about 2% to about 9% w / v of PEG.

[0103] In some embodiments, this disclosure provides an RPA method performed in the presence of about 5% to about 9% w / v PEG (e.g., about 6% to about 9% w / v PEG).

[0104] In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising about 2% to about 8% w / v PEG (e.g., about 5% to about 8% w / v PEG), and / or using a composition comprising about 2% to about 8% w / v PEG (e.g., about 5% to about 8% w / v PEG).

[0105] In some embodiments, this disclosure provides an RPA method performed in the presence of about 2% to about 8% w / v PEG (e.g., about 5% to about 8% w / v PEG).

[0106] In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising about 1% to about 12% w / v trehalose (e.g., about 2% to about 12% w / v trehalose), and / or using a composition comprising about 1% to about 12% w / v trehalose (e.g., about 2% to about 12% w / v trehalose). In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising about 2% to about 12% w / v trehalose (e.g., about 3% to about 12% w / v trehalose), and / or using a composition comprising about 2% to about 12% w / v trehalose (e.g., about 3% to about 12% w / v trehalose). In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising about 5% to about 12% w / v trehalose (e.g., about 6% to about 12% w / v trehalose), and / or using a composition comprising about 5% to about 12% w / v trehalose (e.g., about 6% to about 12% w / v trehalose). In some embodiments, this disclosure provides an RPA method carried out in the presence of a composition comprising about 3% to about 5% w / v trehalose (e.g., about 4% w / v trehalose), and / or using a composition comprising about 3% to about 5% w / v trehalose (e.g., about 4% w / v trehalose).

[0107] In some embodiments, this disclosure provides an RPA method carried out in the presence of about 5% to about 12% w / v trehalose (e.g., about 6% to about 12% w / v trehalose).

[0108] In some embodiments, this disclosure provides an RPA method carried out in the presence of (i) about 5% to about 12% w / v trehalose, such as about 6% to about 12% w / v trehalose, and (ii) about 2% to about 8% w / v PEG, such as about 5% to about 8% w / v PEG.

[0109] In some embodiments, this disclosure provides an RPA method carried out in the presence of (i) about 10% to about 12% w / v trehalose and (ii) about 2% to about 8% w / v PEG, for example about 5% to about 8% w / v PEG.

[0110] In some embodiments, the RPA method further includes the use of a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP, primers, and a template nucleic acid (such as a target nucleic acid). In some embodiments, the RPA method may include the use of one or more of the following (in any combination): at least one recombinase, at least one single-stranded DNA binding protein, at least one DNA polymerase, dNTPs, a buffer, a reducing agent, ATP or an ATP analog, at least one recombinase-loading protein, a first primer and optionally a second primer, a probe, a reverse transcriptase, and a template nucleic acid molecule, such as a single-stranded (e.g., RNA) or double-stranded target nucleic acid. In some embodiments, the RPA reaction may include, for example, a reverse transcriptase. In some embodiments, the RPA reaction does not include a reverse transcriptase.

[0111] In some embodiments, the RPA method of this disclosure includes combining a non-protein component (NPC) (e.g., reaction buffer, dNTPs, ATP, and salt), a protein component (PC) including enzymes required for the RPA method (e.g., DNA polymerase, recombinase, recombinase-loading protein, single-strand binding protein, creatine kinase, nuclease (e.g., exonuclease) and / or reverse transcriptase), and an oligonucleotide component (OC) (e.g., one or more primers and / or one or more probes specific to the target nucleic acid or two or more target nucleic acids (e.g., in a multiplex RPA method)) with a sample containing the target nucleic acid. In some embodiments, these components may be added to the sample in any order, or the components may be included in a “master mixture” and then added to the sample. In some embodiments, trehalose and PEG are present in the NPC at the ratios and / or amounts described herein. Alternatively, trehalose and PEG are present in the master mixture at the ratios and / or amounts described herein. In some embodiments, trehalose and PEG are present in the master mixture in the amounts described herein. In some embodiments, an activator, such as magnesium (e.g., magnesium acetate (MgOAc)), is then added to the reaction, followed by incubation at a specific temperature (e.g., 40°C) to amplify the target nucleic acid.

[0112] In some embodiments, a portion of the NPC and / or master mixture is contacted with the sample to amplify the target nucleic acid present in the sample. For example, but not as a limitation, a portion of the NPC and / or master mixture containing the ratios and / or amounts of trehalose and PEG described herein is contacted with the sample to amplify the target nucleic acid present in the sample. For example, but not as a limitation, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the compositions disclosed herein (e.g., NPC compositions) are added to the isothermal amplification method. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein (e.g., the NPC composition) is added to the isothermal amplification method.

[0113] In some embodiments, the recombinase (e.g., UvsX), the recombinase loader (e.g., UvsY), and the single-stranded DNA-binding protein (e.g., gp32) may be derived from myoviridae bacteriophages. In some embodiments, the myocytoviridae bacteriophages may be, for example, T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanobacterial phage P-SSM2, cyanobacterial phage PSSM4, cyanobacterial phage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rb16, Rb43, phage 31, phage 44RR2.8t, Rb49, phage Rb3, or phage LZ2. In some embodiments, a combination of Rb69 UvsX, Rb69 UvsY, and Rb69 gp32 may be used. In some implementations, a combination of Aeh1 UvsX, Aeh1 UvsY, and Rb69 gp32 may be used. In some implementations, a combination of T4 UvsX, T4 UvsY, and Rb69 gp32 may be used. In some implementations, a combination of T4 UvsX, Rb69 UvsY, and T4 gp32 may be used.

[0114] In some embodiments, the recombinase (e.g., UvsX), recombinase loader (e.g., UvsY), and single-stranded DNA-binding protein (e.g., gp32) may each be a natural, heterozygous, or mutant protein derived from the same or different Myocerviridae phage sources. The natural protein may be a wild-type protein or a natural variant protein. A mutant protein (also called a genetically engineered protein) is a natural protein with a natural or artificial mutation (such as insertion, deletion, substitution, or a combination thereof) located at the N-terminus, C-terminus, or internally (between the N-terminus and C-terminus). A heterozygous protein (also called a chimeric protein) contains sequences from at least two different organisms. For example, but not as a limitation, a heterozygous UvsX protein may contain amino acids from one species (e.g., T4) but a DNA-binding loop from another species (e.g., T6). Compared to the natural protein, the heterozygous protein may contain improved properties. Improved properties may be an increased or faster RPA amplification rate, or a decreased or more controllable RPA amplification rate.

[0115] In some embodiments, the recombinase (e.g., UvsX) may be a mutant UvsX. In some embodiments, the mutant UvsX is Rb69 UvsX, which contains at least one mutation in the Rb69 UvsX amino acid sequence, wherein said mutation is selected from the group consisting of: (a) an amino acid that is not histidine at position 64, a serine at position 64, the addition of one or more glutamate residues at the C-terminus, the addition of one or more aspartic residues at the C-terminus; and combinations thereof. In some embodiments, the mutant UvsX is T6 UvsX, which has at least one mutation in the T6 UvsX amino acid sequence, wherein said mutation is selected from the group consisting of: (a) an amino acid that is not histidine at position 66; (b) a serine at position 66; (c) the addition of one or more glutamate residues at the C-terminus; (d) the addition of one or more aspartic residues at the C-terminus; and (e) combinations thereof.

[0116] In some embodiments, the RPA method of this disclosure is performed using a large-fragment polymerase. In some embodiments, the large-fragment polymerase may be selected from the group consisting of *Escherichia coli* Pol I, *Bacillus subtilis* Pol I, *Staphylococcus aureus* Pol I, and their homologs. In some embodiments, the RPA method is performed in the presence of about 0.01 mg / mL to about 0.5 mg / mL of DNA polymerase, for example, about 0.08 mg / mL to about 0.2 mg / mL of DNA polymerase. In some embodiments, the RPA method is performed in the presence of about 10 units / mL to about 10,000 units / mL of DNA polymerase, for example, about 500 units / mL to about 5,000 units / mL of DNA polymerase.

[0117] In some implementations, the RPA process is performed in the presence of heparin. Heparin can be used as an agent to reduce nonspecific primer noise levels and enhance the ability of E. coli exonuclease III or E. coli exonuclease IV to rapidly remove 3' blocking groups or terminal residues from recombinant intermediates.

[0118] In some embodiments, the dNTPs used in the RPA method of this disclosure include, for example, dATP, dGTP, dCTP, and dTTP. In some embodiments, the ddNTPs used in the RPA method of this disclosure include, for example, ddATP, ddTTP, ddGTP, and ddGTP. In some embodiments, the concentration of each dNTP and / or ddNTP substance that can be used is from about 1 μM to about 500 μM.

[0119] In some embodiments, the RPA method uses blocking primers. Blocking primers are primers that do not allow polymerase extension. When using blocking primers, a deblocking agent is also used to release the primers to allow extension. The deblocking agent can be an endonuclease or an exonuclease, which cleaves the blocking group from the primer. In some embodiments, the deblocking agent includes *E. coli* exonuclease III and *E. coli* endonuclease IV. In some embodiments, the deblocking agent is *E. coli* exonuclease III. In some embodiments, the deblocking agent is *E. coli* endonuclease IV.

[0120] In some embodiments, the RPA method is performed in the presence of two or more primers, such as: (i) at least one or more forward primers, (ii) at least one or more reverse primers, or (iii) at least one or more forward and reverse primers, and / or at least one or more probes. In some embodiments, the RPA method is performed in the presence of at least three primers. In some embodiments, the RPA method is performed in the presence of at least two probes. In some embodiments, the RPA method is performed in the presence of one or more primers and / or probes of about 1 nM to about 1000 nM, for example, one or more primers and / or probes of about 10 nM to about 500 nM. In some embodiments, the RPA method is performed in the presence of one or more primers of about 1 nM to about 1000 nM, for example, one or more primers of about 10 nM to about 500 nM. In some embodiments, the RPA method is performed in the presence of a forward primer of about 1 nM to about 1000 nM, for example, a forward primer of about 10 nM to about 500 nM. In some embodiments, the RPA method is performed in the presence of a reverse primer of about 1 nM to about 1000 nM, for example, a reverse primer of about 10 nM to about 500 nM. In some embodiments, the RPA method is performed in the presence of one or more probes of about 1 nM to about 1000 nM, for example, one or more probes of about 10 nM to about 500 nM, such as detection probes.

[0121] In some embodiments, only one of the nucleic acid primers used in the RPA method of this disclosure is coated with a recombinase / recombinase loader / single-stranded DNA binding protein. That is, RPA can be performed using an uncoated primer and a primer coated with any one or a combination of recombinase, recombinase loader and / or single-stranded DNA binding protein.

[0122] In some embodiments, the RPA method is carried out in the presence of about 1 mM to about 25 mM magnesium ions, such as about 1 mM to about 20 mM, about 1 mM to about 10 mM, or about 1 mM to about 3 mM magnesium ions. In some embodiments, the RPA method is carried out in the presence of about 1 mM to about 25 mM divalent manganese ions, such as about 1 mM to about 20 mM, about 1 mM to about 10 mM, or about 1 mM to about 3 mM divalent manganese ions. In some embodiments, manganese ions replace magnesium ions, and the reaction can be carried out with or without magnesium.

[0123] In some embodiments, the recombinase loader (e.g., UvsY) is omitted. That is, any RPA reaction of this disclosure can be carried out in the absence of a recombinase loader (e.g., UvsY).

[0124] In some embodiments, RNA can be used as an initial template to employ the RPA method of this disclosure. For example, a DNA copy of the RNA template can be first generated using a reverse transcriptase, and then the DNA copy can be subjected to RPA-based nucleic acid amplification to amplify the target nucleic acid derived from an RNA virus. RPA using an RNA template is commonly referred to in the art as reverse transcriptase RPA or RT-RPA. In some embodiments, the reverse transcriptase used in the method of this disclosure can be selected from: OmniScript (Qiagen), SensiScript (Qiagen), MonsterScript (Epicentre), Transcriptor (Roche), HIV RT (Ambion), Superscript III (Invitrogen), ThermoScript (Invitrogen), Thermo-X (Invitrogen), ImProm II (Promega), and EIAV-RT. In some embodiments, the reverse transcriptase is EIAV-RT.

[0125] In some embodiments, reverse transcriptase may be omitted from the RPA reaction. For example, but not as a limitation, any RPA reaction of this disclosure can be performed without reverse transcriptase. In some embodiments, if the target nucleic acid to be analyzed is DNA, the RPA reaction of this disclosure can be performed without reverse transcriptase.

[0126] In some embodiments, ATP or its analogues may be used at concentrations of about 1 and 10 mM. Non-limiting examples of ATP analogues include ATP-γ-S, ATP-β-S, and ddATP.

[0127] In some embodiments, in addition to trehalose and PEG, the following reagents may also be used for the RPA method of this disclosure: buffers (such as Tris-HCl), reducing agents (such as DTT), potassium acetate, second congestion agents, dNTPs, ATP, creatine phosphate, glycerol, creatine kinase, UvsX, UvsY, DNA polymerase, GP32, exonuclease III, BSA, activators (such as magnesium, such as magnesium acetate (MgOAc)), and EIAV. In some embodiments, other reagents may be used, including but not limited to forward primers, reverse primers, probes, and ROX reference dyes.

[0128] In some embodiments, the RPA method of this disclosure may use about 5 mM to about 100 mM Tris-HCl (pH about 6.5-9.0, such as 8.3) or include it in the composition of this disclosure.

[0129] In some embodiments, the RPA method of this disclosure may employ a reducing agent of about 5 mM to about 10 mM (such as DTT), or include it in the composition of this disclosure.

[0130] In some embodiments, the RPA method of this disclosure may use about 50 mM to about 100 mM potassium acetate, or include it in the composition of this disclosure.

[0131] In some embodiments, the RPA method of this disclosure may employ about 1 mM to about 5 mM of dNTPs, or include them in the compositions of this disclosure.

[0132] In some embodiments, the RPA method of this disclosure may use about 1 mM to about 10 mM of ATP, such as about 2 mM to about 5 mM of ATP, or include it in the composition of this disclosure.

[0133] In some embodiments, the RPA method of this disclosure may use about 20 mM to about 100 mM of phosphocreatine, for example about 40 mM to about 100 mM of phosphocreatine, or may include it in the composition of this disclosure.

[0134] In some embodiments, magnesium acetate of about 5 mM to about 40 mM, such as about 10 mM to about 40 mM, may be used in the RPA method of this disclosure, or may be included in the composition of this disclosure.

[0135] In some embodiments, BSA of about 0.01 mg / mL to about 10 mg / mL may be used in the RPA method of this disclosure, or it may be included in the composition of this disclosure.

[0136] In some embodiments, about 5% to about 10% glycerol may be used in the RPA method of this disclosure, or it may be included in the composition of this disclosure.

[0137] In some embodiments, the RPA method of this disclosure may employ creatine kinase at a concentration of about 0.01 mg / mL to about 0.5 mg / mL, for example, creatine kinase at a concentration of about 0.1 mg / mL to about 0.5 mg / mL, or may include it in the composition of this disclosure.

[0138] In some embodiments, UvsX of about 0.1 mg / mL to about 1.0 mg / mL, such as about 0.3 mg / mL to about 1.0 mg / mL, may be used in the RPA method of this disclosure, or it may be included in the composition of this disclosure.

[0139] In some embodiments, UvsY of about 0.01 mg / mL to about 0.25 mg / mL, for example about 0.09 mg / mL to about 0.25 mg / mL, may be used in the RPA method of this disclosure, or may be included in the composition of this disclosure.

[0140] In some embodiments, the RPA method of this disclosure may employ a DNA polymerase of about 0.01 mg / mL to about 0.5 mg / mL, for example, a DNA polymerase of about 0.08 mg / mL to about 0.2 mg / mL, or include it in the composition of this disclosure.

[0141] In some embodiments, GP32 may be used in the RPA method of this disclosure at a concentration of about 0.1 mg / mL to about 2.0 mg / mL, for example, about 0.4 mg / mL to about 0.8 mg / mL, or may be included in the composition of this disclosure.

[0142] In some embodiments, the RPA method of this disclosure may employ exonuclease III at a concentration of about 0.01 mg / mL to about 0.5 mg / mL, for example, about 0.1 mg / mL to about 0.5 mg / mL, or may include it in the composition of this disclosure.

[0143] In some embodiments, the RPA method of this disclosure may employ a reverse transcriptase (such as equine infectious anemia virus reverse transcriptase (EIAV-RT)) of about 0.5 µg / mL to about 100.0 µg / mL, for example, about 0.5 µg / mL to about 1.5 µg / mL, about 1.5 µg / mL to about 20 µg / mL, or about 20 µg / mL to about 70 µg / mL, or may include it in the compositions of this disclosure.

[0144] In some embodiments, in addition to trehalose and PEG, the following reagents may be used (or included in the reagent compositions of this disclosure) at the following concentrations for carrying out the RPA method of this disclosure: about 5 mM to about 100 mM Tris-HCl (pH about 6.5-9.0, e.g., 8.3); about 5 mM to about 10 mM reducing agent (e.g., DTT); about 50 mM to about 100 mM potassium acetate; about 1 mM to about 5 mM dNTPs; about 1 mM to about 10 mM ATP, e.g., about 2 mM to about 5 mM ATP; about 20 mM to about 100 mM creatine phosphate, e.g., about 40 mM to about 100 mM creatine phosphate; about 5 mM to about 40 mM magnesium acetate, e.g., about 10 mM to about 40 mM magnesium acetate; about 0.01 mg / mL to about 10 mg / mL BSA; about 5% to about 10% glycerol; about 0.01 mg / mL to about 0.5 mg / mL BSA. Creatine kinase at approximately 0.1 mg / mL to approximately 0.5 mg / mL; UvsX at approximately 0.1 mg / mL to approximately 1.0 mg / mL, such as approximately 0.3 mg / mL to approximately 1.0 mg / mL; UvsY at approximately 0.01 mg / mL to approximately 0.25 mg / mL, such as approximately 0.09 mg / mL to approximately 0.25 mg / mL; DNA polymerase at approximately 0.01 mg / mL to approximately 0.5 mg / mL, such as approximately 0.08 mg / mL to approximately 0.2 mg / mL; GP32 at approximately 0.1 mg / mL to approximately 2.0 mg / mL, such as approximately 0.4 mg / mL to approximately 0.8 mg / mL; exonuclease III at approximately 0.01 mg / mL to approximately 0.5 mg / mL, such as approximately 0.1 mg / mL to approximately 0.5 mg / mL; and approximately 0.5 mg / mL... Equine infectious anemia virus reverse transcriptase (EIAV-RT) at concentrations from approximately µg / mL to approximately 100.0 µg / mL, such as approximately 0.5 µg / mL to approximately 1.5 µg / mL, approximately 1.5 µg / mL to approximately 20 µg / mL, or approximately 20 µg / mL to approximately 70 µg / mL. In some embodiments, other reagents may be used, including but not limited to forward primers, reverse primers, probes, and ROX reference dyes.

[0145] In some embodiments, the RPA method is carried out in the presence of: (i) about 5% to about 12% w / v trehalose, for example about 6% to about 12% w / v trehalose; and (ii) about 2% to about 8% w / v PEG, for example about 5% to about 8% w / v PEG; and one or more of the following: about 5 mM to about 10 mM of a reducing agent (e.g., DTT); about 50 mM to about 100 mM of potassium acetate; about 1 mM to about 5 mM of dNTPs; about 1 mM to about 10 mM of ATP, for example about 2 mM to about 5 mM of ATP; about 20 mM to about 100 mM of phosphocreatine, for example about 40 mM to about 100 mM of phosphocreatine; about 5 mM to about 40 mM of magnesium acetate, for example about 10 mM to about 40 mM of magnesium acetate; about 0.01 mg / mL to about 10 BSA at a concentration of approximately 5% to approximately 10%; creatine kinase at a concentration of approximately 0.01 mg / mL to approximately 0.5 mg / mL, e.g., approximately 0.1 mg / mL to approximately 0.5 mg / mL; uvsX at a concentration of approximately 0.1 mg / mL to approximately 1.0 mg / mL, e.g., approximately 0.3 mg / mL to approximately 1.0 mg / mL; uvsY at a concentration of approximately 0.01 mg / mL to approximately 0.25 mg / mL, e.g., approximately 0.09 mg / mL to approximately 0.25 mg / mL; DNA polymerase at a concentration of approximately 0.01 mg / mL to approximately 0.5 mg / mL, e.g., approximately 0.08 mg / mL to approximately 0.2 mg / mL; GP32 at a concentration of approximately 0.1 mg / mL to approximately 2.0 mg / mL, e.g., approximately 0.4 mg / mL to approximately 0.8 mg / mL; approximately 0.01 mg / mL; uvsX at a concentration of approximately 0.01 mg / mL to approximately 1.0 mg / mL; uvsX at a concentration of approximately 0.01 mg / mL to approximately 0.5 mg / mL, e.g., approximately 0.01 mg / mL; uvsY at a concentration of approximately 0.01 mg / mL to approximately 0.25 mg / mL; DNA polymerase at a concentration of approximately 0.01 mg / mL to approximately 0.5 mg / mL, e.g., approximately 0.08 mg / mL; uvsY at a concentration of approximately 0.01 ... Exonuclease III at a concentration of approximately 0.5 mg / mL to approximately 0.1 mg / mL to approximately 0.5 mg / mL; and equine infectious anemia virus reverse transcriptase (EIAV-RT) at a concentration of approximately 0.5 µg / mL to approximately 100.0 µg / mL, such as EIAV-RT at a concentration of approximately 0.5 µg / mL to approximately 1.5 µg / mL, approximately 1.5 µg / mL to approximately 20 µg / mL, or approximately 20 µg / mL to approximately 70 µg / mL.

[0146] In some embodiments, the RPA method is carried out in the presence of: (i) about 5% to about 12% w / v trehalose, for example about 6% to about 12% w / v trehalose; (ii) about 2% to about 8% w / v PEG, for example about 5% to about 8% w / v PEG; (iii) about 20 mM to about 100 mM creatine phosphate, for example about 40 mM to about 100 mM creatine phosphate; (iv) about 5 mM to about 40 mM magnesium acetate, for example about 10 mM to about 40 mM magnesium acetate; (v) about 0.01 mg / mL to about 0.5 mg / mL creatine kinase, for example about 0.1 mg / mL to about 0.5 mg / mL creatine kinase; (vi) about 0.1 mg / mL to about 1.0 mg / mL UvsX, for example about 0.3 mg / mL to about 1.0 mg / mL UvsX; (vii) about 0.01 mg / mL creatine kinase; (viii) about 0.01 mg / mL creatine kinase; (iv) about 5 mM to about 40 mM magnesium acetate; (v) about 0.01 mg / mL creatine kinase; (vii) about 0.01 mg / mL creatine kinase; (viii ... (viii) UvsY at a concentration of about 0.01 mg / mL to about 0.5 mg / mL, for example, about 0.08 mg / mL to about 0.2 mg / mL; (ix) GP32 at a concentration of about 0.1 mg / mL to about 2.0 mg / mL, for example, about 0.4 mg / mL to about 0.8 mg / mL; and (x) exonuclease III at a concentration of about 0.01 mg / mL to about 0.5 mg / mL, for example, about 0.1 mg / mL to about 0.5 mg / mL.

[0147] In some embodiments, the single RPA reaction for amplifying and detecting a single target nucleic acid (such as that from a pathogen or pathogenic agent (e.g., HIV-1, HIV-2, HCV, or HBV)) may, in addition to trehalose and PEG, contain the following reagents used in the following concentrations: 50 mM Tris-HCl (1M, pH 8.3), 5 mM DTT, 100 mM potassium acetate, 5.5% 20% polyethylene glycol, 1.8 mM 100 mM dNTP, 2.5 mM ATP, 50 mM creatine phosphate, 420 nM forward primer, 420 nM reverse primer, 120 nM Exo probe, 15 nM ROX reference dye, 6.5% glycerol, 0.1 mg / ml creatine kinase, 0.3 mg / ml UvsX, 0.09 mg / ml UvsY, 0.0798 The following are listed: 0.48 mg / ml DNA polymerase, 0.1 mg / ml Gp32, 0.02 mg / ml bovine serum albumin (BSA), 0.0008 mg / ml EIAV reverse transcriptase and / or 14 mM magnesium acetate.

[0148] In some embodiments, the multiplex RPA reaction for amplifying and detecting at least two target nucleic acids (e.g., for detecting HIV-1 and HBV) may, in addition to trehalose and PEG, contain the following reagents used in the following concentrations: 50 mM Tris-HCl (1M, pH 8.3), 5 mM DTT, 100 mM potassium acetate, 5.5% 20% polyethylene glycol, 2.7 mM 100 mM dNTPs, 3.5 mM ATP, 50 mM creatine phosphate, 157.50 nM HIV-1 INT forward primer, 236.37 nM HIV-1 INT reverse primer, 90 nM HIV-1 INT Exo probe, 39.37 nM HIV-1 LTR forward primer, 39.37 nM HIV-1 LTR reverse primer, 22.5 nM HIV-1 INT Exo probe, 86.13 HBV forward primer (nM), HBV reverse primer (86.13nM), HBV Exo probe (90 nM), ROX reference dye (45 nM), glycerol (8.4%), creatine kinase (0.1 mg / ml), UvsX (0.375 mg / ml), UvsY (0.0675 mg / ml), DNA polymerase (0.1396 mg / ml), Gp32 (1.2 mg / ml), exonuclease III (0.1 mg / ml), bovine serum albumin (BSA) (0.02 mg / ml), EIAV reverse transcriptase (0.0016 mg / ml), and / or magnesium acetate (14 mM).

[0149] In some embodiments, the multiplex reaction for amplifying and detecting at least two target nucleic acids (e.g., for detecting HIV-1 and HBV), in addition to trehalose and PEG, may contain the following reagents in concentrations: 50 mM Tris-HCl (1M, pH 8.3), 5 mM DTT, 100 mM potassium acetate, 5.5% 20% polyethylene glycol, 2.7 mM 100 mM dNTPs, 3.5 mM ATP, 50 mM creatine phosphate, 157.50 nM HIV-1 forward primer, 236.37 nM HIV-1 reverse primer, 90 nM HIV-1 EXO probe, 86.13 nM HBV forward primer, 86.13 nM HBV reverse primer, 90 nM HBV EXO probe, 45 nM ROX reference dye, 8.4% glycerol, 0.1 0.375 mg / ml creatine kinase, 0.0675 mg / ml UvsX, 0.1396 mg / ml UvsY, 1.2 mg / ml Gp32, 0.1 mg / ml exonuclease III, 0.02 mg / ml bovine serum albumin (BSA), 0.0016 mg / ml EIAV reverse transcriptase and / or 14 mM magnesium acetate.

[0150] In some embodiments, the RPA method of this disclosure includes contacting a sample containing a target nucleic acid with a reagent composition comprising about 5% to about 12% w / v trehalose (e.g., about 6% to about 12% w / v trehalose) and about 2% to about 8% w / v PEG (e.g., about 5% to about 8% w / v PEG). In some embodiments, the RPA method of this disclosure includes: (a) contacting a sample containing multiple nucleic acids with a reagent composition comprising: (i) about 5% to about 12% w / v trehalose, e.g., about 6% to about 12% w / v trehalose, and (ii) about 2% to about 8% w / v PEG, e.g., about 5% to about 8% w / v PEG, to generate a reaction mixture; and (b) incubating the reaction mixture to amplify the target nucleic acid (if present in multiple nucleic acids). In some embodiments, the reagent composition may also comprise one or more reagents disclosed herein. For example, but not as a limitation, the reagent composition may further comprise: (iii) about 20 mM to about 100 mM creatine phosphate, for example, about 40 mM to about 100 mM creatine phosphate; (iv) about 5 mM to about 40 mM magnesium acetate, for example, about 10 mM to about 40 mM magnesium acetate; (v) about 0.01 mg / mL to about 0.5 mg / mL creatine kinase, for example, about 0.1 mg / mL to about 0.5 mg / mL creatine kinase; (vi) about 0.1 mg / mL to about 1.0 mg / mL UvsX, for example, about 0.3 mg / mL to about 1.0 mg / mL UvsX; (vii) about 0.01 mg / mL to about 0.25 mg / mL UvsY, for example, about 0.09 mg / mL to about 0.25 mg / mL UvsY; (viii) about 0.01 mg / mL to about 0.5 mg / mL creatine kinase. (ix) about 0.1 mg / mL of DNA polymerase, for example about 0.4 mg / mL to about 0.8 mg / mL of DNA polymerase; (ix) about 0.1 mg / mL to about 2.0 mg / mL of GP32, for example about 0.4 mg / mL to about 0.8 mg / mL of GP32; and (x) about 0.01 mg / mL to about 0.5 mg / mL of exonuclease III, for example about 0.1 mg / mL to about 0.5 mg / mL of exonuclease III.

[0151] In some embodiments, the RPA method of this disclosure includes contacting a sample containing a target nucleic acid with a composition disclosed herein, such as a composition containing trehalose and PEG in a wt% ratio of about 0.25 to about 0.79. In some embodiments, the RPA method of this disclosure includes contacting a sample containing a target nucleic acid with a composition disclosed herein, such as a composition containing trehalose and PEG in a wt% ratio of about 0.3 to about 0.79. In some embodiments, the RPA method of this disclosure includes contacting a sample containing a target nucleic acid with a composition disclosed herein, such as a composition containing trehalose and PEG in a wt% ratio of about 0.45 to about 0.75 (e.g., a wt% ratio of about 0.5 to about 0.7). In some embodiments, the RPA method of this disclosure includes contacting a sample containing a target nucleic acid with a composition disclosed herein, such as a composition containing trehalose and PEG in a wt% ratio of about 0.3 to about 0.45. In some embodiments, this disclosure provides an RPA method comprising contacting a sample containing a target nucleic acid with a composition disclosed herein, for example, a composition containing trehalose and PEG at a wt% ratio of about 0.79 or less. In some embodiments, this disclosure provides an RPA method comprising contacting a sample containing a target nucleic acid with a composition disclosed herein, for example, a composition containing trehalose and PEG at a wt% ratio of about 0.75 or less (e.g., about 0.65 or less, about 0.55 or less, about 0.45 or less, or about 0.35 or less). In some embodiments, the RPA method of this disclosure for amplifying target nucleic acids includes: (a) contacting a sample (e.g., a solution) containing multiple nucleic acids with a composition containing trehalose and PEG in a wt% ratio of about 0.3 to about 0.79 (e.g., about 0.45 to about 0.75, or about 0.5 to about 0.7, or about 0.3 to about 0.45) to generate a reaction mixture; and (b) incubating the reaction mixture to amplify the target nucleic acid (if it is present in multiple nucleic acids). In some embodiments, the RPA method of this disclosure for amplifying target nucleic acids includes: (a) contacting a composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.79 (e.g., about 0.45 to about 0.75, or about 0.5 to about 0.7, or about 0.3 to about 0.45) with at least one additional composition comprising one or more other RPA reagents, and subsequently contacting the mixture with a sample (e.g., a solution) containing a plurality of nucleic acids to generate a reaction mixture; and (b) incubating the reaction mixture to amplify the target nucleic acid (if present in a plurality of nucleic acids).

[0152] In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition disclosed herein is added to a sample or other RPA reagent to produce a PEG concentration of about 2% to about 9% w / v in the RPA method, for example, about 5% to about 9% PEG or about 5% to about 8% PEG. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to a sample or other RPA reagent to produce an RPA method with a PEG concentration of about 2% to about 9% w / v, for example, about 5% to about 9% PEG or about 5% to about 8% PEG. In some embodiments, the RPA reaction or composition comprises about 2% to about 8% w / v PEG, for example, about 5% to about 8% w / v PEG.

[0153] In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition of this disclosure are added to a sample or other RPA reagent to reduce the trehalose concentration in the composition to about 2% to about 6% w / v trehalose. In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition of this disclosure are added to a sample or other RPA reagent to reduce the trehalose concentration in the composition to about 1.5% to about 6% w / v trehalose. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to a sample or other RPA reagent to reduce the trehalose concentration in the composition to about 2% to about 6% w / v trehalose. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to a sample or other RPA reagent to reduce the trehalose concentration in the composition to about 1.5% to about 6% w / v trehalose. In some embodiments, the RPA reaction or composition comprises about 2% to about 6% w / v or about 2% to about 5% w / v trehalose, for example, about 2% w / v trehalose or about 3% w / v trehalose.

[0154] In some embodiments, the RPA reaction or composition contains about 3% to about 6% w / v or about 3% to about 5% w / v of trehalose, for example, about 4% w / v of trehalose.

[0155] In some embodiments, the composition may comprise one or more RPA reagents disclosed herein, such as reagents required for performing and / or supporting RPA methods. Non-limiting examples of such reagents include recombinases, single-stranded DNA-binding proteins, recombinase-loading proteins, creatine kinases, nucleases, reverse transcriptases, DNA polymerases, dNTPs, buffers, congestants, reducing agents, ATP or ATP analogs, a first primer, a second primer, and a probe.

[0156] In some embodiments, the method of this disclosure may further include detecting the presence and / or absence of the target nucleic acid. In some embodiments, the method of this disclosure may further include detecting the amount of the target nucleic acid.

[0157] In some embodiments, the reaction volume of the RPA method of this disclosure can be about 5 μl, about 10 μl, about 20 μl, about 30 μl, about 50 μl, about 75 μl, about 100 μl, about 300 μl, about 1 ml, about 3 ml, about 10 ml, about 30 ml, about 50 ml, or about 100 ml. In some embodiments, the reaction volume of the RPA method of this disclosure can be about 50 μl to about 100 μl.

[0158] In some embodiments, the target nucleic acid in the RPA reaction can be of any concentration. For example, but not as a limitation, the target nucleic acid may be less than about 10,000 copies, less than about 1,000 copies, less than about 100 copies, less than about 10 copies, or even 1 copy. In some embodiments, the isothermal amplification method of this disclosure can result in 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, or 1,000,000-fold amplification of the target nucleic acid in the reaction.

[0159] In some embodiments, the reaction temperature of the RPA method of this disclosure is between about 20°C and about 50°C, about 20°C and about 40°C, about 20°C and about 30°C, or about 37°C and about 42°C. In some embodiments, the reaction temperature is about 40°C.

[0160] In some embodiments, the RPA method of this disclosure begins with the addition of reagents sufficient to initiate the RPA method, and the reaction time is approximately 10 minutes to approximately 3 hours, approximately 10 minutes to approximately 2 hours, approximately 10 minutes to approximately 1 hour, approximately 10 minutes to approximately 30 minutes, approximately 10 minutes to approximately 25 minutes, approximately 10 minutes to approximately 20 minutes, or even approximately 10 minutes to approximately 15 minutes. In some embodiments, the RPA method of this disclosure begins with the addition of reagents sufficient to initiate RPA amplification, and the reaction time is approximately 1 minute to approximately 20 minutes, approximately 5 minutes to approximately 20 minutes, approximately 8 minutes to approximately 20 minutes, approximately 1 minute to approximately 10 minutes, or approximately 5 minutes to approximately 10 minutes. In some embodiments, the reaction time of the RPA method of this disclosure is approximately 1 minute. In some embodiments, the reaction time of the RPA method of this disclosure is approximately 5 minutes. In some embodiments, the reaction time of the RPA method of this disclosure is approximately 20 minutes. In some embodiments, the RPA reaction time is sufficient to obtain results, for example, for detecting target nucleic acids.

[0161] B. NEAR Method

[0162] In some embodiments, the isothermal amplification method is NEAR. In some embodiments, this disclosure provides a NEAR method and / or a reagent composition for a NEAR method, the composition comprising trehalose and PEG in the ratios and amounts described herein. In some embodiments, this disclosure provides a NEAR method performed in the presence of a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.79%, and / or using a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.3 to about 0.79%. For example, but not as a limitation, this disclosure provides a NEAR method performed in the presence of a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.45 to about 0.75 (e.g., about 0.5 to about 0.7), and / or using a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.45 to about 0.75 (e.g., about 0.5 to about 0.7).

[0163] In some embodiments, this disclosure provides a NEAR method performed in the presence of a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.79 or less, and / or using a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.79 or less. In some embodiments, this disclosure provides a NEAR method performed in the presence of a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.75 or less (e.g., 0.65 or less or 0.55 or less), and / or using a reagent composition comprising trehalose and PEG in a wt% ratio of about 0.75 or less (e.g., 0.65 or less or 0.55 or less).

[0164] In some embodiments, this disclosure provides a NEAR method performed in the presence of about 2% to about 9% w / v PEG. In some embodiments, this disclosure provides a NEAR method performed in the presence of about 2% to about 8% w / v PEG (e.g., about 5% to about 8% w / v PEG).

[0165] In some embodiments, this disclosure provides a NEAR method carried out in the presence of about 1% to about 12% w / v trehalose (e.g., about 2% to about 12% w / v trehalose). In some embodiments, this disclosure provides a NEAR method carried out in the presence of about 5% to about 12% w / v trehalose. In some embodiments, this disclosure provides a NEAR method carried out in the presence of about 3% to about 5% w / v trehalose (e.g., about 4% w / v trehalose).

[0166] In NEAR, a target nucleic acid sequence having a sense strand and an antisense strand is contacted with a pair of amplified oligonucleotides. The first amplified oligonucleotide comprises a nucleic acid sequence containing a recognition region at its 3' end complementary to the 3' end of the antisense strand of the target sequence, a nicking enzyme site upstream of the recognition region, and a stability region upstream of the nicking enzyme site (see, for example, U.S. Patents 9,689,031; 9,617,586; 9,562,264; and 9,562,263, each of which is incorporated herein by reference in its entirety). The second amplified oligonucleotide comprises a nucleotide sequence containing a recognition region at its 3' end complementary to the 3' end of the sense strand of the target sequence, a nicking enzyme site upstream of the recognition region, and a stability region upstream of the nicking enzyme site. Two nicking enzymes are provided. One nicking enzyme is capable of nicking the nicking enzyme site of the first amplified oligonucleotide but not of nicking the target sequence. The other nicking enzyme is capable of nicking the nicking enzyme site of the second amplified oligonucleotide but not of nicking the target sequence. Under amplification conditions, amplification is performed using a DNA polymerase, which involves multiple extension cycles of amplifying oligonucleotides to generate double-stranded nickase sites, which are nicked by the nickase to produce amplified products. See, for example, U.S. Patent Nos. 9,689,031; 9,617,586; 9,562,264; 9,562,263; and 10,851,406 and U.S. Patent Application Nos. 15 / 467,893 and 16 / 243 / 829, each of which is incorporated herein by reference in its entirety.

[0167] In some embodiments, the reaction is carried out under isothermal conditions using only two templates for initiation, one or two nicking enzymes, and a polymerase. In exemplary, non-limiting embodiments, the polymerase and nicking enzyme are thermophilic, and the reaction temperature is significantly above the melting temperature of the hybridization target region. The nicking enzyme nicks only one strand in the double helix, eliminating the need for incorporation of modified nucleotides as in strand substitution methods. In some embodiments, the method can amplify RNA without a separate reverse transcription step, although if desired, conversion of RNA to DNA via reverse transcription can be used.

[0168] In some embodiments, the method includes: contacting a target DNA molecule containing a double-stranded target sequence (the target sequence having a sense strand and an antisense strand) with a forward template and a reverse template, wherein the forward template includes a nucleic acid sequence comprising a recognition region complementary to the 3' end of the antisense strand of the target sequence, a nicking enzyme site upstream of the recognition region, and a stable region upstream of the nicking enzyme site; the reverse template includes a nucleotide sequence comprising a recognition region complementary to the 3' end of the sense strand of the target sequence, a nicking enzyme site upstream of the recognition region, and a stable region upstream of the nicking enzyme site; providing a first nicking enzyme capable of nicking at the nicking enzyme site on the forward template but not within the target sequence; providing a second nicking enzyme capable of nicking at the nicking enzyme site on the reverse template but not within the target sequence; and providing a DNA polymerase; wherein amplification is performed under conditions in which the polymerase extends the forward and reverse templates along the target sequence to generate a double-stranded nicking enzyme site, and the nicking enzyme nicks at the nicking enzyme site to generate an amplification product.

[0169] In some embodiments, the DNA polymerase is a thermophilic polymerase. In other embodiments, the polymerase and the nicking enzyme are stable at temperatures up to 37°C, 42°C, 60°C, 65°C, 70°C, 75°C, 80°C, or 85°C. In some embodiments, the polymerase is stable at temperatures up to 60°C. In some embodiments, the polymerase may be selected, for example, from the group consisting of: Bst (large fragment), 9°N, VentR® (exonuclease) DNA polymerase, THERMINATOR, and THERMINATOR II (New England Biolabs).

[0170] In some embodiments, the nicking enzyme may nick upstream of the nicking enzyme binding site, or the nicking enzyme may nick downstream of the nicking enzyme binding site. In some embodiments, the forward and reverse templates contain nicking enzyme sites recognized by the same nicking enzyme, and the first and second nicking enzymes are identical. In some embodiments, the nicking enzyme may be selected, for example, from the group consisting of: Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10I, and Nt.Bpu10I.

[0171] In some implementations, the target sequence contains 1 to 5 more nucleotides than the sum of the nucleotides in the forward template recognition region and the reverse template recognition region.

[0172] In some embodiments, the concentrations of the provided forward template and reverse template are the same. In some embodiments, the ratio of the provided forward template to the reverse template ranges from 1:100 to 100:1.

[0173] In some embodiments, the NEAR reaction time, starting from the addition of sufficient reagent to initiate NEAR amplification, can be approximately 10 minutes to approximately 3 hours, approximately 10 minutes to approximately 2 hours, approximately 10 minutes to approximately 1 hour, approximately 10 minutes to approximately 30 minutes, approximately 8 minutes to approximately 25 minutes, approximately 8 minutes to approximately 20 minutes, or even approximately 8 minutes to approximately 15 minutes. In some embodiments, the NEAR reaction time, starting from the addition of sufficient reagent to initiate NEAR amplification, can be approximately 1 minute to approximately 20 minutes, approximately 5 minutes to approximately 20 minutes, approximately 8 minutes to approximately 20 minutes, approximately 1 minute to approximately 10 minutes, or approximately 5 minutes to approximately 10 minutes.

[0174] C. Nucleic acid testing

[0175] The nucleic acid detection methods used herein are employed to determine the presence (e.g., presence or absence) of a target nucleic acid or multiple different target nucleic acids in a sample. In some embodiments, nucleic acid detection is used to quantify the amount of nucleic acid or multiple different nucleic acids in a sample. As illustrated herein, the nucleic acid detection methods of this disclosure can be configured to detect a target nucleic acid or multiple different target nucleic acids using any of a variety of suitable detection techniques or combinations of suitable detection techniques.

[0176] In some embodiments, after amplifying one or more target nucleic acids present in a sample, the methods of this disclosure can be configured to detect the amplified nucleic acid, for example, by hybridization. Such detection may include, for example but not limited to, hybridizing a probe oligonucleotide that is sufficiently complementary to the amplified target nucleic acid to facilitate the detection of the target nucleic acid. In some embodiments, after the probe oligonucleotide hybridizes with the target nucleic acid, the method includes detecting the hybridization of the probe oligonucleotide with the target nucleic acid. Such detection may be achieved, for example but not limited to, by observing a signal from a detectable marker, wherein: (i) the presence of one or more signals indicates hybridization of the probe oligonucleotide with the target nucleic acid and indicates the presence of the target nucleic acid in the sample; and (ii) the absence of a signal indicates the absence of the target nucleic acid in the sample. Depending on the type of detectable marker, various suitable methods can be used for the detection of signals from the probe oligonucleotide.

[0177] In some embodiments, nucleic acid amplification (e.g., using the isothermal amplification method described herein) and nucleic acid detection can be performed simultaneously, for example, during the amplification and detection process. In some embodiments, the amplification and detection process disclosed herein includes simultaneously amplifying and detecting nucleic acids in a sample (e.g., elution buffer). In some embodiments, the amplification and detection process begins with incubation of the elution buffer with reagents sufficient to initiate the amplification of the target nucleic acid in the sample (using the isothermal amplification method of this disclosure, if present) and ends when a result is determined in the sample, for example, the target nucleic acid is detected in the elution buffer or is not detected in the sample.

[0178] In some implementations, the amplified nucleic acid may be detected using optical detection, digital detection, and / or other detection methods known in the art.

[0179] i. Optical inspection

[0180] In some implementations, the detection of amplified nucleic acids can be performed using optical detection. For example, but not as a limitation, the detection of amplified target nucleic acids can be mediated by the binding of labeled probes or by the incorporation of labels into copies of amplified target nucleic acids.

[0181] In addition, or alternatively, and depending on other aspects of the disclosed subject matter, detection may be mediated by observation of fluorescent markers (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein (e.g., FAM), 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-coated cadmium selenide), Fluor Orange 560 fluorophore, Quasar 670 fluorophore, and Quasar 705 fluorophore). Introductions to labeling, labeling procedures, and labeling detection can be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd Edition, Springer Verlag, NY (1997) and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996) (the latter being a collection of manuals and catalogues published by Molecular Probes, Inc., Eugene, Oregon). Fluorescent labeling can be used for FPIA (see, for example, U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated herein by reference in their entirety).

[0182] Furthermore, or alternatively, and according to other aspects of the disclosed subject matter, optical detection is performed in response to the presence of an analyte by fluorescence, chemiluminescence, or other signal generation. Many assays involve measuring the intensity of a light signal generated in the total volume of the reaction mixture. The generated light signal can be measured optically, wherein the generated light signal is emitted by a large number of molecules. Typically, as described herein, the assay may involve mixing a sample suspected of containing a target nucleic acid (e.g., an amplified target nucleic acid as described herein) with a reagent containing a labeled probe capable of hybridizing with the target nucleic acid, thereby forming a reaction mixture. After removing unbound probes from the reaction mixture (typically by a washing step), the signal attributed to the label is then measured. In some embodiments, the presence of a detectable signal is sufficient to confirm the presence of the target nucleic acid in the sample. In some embodiments, the signal derived from the total volume of the reaction mixture is measured and then compared to a calibration curve to determine the concentration of the target nucleic acid in the sample.

[0183] Furthermore, or alternatively, and according to other aspects of the disclosed subject matter, optical detection strategies include using probes labeled with a detectable marker and a “quenching molecule,” wherein the quenching molecule is capable of interacting with the detectable marker to reduce or eliminate the signal emitted by the detectable marker. For example, but not as a limitation, the detection probe used in the methods of this disclosure may have a covalently linked fluorescent portion (e.g., attached to the 5' end of the probe) and a quenching molecule (e.g., at the 3' end of the probe). In the absence of a target sequence, the probe adopts a conformation in which the quencher is sufficiently close to the excited-state fluorophore to absorb its energy before it can be emitted fluorescently. When the probe binds to a complementary sequence in its target, the fluorophore and the quencher are positioned at a sufficiently large distance to allow for fluorescence emission and detection. In some embodiments, the quencher may be selected from any suitable quencher known in the art, such as, for example, BLACK HOLE QUENCHER. ® 1 (BHQ-1 ® ), BLACKHOLE QUENCHER ® 2 (BHQ-2 ® ), BLACK HOLE QUENCHER ® -1-dT (BHQ-1 dT ® ), BLACK HOLEQUENCHER ® -2-dT (BHQ-2dT ® ), IOWA BLACK ® FQ and IO WA BLACK ® RQ. For example, but not as a limitation, the oligonucleotide probes used in the methods disclosed herein may contain a FAM fluorophore and BHQ-1 dT.® Quenching agent or BHQ-2dT ® Quenching agent. In some embodiments, the oligonucleotide probes used in the methods of this disclosure may include Quasar 670 fluorophores and BHQ-1. ® Quenching agent or BHQ-2 ® Quenching agent. In some embodiments, the oligonucleotide probes used in the methods of this disclosure may include Quasar 670 fluorophores and BHQ-1 dT. ® Quenching agent or BHQ-2dT ® Quenching agent.

[0184] In addition, or alternatively, and depending on other aspects of the disclosed subject matter, specific probes (e.g., probes for specific target nucleic acids and / or internal controls) are each labeled with different fluorophores, thereby allowing the simultaneous detection of multiple amplified products.

[0185] Furthermore, or alternatively, and according to other aspects of the disclosed subject matter, light-emitting diodes (LEDs) and / or lasers can be used for excitation, and emission detection can be performed using any suitable detector to measure light intensity. A fluorescence optical detection “scanner” can be used, which uses a focused laser beam to scan the chip surface, thereby allowing the detection of emitted fluorescence. Exemplary fluorescence scanners are described, for example, in U.S. Patent Nos. 5,837,475 and 5,945,679. Scanners that integrate a confocal excitation and detection system into an epifluorescence microscope are known. The system in the scanner used to detect emitted fluorescence is typically a “single-channel system,” i.e., a single photocell or secondary electron multiplier (photomultiplier tube). Two-dimensional detection systems, such as, for example, charge-coupled device (CCD) cameras, are also used to detect fluorescence or chemiluminescence in samples. Commercially available systems have optical imaging systems that utilize lens optics to project a bonding surface, provided with chemiluminescent or fluorescent labels, onto a CCD sensor; or have a combination of an image intensifier and a CCD camera.

[0186] ii. Digital detection

[0187] In some implementations, the detection of amplified nucleic acids can be performed using digital detection methods. Since each individual target nucleic acid (as the endpoint entity) can be detected in principle in the context of digital detection, the components and methods associated with digital detection can significantly improve the detection sensitivity of sample analysis compared to systems using analog optical detection. Therefore, digital detection can be performed using lower concentrations of analytes (e.g., target nucleic acids), which allows for shorter sample processing times for detection. Furthermore, or alternatively, detection can be performed using smaller sample volumes, less reagent material, less conjugate material, fewer particles, or any combination thereof, which reduces the cost per assay. Thus, and as described herein, sample preparation time can be improved, at least in part by reducing sample handling involved (e.g., faster washing time) and / or by using fewer sample volumes, less reagent or conjugate material, and / or fewer particles or beads to obtain suitable analyte concentrations for detection, thereby achieving improved reaction kinetics. Assays using fewer sample volumes and / or reagent material can be performed using smaller equipment, which reduces the space required for laboratory systems to perform assays, as discussed further herein. Furthermore, or as a further alternative, increasing detection sensitivity can provide additional benefits when used with multiplex analysis. For example, and without limitation, when multiple analytes and their corresponding signals are combined into a single multiplex assay, the noise levels associated with the detection of each analyte signal can be multiplied to obtain the total noise level of the multiplex analysis system. By increasing the detection sensitivity of each detected signal, the increased sensitivity can be multiplied to further reduce the total noise level of the multiplex analysis system.

[0188] Digital detection offers improved sensitivity, at least in part, due to the reduction of noise relative to the measured signal during detection, resulting in a higher signal-to-noise ratio (SNR). Such improved SNR is possible by coupling the analyte of interest (e.g., a specific target nucleic acid) to an independently detectable endpoint entity. For example, but not limited to, the amplified target nucleic acid can be immobilized to a microparticle and labeled with a detectable conjugate, where the conjugate is a detectable endpoint entity because it emits an independently detectable signal, either directly or through transformation of the substrate.

[0189] In some embodiments, the detection operation employs a digital nanopore detection method. In some embodiments, a support medium (such as, but not limited to, microparticles, beads, or other markers) may be mixed with the sample to allow for digital detection after amplification. In some embodiments, a reagent combination comprising antibodies and coated microparticles may be used.

[0190] For example, but not as a limitation, digital nanopore detection methods containing microparticles may employ anti-digoxin microparticles. In some embodiments, digital nanopore detection containing microparticles may be performed in a formulation comprising: Tris-HCl, NaCl, BSA, Tergitol 15-s-40, sodium azide, and 0.02% anti-digoxin μP (microparticles). For example, but not as a limitation, digital nanopore detection containing microparticles may be performed in an environment containing approximately 50 mM Tris-HCl (pH approximately 8.0); approximately 150 mM NaCl; approximately 0.2% BSA; approximately 0.5% Tergitol 15-s-40; approximately 0.08% sodium azide; and approximately 0.02% anti-digoxin μP (microparticles). The solution may be washed, for example, to remove excess reagents and / or unbound analytes. Each washing step may be performed any suitable number of times, including one, two, three or more washes, and each wash may be performed in a single chamber or location, or in different chambers or locations. For example, but not limited to, as illustrated herein, three washes may be performed.

[0191] Furthermore, or alternatively, and depending on other aspects of the disclosed subject matter, a conjugate may be added to bind to an analyte of interest in the sample. In some embodiments, a conjugate (e.g., alkaline phosphatase-SA) may be added to the sample. In some embodiments, other reagents (including, but not limited to, Tris-HCl, NaCl, MgCl2, ZnCl2, fish gelatin, rabbit IgG, saponins, calf serum, goat IgG, and sodium azide) may be added to the sample. For example, and not limited to, the conjugate may contain one or more reagents or enzymes selected or configured to react with the analyte of interest to generate a signal for detection by a detection component. In some embodiments, the digital nanopore detection method employs conjugates in the following context: approximately 3000 pM alkaline phosphatase-SA; approximately 100 mM Tris-HCl (pH approximately 7.5); approximately 500 mM NaCl; approximately 1 mM MgCl2; approximately 0.1 mM ZnCl2; approximately 8.9 g / L fish gelatin; approximately 30 μg / mL rabbit IgG; approximately 0.1% saponins; approximately 10% fetal bovine serum; approximately 5 mg / mL goat IgG; and approximately 0.1% sodium azide. The solution may be washed, for example, to remove excess conjugates that are not bound to the analyte of interest. Washing may be performed any suitable number of times for each washing step, including one, two, three, or more washes, and each wash may be performed in a single chamber or location, or in different chambers or locations.

[0192] Furthermore, or alternatively, and according to other aspects of the disclosed subject matter, the detection of microparticles bound to the analyte and conjugate may be performed in a single chamber or location, or in different chambers or locations. For illustrative purposes and not to limit, the detection chamber or location may include a surface and a detection region. Any suitable technique may be used to add microparticles to the detection chamber or location, including but not limited to pipetting, magnetics, or dielectrophoresis. In some embodiments, the digital nanopore detection method will employ a detection substrate, such as AJ Phos. In some embodiments, other reagents may be added, including but not limited to DEA, MgCl2, and Tween 20, and combined with the detection substrate. For example, but not as a limitation, the digital nanopore detection method will employ a detection substrate in the following contexts: approximately 200 µM of AJ Phos; approximately 1 M of DEA; approximately 1 mM of MgCl2; and approximately 0.05% Tween 20. As illustrated herein, the detection region may include one or more nanopores. Microparticles may be transferred to the detection region, for example, and as illustrated herein, to a nanopore array. Particles can be transferred to nanopores using any suitable technique, including but not limited to pipetting, magnetometry, or dielectrophoresis. In some embodiments, an oil (e.g., FC-40 oil of 3 mM guaiac blue) is added to seal the nanopores. In some embodiments, a dye may be added to improve contrast or otherwise enhance optical conditions for the detection of analytes of interest within the nanopores. In some embodiments, digital nanopore detection methods involving particles will employ dyes in the following contexts: approximately 0.1% Tween 20; approximately 10 mM PBS; and approximately 50 mM Nigrosine. In some embodiments, one or more particle images are captured and analyzed to determine the presence or absence of the analyte of interest in the sample and / or the concentration of the analyte of interest.

[0193] D. Sample

[0194] In some embodiments, the target nucleic acid amplified using the disclosed methods can be isolated from a sample, such as a sample from a subject. The target nucleic acid can be isolated from the sample by any method known in the art. Non-limiting examples of methods for isolating nucleic acids from a sample are disclosed in International Patent Application PCT / US2022 / 027067 (which is incorporated herein by reference in its entirety). For example, but not as a limitation, nucleic acids can be isolated from the sample using magnetic microparticles (e.g., copper-titanium microparticles), such as those described in PCT / US2022 / 027067. Figure 4 As shown in 9 and 10.

[0195] In some embodiments, the sample may be a tissue sample. In some embodiments, the sample may be obtained from preserved tissue (e.g., fixed tissue), frozen tissue, or fresh tissue (e.g., fresh tissue sample). Non-limiting examples of tissue include the eye, muscle, skin, tendon, vein, artery, heart, spleen, lymph nodes, bone, bone marrow, lung, bronchus, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonads, testes, ovaries, uterus, fallopian tubes, thymus, pituitary gland, thyroid gland, adrenal gland, or parathyroid gland tissue. In some embodiments, the tissue may be cancerous tissue, such as tumor tissue.

[0196] In some embodiments, the sample is a biological fluid sample. In some embodiments, the biological fluid sample is bodily secretions. Non-limiting examples of biological fluid and bodily secretion samples include blood (e.g., whole blood, lysed whole blood, serum, or plasma), saliva or oral fluid, sweat, tears, mucus, urine, lymph, cerebrospinal fluid, tissue fluid, bronchoalveolar lavage fluid, nasopharyngeal fluid, or any other sample suitable for analysis using the methods and techniques described herein. In some embodiments, the sample may be a nasal swab (e.g., a nasal swab in a buffer (e.g., at least partially coated with a target nucleic acid)) and / or a sample obtained using a nasal swab (e.g., a nasopharyngeal fluid sample).

[0197] In some embodiments, the biological fluid sample is whole blood. As used herein, “whole blood” means blood that has not yet had any components removed (blood containing both liquid and solid components). Transfusing whole blood or its red blood cell (RBC) component can increase its oxygen-carrying capacity by effectively increasing the patient’s RBC count, thereby increasing the amount of available oxygen-carrying hemoglobin. In addition to its oxygen-carrying capacity, whole blood transfusion can be a source of platelets, which contribute to blood clotting. In some embodiments, for clinical use, platelet transfusion can be used to treat thrombocytopenia, certain cancers, aplastic anemia, and bone marrow transplantation.

[0198] In some embodiments, the biological fluid sample is lysed whole blood. As used herein, "lysed whole blood" means blood (containing both liquid and solid components) for which no components have been removed, but in which RBCs have been lysed by exposure to a buffer containing, for example, ammonium chloride, potassium carbonate, and EDTA. Ammonium chloride (which lyses RBS) has minimal effect on lymphocytes.

[0199] In some embodiments, the biological fluid sample is plasma. Plasma is the aqueous portion of blood remaining after centrifugation to remove cellular components. In some embodiments, plasma may contain albumin, clotting factors, fibrinolytic proteins, immunoglobulins, and other proteins. In some embodiments, products derived from plasma donation can be used to treat hemorrhagic conditions and / or life-threatening wounds / bleeding.

[0200] In some embodiments, the biological fluid sample is serum. As used herein, "serum" means the clear fraction of plasma that does not contain fibrinogen, cells, or any solid components.

[0201] In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is a vertebrate or invertebrate, such as a human or a non-human animal, such as a mammal. In some embodiments, non-human animal subjects include rodents, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, apes, and monkeys. In some embodiments, the subject is a human.

[0202] III. Composition

[0203] This disclosure also provides compositions for performing the methods of this disclosure. For example, but not as a limitation, this disclosure provides compositions comprising one or more reagents, such as reagent compositions, for performing the isothermal application methods of this disclosure.

[0204] In some embodiments, the compositions of this disclosure comprise the ratios and amounts described herein, such as the ratios and amounts of trehalose and PEG described in Section II. In some embodiments, the compositions of this disclosure comprise in wt% ratios of about 0.79 or less, or about 0.75 or less, for example, about 0.74 or less, about 0.73 or less, about 0.72 or less, about 0.71 or less, about 0.7 or less, about 0.69 or less, about 0.68 or less, about 0.67 or less, about 0.66 or less, about 0.65 or less, about 0.64 or less, about 0.63 or less, about 0.62 or less, about 0.61 or less, about 0.60 or less, about 0.59 or less, about 0.58 or less, about 0.57 or less, about 0.56 or less, about 0.55 or less, about 0.54 or less, about... The composition comprises 0.53 or less, about 0.52 or less, about 0.51 or less, about 0.50 or less, about 0.49 or less, about 0.48 or less, about 0.47 or less, about 0.46 or less, about 0.45 or less, about 0.44 or less, about 0.43 or less, about 0.42 or less, about 0.41 or less, about 0.40 or less, about 0.39 or less, about 0.38 or less, about 0.37 or less, about 0.36 or less, about 0.35 or less, about 0.34 or less, about 0.33 or less, about 0.32 or less, or about 0.31 or less trehalose and PEG. In some embodiments, the compositions of this disclosure comprise trehalose and PEG in a wt% ratio of about 0.65 or less or 0.55 or less.

[0205] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.75 or less.

[0206] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.65 or less.

[0207] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.60 or less.

[0208] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.55 or less.

[0209] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.45 or less.

[0210] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.35 or less.

[0211] In some embodiments, the compositions of this disclosure comprise a wt% ratio not greater than about 0.79 or not greater than about 0.75, for example, not greater than about 0.74, not greater than about 0.73, not greater than about 0.72, not greater than about 0.71, not greater than about 0.7, not greater than about 0.69, not greater than about 0.68, not greater than about 0.67, not greater than about 0.66, not greater than about 0.65, not greater than about 0.64, not greater than about 0.63, not greater than about 0.62, not greater than about 0.60, not greater than about 0.59, not greater than about 0.58, not greater than about 0.57, not greater than about 0.56, not greater than about 0.55, not greater than about 0.54, not greater than about 0. 53. Trehalose and PEG not greater than about 0.52, 0.51, 0.50, 0.49, 0.48, 0.47, 0.46, 0.45, 0.44, 0.43, 0.42, 0.41, 0.40, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, or 0.31.

[0212] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of no more than about 0.75.

[0213] In some embodiments, the compositions of this disclosure contain trehalose and PEG in a wt% ratio of no more than about 0.65.

[0214] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of no more than about 0.55.

[0215] In some embodiments, the compositions of this disclosure contain trehalose and PEG in a wt% ratio of no more than about 0.45%.

[0216] In some embodiments, the compositions of this disclosure contain trehalose and PEG in a wt% ratio of no more than about 0.35. In some embodiments, the compositions of this disclosure contain trehalose and PEG in a wt% ratio of about 0.3 to about 0.79, for example, about 0.3 to about 0.7, about 0.3 to about 0.5, or about 0.3 to about 0.35. In some embodiments, the compositions of this disclosure comprise trehalose and PEG in a wt% ratio of about 0.45 to about 0.75, for example, about 0.45 to about 0.75, about 0.55 to about 0.75, about 0.55 to about 0.75, about 0.6 to about 0.75, about 0.65 to about 0.75, about 0.7 to about 0.75, about 0.4 to about 0.7, about 0.4 to about 0.65, about 0.4 to about 0.65, about 0.4 to about 0.55, about 0.4 to about 0.45, about 0.5 to about 0.7, about 0.45 to about 0.55, about 0.45 to about 0.65, about 0.55 to about 0.65, about 0.5 to about 0.55, about 0.6 to about 0.65, or about 0.7 to about 0.75. In some embodiments, the compositions of this disclosure comprise trehalose and PEG in a wt% ratio of about 0.3 to about 0.7. In some embodiments, the compositions of this disclosure comprise trehalose and PEG in a wt% ratio of about 0.5 to about 0.75. In some embodiments, the compositions of this disclosure comprise trehalose and PEG in a wt% ratio of about 0.5 to about 0.7.

[0217] In some embodiments, the compositions disclosed herein contain trehalose and PEG in a wt% ratio of about 0.3 to about 0.75.

[0218] In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition of this disclosure are added to an isothermal amplification method, for example, added to a solution to prepare a master mixture for performing the isothermal amplification method. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 2% to about 9% w / v, for example, about 5% to about 9% w / v or about 6% to about 9% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 2% to about 8.5% w / v, for example, about 5% to about 8.5% w / v or about 6% to about 8.5% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 2% to about 8.5% w / v, for example, about 5% to about 8.5% w / v or about 6% to about 8.5% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 5% to about 8.5% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 6.3% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 6.5% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 7%. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 7.7%. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 8.4%. For example, but not as a limitation, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition of this disclosure may be added to an isothermal amplification process to produce a PEG concentration of about 5% to about 8.5% w / v.In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition disclosed herein are added to the isothermal amplification process to produce PEG with a reaction concentration of about 5% to about 9% w / v. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to the isothermal amplification process to produce PEG with a reaction concentration of about 5% to about 9% w / v. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to the isothermal amplification process to produce a medium PEG with a reaction concentration of about 5% to about 8.5% w / v.

[0219] In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain PEG at a concentration of about 10% to about 30% w / v, thereby producing a PEG concentration of about 5% to about 8.5% w / v. In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain PEG at a concentration of about 10% to about 25% w / v, thereby producing a PEG concentration of about 5% to about 8.5% w / v. In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain PEG at a concentration of about 10% to about 20% w / v, thereby producing a PEG concentration of about 5% to about 8.5% w / v.

[0220] In some embodiments, this disclosure provides a composition comprising about 10% to about 30% w / v of PEG.

[0221] In some embodiments, this disclosure provides a composition comprising about 10% to about 25% w / v of PEG, for example, about 30% w / v of PEG.

[0222] In some embodiments, this disclosure provides a composition comprising about 10% to about 20% w / v of PEG.

[0223] In some embodiments, this disclosure provides a composition comprising about 2% to about 9% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 2% to about 8.5% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 4% to about 6% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 5% to about 7% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 6% to about 7% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 6% to about 8% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 7% to about 8% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 5.5% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 6.5% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 7.7% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 5% to about 8% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 8% to about 9% w / v of PEG. In some embodiments, this disclosure provides a composition comprising about 8% to about 8.5% w / v of PEG.

[0224] In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the composition of this disclosure are added to the isothermal amplification method, for example, to reduce the concentration of trehalose provided by the composition. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification method (e.g., diluted), produces a concentration of trehalose from about 1.5% to about 6% w / v. In some embodiments, this disclosure provides a composition that, when added to an isothermal amplification method (e.g., diluted), produces a concentration of trehalose from about 2% to about 6% w / v. For example, but not as a limitation, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the compositions disclosed herein may be added to the isothermal amplification method, for example, to reduce the trehalose concentration provided by the composition to about 2% to about 6% w / v, for example, about 2% to about 4% trehalose concentration. In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the compositions disclosed herein may be added to the isothermal amplification method, for example, to reduce the trehalose concentration provided by the composition to about 1.5% to about 6% w / v, for example, about 1.5% to about 4% trehalose concentration. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to the isothermal amplification method, for example, to reduce the trehalose concentration provided by the composition to about 2% to about 6% w / v trehalose concentration, for example, about 2% to about 4% trehalose concentration. In some embodiments, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the composition disclosed herein are added to the isothermal amplification method, for example, to reduce the trehalose concentration provided by the composition to about 1.5% to about 6% w / v, such as about 1.5% to about 4% trehalose concentration.

[0225] In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain trehalose at a concentration of about 5% to about 30% w / v, said trehalose being diluted, for example, to a concentration of about 2% to about 6% w / v when added to an isothermal amplification method. In some embodiments, the reagent composition comprising one or more non-protein components may contain trehalose at a concentration of about 10% to about 30% w / v (e.g., about 15% to about 30% w / v), said trehalose being reduced to a concentration of about 2% to about 6% w / v when added to an isothermal amplification method. In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain trehalose at a concentration of about 5% to about 25% w / v, said trehalose being diluted to a concentration of about 2% to about 6% w / v when added to an isothermal amplification method. In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain trehalose at a concentration of about 5% to about 20% w / v, said trehalose being diluted, for example, to a concentration of about 2% to about 6% w / v when added to an isothermal amplification method. In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain trehalose at a concentration of about 5% to about 15% w / v, said trehalose being diluted, for example, to a concentration of about 2% to about 6% w / v when added to an isothermal amplification method. In some embodiments, the reagent composition (e.g., a reagent composition comprising one or more non-protein components) may contain trehalose at a concentration of about 5% to about 10% w / v, said trehalose being diluted, for example, to a concentration of about 2% to about 6% w / v when added to an isothermal amplification method.

[0226] In some embodiments, this disclosure provides a composition comprising about 5% to about 15% w / v trehalose, for example about 5% to about 7% trehalose. In some embodiments, this disclosure provides a composition comprising about 5.9% w / v trehalose.

[0227] In some embodiments, this disclosure provides a composition comprising about 5% to about 20% w / v trehalose, for example about 10% to about 20% trehalose. In some embodiments, this disclosure provides a composition comprising about 15% w / v trehalose.

[0228] In some embodiments, this disclosure provides a composition comprising about 7% to about 10% w / v trehalose, for example, about 7% to about 10% trehalose. In some embodiments, this disclosure provides a composition comprising about 8.15% w / v trehalose.

[0229] In some embodiments, this disclosure provides a composition comprising about 5% to about 6% w / v trehalose, for example about 5.5% to about 6% trehalose. In some embodiments, this disclosure provides a composition comprising about 5.7% w / v trehalose.

[0230] In some embodiments, this disclosure provides a composition comprising about 3% to about 5% w / v trehalose, for example about 3.5% to about 4.5% trehalose. In some embodiments, this disclosure provides a composition comprising about 4% w / v trehalose.

[0231] In some embodiments, this disclosure provides a composition comprising about 1% to about 2% w / v trehalose, for example about 1.5% to about 2% trehalose. In some embodiments, this disclosure provides a composition comprising about 1.95% w / v trehalose.

[0232] In some embodiments, this disclosure provides a composition comprising about 2% to about 3% w / v trehalose, for example about 2.5% to about 3% trehalose. In some embodiments, this disclosure provides a composition comprising about 2.75% w / v trehalose.

[0233] In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise one or more of the following (in any combination): at least one recombinase, at least one single-stranded DNA-binding protein, at least one DNA polymerase, dNTP, buffer, reducing agent, ATP or ATP analog, at least one recombinase-loading protein, a first primer and optionally a second primer, a probe, a nuclease (e.g., an exonuclease), creatine kinase, reverse transcriptase, and a template nucleic acid molecule (e.g., single-stranded (such as RNA) or double-stranded nucleic acid).

[0234] In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise non-protein components present in the isothermal reaction. In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise dNTPs, buffers, salts, and / or ATP or ATP analogs.

[0235] In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise non-protein components, including but not limited to dNTPs, buffers, ATP, salts, and creatine phosphate. In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise NTPs, buffers (e.g., Tris buffer), ATP, salts (e.g., potassium acetate), and creatine phosphate.

[0236] In some embodiments, the compositions of this disclosure (e.g., reagent compositions) comprise dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.79 or less, for example, about 0.75 or less. In some embodiments, the compositions of this disclosure (e.g., reagent compositions) comprise dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.79 or less, for example, about 0.75 or less, and wherein trehalose is present in the composition at a concentration of about 5% to about 20% w / v (e.g., about 5% to about 15% w / v), and PEG is present in the composition at a concentration of about 10% to about 30% w / v (about 15% to about 25% w / v).

[0237] In some embodiments, the compositions of this disclosure (e.g., reagent compositions) comprise dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.3 to about 0.79, for example, about 0.3 to about 0.75. In some embodiments, the compositions of this disclosure (e.g., reagent compositions) comprise dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.3 to about 0.79 (e.g., about 0.3 to about 0.75), and wherein trehalose is present at a concentration of about 5% to about 20% w / v (e.g., about 5% to about 15% w / v), and PEG is present at a concentration of about 10% to about 30% w / v (e.g., about 15% to about 25% w / v).

[0238] In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP, and primers. In some embodiments, the compositions of this disclosure (e.g., reagent compositions) further comprise a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP, primers, and template nucleic acid. In some embodiments, the compositions of this disclosure do not contain reverse transcriptase.

[0239] In some embodiments, this disclosure also provides an additional reagent composition (e.g., a second reagent composition) comprising a protein component (PC) (i.e., an enzyme) required for the RPA method (e.g., DNA polymerase, recombinase, recombinase-loading protein, single-strand binding protein, creatine kinase, nuclease (e.g., exonuclease), and / or reverse transcriptase). In some embodiments, this disclosure also provides an additional reagent composition, such as a second or third reagent composition comprising an oligonucleotide component (OC) required for the RPA method (e.g., one or more primers and / or one or more probes specific to one or two or more target nucleic acids). In some embodiments, trehalose may also be present in the reagent composition comprising the oligonucleotide component (OC). In some embodiments, trehalose may also be present in the reagent composition comprising the protein component (PC). In some embodiments, PEG is present only in the reagent composition comprising the non-protein component (NPC).

[0240] In some embodiments, the methods of this disclosure (e.g., the isothermal amplification method of this disclosure) include preparing a reagent composition using a composition of this disclosure (e.g., a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less (e.g., the composition has a trehalose to PEG weight percentage (wt%) ratio of about 0.3 to about 0.75)), and contacting the target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the method may further include using one or more additional compositions to prepare the reagent composition. In some embodiments, one or more additional compositions may include protein components (e.g., DNA polymerase, recombinase, recombinase-loading protein, single-stranded binding protein, creatine kinase, nuclease (e.g., exonuclease) and / or reverse transcriptase) and / or oligonucleotide components (e.g., one or more primers and / or one or more probes that are specific to one or two or more target nucleic acids). In some embodiments, one or more additional compositions may further comprise trehalose. For example, but not as a limitation, the isothermal amplification method of this disclosure includes: (a) preparing a reagent composition using: (i) a composition of this disclosure, such as a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a trehalose to PEG weight percentage (wt%) ratio of about 0.79 or less (e.g., the composition has a trehalose to PEG weight percentage (wt%) ratio of about 0.3 to about 0.75); (ii) a second composition comprising one or more protein compositions; and (iii) a third composition comprising one or more oligonucleotide components; and (b) contacting a target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the method may further include contacting the reagent composition and the target nucleic acid with an activator. In some embodiments, the second composition further comprises trehalose. In some embodiments, the third composition further comprises trehalose.

[0241] In some embodiments, the methods of this disclosure (e.g., the isothermal amplification method of this disclosure) include: providing a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less (e.g., the composition has a weight percentage (wt%) ratio of about 0.3 to about 0.75 for trehalose); and (ii) contacting a target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the reagent composition may be further prepared using one or more additional compositions. In some embodiments, one or more additional compositions may include protein components (e.g., DNA polymerase, recombinase, recombinase-loading protein, single-stranded binding protein, creatine kinase, nuclease (e.g., exonuclease) and / or reverse transcriptase) and / or oligonucleotide components (e.g., one or more primers and / or one or more probes that are specific to one or two or more target nucleic acids). For example, but not as a limitation, the isothermal amplification method of this disclosure includes: (a) providing a reagent composition, wherein the reagent composition is prepared using: (i) a composition of this disclosure, such as a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less (e.g., the composition has a weight percentage (wt%) ratio of about 0.3 to about 0.75 for trehalose); (ii) a second composition comprising one or more protein compositions; and (iii) a third composition comprising one or more oligonucleotide components; and (b) contacting a target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the second composition further comprises trehalose. In some embodiments, the third composition further comprises trehalose. In some embodiments, the method may further include contacting the reagent composition and the target nucleic acid with an activator.

[0242] IV. Instructions for Use

[0243] In some embodiments, the methods of this disclosure can be used to detect the presence or absence of a target nucleic acid in a sample. In some embodiments, the methods of this disclosure can be used to quantify the amount of the target nucleic acid in a sample.

[0244] In some embodiments, the methods of this disclosure can be used to amplify and detect target nucleic acids associated with a disease or condition, for example, to determine whether a subject has the disease or condition. In some embodiments, the methods of this disclosure can be used to amplify and detect target nucleic acids as biomarkers of a disease or condition. For example, but not as a limitation, this disclosure can be used to determine whether a subject has a disease or condition (e.g., cancer) or is at risk of developing a disease or condition (e.g., cancer). In some embodiments, the methods of this disclosure can be used to determine whether a subject has a certain hereditary condition, for example, by determining whether the subject has a gene mutation associated with that hereditary condition. In some embodiments, the methods of this disclosure can be used to amplify and detect target nucleic acids derived from pathogens, for example, to determine whether a subject is infected with that pathogen.

[0245] In some embodiments, this disclosure can be used to determine a subject's blood type (blood group).

[0246] In some embodiments, the methods of this disclosure can be used to screen blood samples. In some embodiments, the methods of this disclosure can be used to screen samples originating from a single individual or multiple individuals. In some embodiments, blood sample screening can be used in relation to material donations, such as plasma, platelets, red blood cells, and whole blood. In some embodiments, the blood sample being screened is a whole blood sample. In some embodiments, the blood sample being screened is a lysed whole blood sample. In some embodiments, the blood sample being screened is a serum sample. In some embodiments, the blood sample being screened is a plasma sample.

[0247] In some embodiments, the target nucleic acid is bacterial, eukaryotic, or viral nucleic acid. In some embodiments, the target nucleic acid is bacterial nucleic acid. In some embodiments, the target nucleic acid is eukaryotic nucleic acid. In some embodiments, the target nucleic acid is viral nucleic acid.

[0248] In some implementations, the target nucleic acid is derived from SARS-CoV-2 (COVID-19), coronavirus, HIV (e.g., HIV-1 and / or HIV-2), hepatitis B (HBV), hepatitis C (HCV), hepatitis A (HAV), hepatitis E (HEV), cytomegalovirus (CMV), parvovirus B19, Creutzfeldt-Jakob disease (vCJD), chlamydia, gonorrhea, West Nile virus (WNV), Zika virus (ZIKV), dengue fever, chikungunya, influenza (e.g., influenza A virus, influenza B virus, or influenza C virus), Babesia, malaria, rubella, varicella-zoster virus, herpes simplex, poliomyelitis, syphilis, smallpox, cowpox, rabies, human T-lymphovirus (HTLV), Usutu virus, or Epstein-Barr virus. In some embodiments, the target nucleic acid is selected from the group consisting of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV), hepatitis E virus (HEV), and combinations thereof. In some embodiments, the target nucleic acid is a nucleic acid derived from HCV. In some embodiments, the target nucleic acid is a nucleic acid derived from HIV, such as HIV-1 and / or HIV-2. In some embodiments, the target nucleic acid is a nucleic acid derived from HBV. In some embodiments, the target nucleic acid is a nucleic acid derived from dengue fever. In some embodiments, the target nucleic acid is a nucleic acid derived from chikungunya.

[0249] In some implementations, the target nucleic acid is a nucleic acid derived from one or more new or emerging pathogens, viruses, and / or agents.

[0250] In some implementations, if the virus is an RNA-based virus, such as HIV-1 and HCV, the nucleic acid to be detected will be RNA. If the virus is a DNA-based virus, such as HBV, the nucleic acid to be detected will be DNA. In some implementations, the method can detect ribosomal RNA of the parasite *Babesia*.

[0251] In some embodiments, the methods of this disclosure can be used to detect (e.g., present or absent) or quantify two or more target nucleic acids in a sample via multiplex analysis, such as multiple target nucleic acids. In some embodiments, higher-order multiplex amplification may be employed in the methods of this disclosure, enabling the detection of the presence of 3, 4, 5, 6, 7, 8, 9, 10 or more target nucleic acids in a single sample. In some embodiments, each target nucleic acid originates from a different pathogen, pathogenic factor, gene, or mRNA. As used herein, “multiplex analysis” refers to the simultaneous screening of two or more target nucleic acids, for example, in cases where each target nucleic acid originates from a pathogen or pathogenic factor. As used herein, “multiplex analysis” encompasses the simultaneous screening of two or more target nucleic acids in a single reaction vessel (e.g., an amplification vessel), as well as the screening of two or more target nucleic acids in separate reaction vessels (e.g., in cases where sample eluent has been aliquoted into two or more separate reaction vessels (e.g., amplification vessels)).

[0252] In some embodiments, the methods of this disclosure can be used for multiplex analysis of HIV-1, HIV-2, HCV, and / or HBV. In some embodiments, the methods of this disclosure can be used for multiplex analysis of HIV-1 and HIV-2. In some embodiments, the methods of this disclosure can be used for multiplex analysis of HIV-1, HIV-2, and HCV. In some embodiments, the methods of this disclosure can be used for multiplex analysis of HIV-1, HIV-2, and HBV. In some embodiments, the methods of this disclosure can be used for multiplex analysis of HCV and HBV. In some embodiments, the methods of this disclosure can be used for multiplex analysis of HIV-1, HIV-2, HCV, and HBV.

[0253] In some embodiments, the methods of this disclosure can be used for multiplex analysis of Zika virus, WNV, chikungunya virus, and / or dengue virus. In some embodiments, the methods of this disclosure can be used for multiplex analysis of Zika virus and WNV. In some embodiments, the methods of this disclosure can be used for multiplex analysis of chikungunya virus and dengue virus. In some embodiments, the methods of this disclosure can be used for multiplex analysis of chikungunya virus and WNV. In some embodiments, the methods of this disclosure can be used for multiplex analysis of Zika virus and dengue virus.

[0254] In some implementations, the methods disclosed herein can be used for multiplex analysis of Babesia and malaria.

[0255] In some implementations, the methods disclosed herein can be used for multiplex analysis of parvovirus B19 and HAV.

[0256] In some embodiments, the methods of this disclosure, such as the isothermal amplification method of this disclosure, include: (i) providing a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.75 or less (e.g., the composition has a trehalose to PEG weight percentage (wt%) ratio of about 0.3 to about 0.75 or about 0.3 to about 0.65); and (ii) contacting a sample containing a target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the target nucleic acid is derived from one or more of HIV-1, HIV-2, HCV, and HBV. In some embodiments, the isothermal amplification method is a multiplex reaction, and the target nucleic acid is derived from HIV-1, HIV-2, HCV, and HBV. In some embodiments, the composition may comprise about 10% to about 20% w / v trehalose (e.g., about 15% w / v trehalose) and about 15% to about 25% w / v PEG. In some embodiments, the composition may further comprise dNTPs, buffers, salts, and / or ATP or ATP analogs.

[0257] In some embodiments, the methods of this disclosure, such as the isothermal amplification method of this disclosure, include: (i) providing a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.75 or less (e.g., the composition has a trehalose to PEG weight percentage (wt%) ratio of about 0.3 to about 0.60 or about 0.3 to about 0.50); ​​and (ii) contacting a sample containing a target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the target nucleic acid is derived from Zika virus. In some embodiments, the composition may comprise about 5% to about 10% w / v trehalose and about 15% to about 25% w / v PEG. In some embodiments, the composition may further comprise dNTPs, buffers, salts, and / or ATP or ATP analogs.

[0258] In some embodiments, the methods of this disclosure, such as the isothermal amplification method of this disclosure, include: (i) providing a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.75 or less (e.g., the composition has a trehalose to PEG weight percentage (wt%) ratio of about 0.3 to about 0.50 or about 0.3 to about 0.40); and (ii) contacting a sample containing a target nucleic acid with the reagent composition to amplify the target nucleic acid. In some embodiments, the target nucleic acid is derived from dengue virus. In some embodiments, the target nucleic acid is derived from chikungunya virus. In some embodiments, the target nucleic acid is derived from both chikungunya virus and dengue virus. In some embodiments, the composition may comprise about 5% to about 10% w / v trehalose and about 15% to about 25% w / v PEG. In some embodiments, the composition may further comprise dNTPs, buffers, salts, and / or ATP or ATP analogs.

[0259] V. Reagent Kit

[0260] This disclosure also provides kits for performing the methods of this disclosure. In some embodiments, this disclosure provides kits comprising materials for performing the methods of this disclosure. In some embodiments, the kits of this disclosure include containers containing reagents for performing the methods of this disclosure, such as reagent compositions comprising trehalose and PEG, for example, in ratios and amounts as described herein.

[0261] In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.3 to about 0.79. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.45 to about 0.75, for example, about 0.5 to about 0.7, about 0.45 to about 0.65, or about 0.55 to about 0.55. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.3 to about 0.45, for example, about 0.3 to about 0.4, about 0.3 to about 0.35, or about 0.4 to about 0.45. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.79 or less, or about 0.75 or less, for example, 0.65 or less, 0.55 or less, 0.45 or less, or 0.35 or less. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.65 or less. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.55 or less. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.45 or less. In some embodiments, the kit comprises a reagent composition containing trehalose and PEG in a wt% ratio of about 0.35 or less.

[0262] In some embodiments, the kit of this disclosure provides a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 2% to about 8.5% w / v, for example, about 5% to about 8% w / v, about 6% to about 8% w / v, about 7% to about 8% w / v, or about 8% to about 8.5% w / v. In some embodiments, the kit comprises a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 2% to about 8.5% w / v, for example, about 5% to about 8% w / v, about 6% to about 8% w / v, about 7% to about 8% w / v, or about 8% to about 8.5% w / v. In some embodiments, the kit comprises a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 5% to about 8.5% w / v. In some embodiments, the kit contains a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 6.3%. In some embodiments, the kit contains a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 6.5% w / v. In some embodiments, the kit contains a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 7%. In some embodiments, the kit contains a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 7.4%. In some embodiments, the kit contains a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 8.4%. For example, but not as a limitation, when about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the reagent composition in the kit of this disclosure is added to the isothermal amplification method, it produces a PEG reaction concentration of about 5% to about 8.5% w / v. In some embodiments, when about 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, or 75% or less of the reagent composition in the kit of this disclosure is added to the isothermal amplification method, it produces a PEG reaction concentration of about 5% to about 8.5% w / v. For example, but not as a limitation, the reagent composition may contain about 10% to about 30% w / v of PEG to produce a PEG reaction concentration of about 5% to about 8.5% w / v.In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 10% to about 30% w / v of PEG. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 10% to about 25% w / v of PEG, for example, about 30% w / v of PEG. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 10% to about 20% w / v of PEG.

[0263] In some embodiments, the kit comprises a reagent composition containing about 2% to about 8.5% w / v PEG, such as about 5% to about 8% w / v PEG, about 6% to about 8% w / v PEG, about 7% to about 8% w / v PEG, about 8% to about 8.5% w / v PEG, about 6% to about 7% w / v PEG, or about 5% to about 7% w / v PEG. In some embodiments, the kit comprises a reagent composition containing about 5.5% w / v PEG. In some embodiments, the kit comprises a reagent composition containing about 6.3% w / v PEG. In some embodiments, the kit comprises a reagent composition containing about 6.5% w / v PEG. In some embodiments, the kit comprises a reagent composition containing about 7.4% w / v PEG. In some embodiments, the kit comprises a reagent composition containing about 7.7% w / v PEG. In some embodiments, the kit comprises a reagent composition containing about 8.4% w / v PEG. In some embodiments, PEG has a molecular weight between about 30,000 Daltons and about 40,000 Daltons, for example about 35,000 Daltons.

[0264] In some embodiments, the kit contains a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a trehalose concentration of about 2% to about 6% w / v. For example, but not as a limitation, when about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the reagent composition in the kit of this disclosure is added to an isothermal amplification method, the trehalose concentration provided by the composition will be reduced to about 2% to about 6% w / v, for example, about 2% to about 4% trehalose concentration. In some embodiments, when about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, or about 75% or less of the reagent composition from the kit of this disclosure are added to the isothermal amplification method, the trehalose concentration provided by the composition will be reduced to about 2% to about 6% w / v trehalose, for example, about 2% to about 4% trehalose concentration. For example, but not as a limitation, the reagent composition may contain about 10% to about 30% w / v (e.g., about 15% to about 30% w / v) trehalose, for example, when added to the isothermal amplification method, the trehalose concentration is reduced to about 2% to about 6% w / v trehalose concentration. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 5% to about 15% w / v trehalose (e.g., about 5% to about 7% trehalose). In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 5.9% w / v trehalose. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 5% to about 20% w / v trehalose (e.g., about 10% to about 20% trehalose). In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 15% w / v trehalose. In some embodiments, this disclosure provides a reagent composition containing about 7% to about 10% w / v trehalose, for example, about 7% to about 10% trehalose. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 8.15% w / v trehalose. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 5% to about 6% w / v trehalose, for example, about 5.5% to about 6% trehalose. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 5.7% w / v trehalose.

[0265] In some embodiments, the kit comprises a reagent composition containing about 3% to about 5% w / v trehalose, for example about 3.5% to about 4.5% w / v trehalose. In some embodiments, the kit comprises a reagent composition containing about 4% w / v trehalose.

[0266] In some embodiments, the kit comprises a reagent composition containing about 2% to about 3% w / v trehalose, for example, about 2.5% w / v to about 3% w / v trehalose. In some embodiments, the kit comprises a reagent composition containing about 2.75% w / v trehalose.

[0267] In some embodiments, the kit comprises a reagent composition containing about 1% to about 2% w / v trehalose, for example, about 1.5% w / v to about 2% w / v trehalose. In some embodiments, the kit comprises a reagent composition containing about 1.95% w / v trehalose.

[0268] In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 10% to about 20% w / v PEG and about 5% to about 20% w / v trehalose. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 15% to about 20% w / v PEG and about 8% to about 20% w / v trehalose.

[0269] In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 5% to about 10% w / v PEG and about 6% to about 12% w / v trehalose. In some embodiments, this disclosure provides a kit comprising a reagent composition containing about 6% to about 10% w / v PEG and about 6% to about 12% w / v trehalose.

[0270] In some embodiments, the reagent composition may further comprise one or more of the following components (in any combination), and / or the kit of this disclosure may comprise one or more additional reagent compositions comprising one or more of the following components (in any combination): at least one recombinase, at least one single-stranded DNA-binding protein, at least one DNA polymerase, dNTPs, buffers, reducing agents, ATP or ATP analogs, at least one recombinase-loading protein, a first primer and optionally a second primer, a probe, reverse transcriptase, creatine kinase, nucleases, and a template nucleic acid molecule, such as single-stranded (e.g., RNA) or double-stranded nucleic acid. In some embodiments, the compositions of this disclosure further comprise dNTPs, buffers, salts, ATP or ATP analogs, and / or congestants.

[0271] In some embodiments, the compositions of this disclosure further comprise dNTPs, buffers, salts, ATP, and / or ATP analogs. In some embodiments, the kits of this disclosure may comprise reagent compositions that further comprise non-protein components, including but not limited to dNTPs, buffers, ATP, salts, and creatine phosphate. In some embodiments, the kits of this disclosure may comprise reagent compositions that further comprise dNTPs, buffers (e.g., Tris buffer), ATP, salts (e.g., potassium acetate), and creatine phosphate. In some embodiments, the compositions of this disclosure further comprise recombinases, single-stranded binding proteins, polymerases, dNTPs, ATP, primers, and template nucleic acids. In some embodiments, the compositions of this disclosure do not contain reverse transcriptase. Alternatively, such reagents may be present in separate containers from trehalose and PEG. For example, but not as a limitation, the kits of this disclosure may include a second composition (e.g., a second reagent composition in a second container) containing an enzyme for isothermal reactions, said enzyme including, but not limited to: at least one recombinase (e.g., UvsX), at least one single-stranded DNA-binding protein (e.g., gp32), at least one DNA polymerase (e.g., Pol I), at least one recombinase-loading protein (e.g., UvsY), an exonuclease (e.g., exonuclease III), creatine kinase, and / or at least one reverse transcriptase (e.g., EIAV-RT). In some embodiments, the second reagent composition does not contain reverse transcriptase. In some embodiments, the kits of this disclosure may include a third composition (e.g., a third reagent composition in a third container) containing one or more primers (e.g., a first primer and a second primer) and one or more probes. In some embodiments, the kits of this disclosure may include a fourth composition (e.g., a fourth reagent composition in a fourth container) containing an activator, such as MgOAc. In some embodiments, trehalose may also be present in the enzyme-containing reagent composition. In some embodiments, trehalose may also be present in a reagent composition comprising one or more primers (e.g., a first primer and a second primer) and one or more probes.

[0272] In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.79 or less. In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.70 or less. In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.6 or less. In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.45 or less. In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.35 or less.

[0273] In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.3 to about 0.79, for example, about 0.3 to about 0.45, about 0.45 to about 0.65, about 0.5 to about 0.55, about 0.65 to about 0.70, or about 0.7 to about 0.79. In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) primarily composed of dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.3 to about 0.79. In some embodiments, the kit of this disclosure may include a reagent composition (e.g., in a first container) comprising: dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition in a wt% ratio of about 0.3 to about 0.79.

[0274] In some embodiments, the reagents and / or compositions of this disclosure may be lyophilized.

[0275] Examples of suitable containers include, but are not limited to, bottles, test tubes, vials, and microplates. Containers can be made of a variety of materials, such as glass or plastic.

[0276] In some embodiments, the kit may also include a packaging insert providing instructions for using the components provided in the kit. For example, the kit of this disclosure may include a packaging insert providing instructions for performing the methods of this disclosure.

[0277] In some implementations, the kit may include additional materials required from a commercial and user perspective, including additional buffers and diluents.

[0278] VI. System

[0279] This disclosure further provides a system for performing the methods of this disclosure. In some embodiments, the system is an automated system. In some embodiments, an automated system that can be used to perform the methods of this disclosure may include a sample preparation region (such as a region for isolating nucleic acids from a sample), a nucleic acid amplification region, and a nucleic acid detection region. In some embodiments, the nucleic acid amplification region and the nucleic acid detection region are the same. An automated system for performing the methods of this disclosure is provided in International Patent Application PCT / US2022 / 027067 (which is incorporated herein by reference in its entirety). For example, but not as a limitation, the system of Figures 68A-68D of PCT / US2022 / 027067 can be used to perform the methods of this disclosure.

[0280] In some embodiments, the systems of this disclosure include containers and / or reservoirs containing one or more compositions disclosed herein for performing isothermal amplification reactions (e.g., RPA methods). In some embodiments, the systems of this disclosure may include at least one container or reservoir containing a composition (e.g., a reagent composition) comprising trehalose and PEG in the ratio and amount described herein.

[0281] In some embodiments, the system of this disclosure may include at least one container or reservoir containing a composition (e.g., a reagent composition) comprising trehalose and PEG in a wt% ratio of about 0.25 to about 0.79 or about 0.3 to about 0.79, such as about 0.3 to about 0.45, about 0.45 to about 0.65, about 0.5 to about 0.55, about 0.65 to about 0.70, or about 0.7 to about 0.79. In some embodiments, the system may include at least one container or reservoir containing a composition (e.g., a reagent composition) comprising trehalose and PEG in a wt% ratio of about 0.45 to about 0.75, such as about 0.5 to about 0.7, about 0.45 to about 0.65, about 0.55 to about 0.55, or about 0.7 to about 0.75. In some embodiments, the system disclosed herein may include at least one container or reservoir comprising a composition (e.g., a reagent composition) containing trehalose and PEG in a wt% ratio of about 0.79 or less, or about 0.75 or less, for example, 0.65 or less, 0.55 or less, 0.45 or less, or 0.35 or less.

[0282] In some embodiments, the system of this disclosure may include at least one container or reservoir comprising a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 2% to about 8.5% w / v, such as about 5% to about 8% w / v, about 6% to about 8% w / v, or about 8% to about 8.5% w / v. In some embodiments, the system comprises a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 2% to about 8.5% w / v, such as about 5% to about 8% w / v, about 6% to about 8% w / v, or about 8% to about 8.5% w / v. In some embodiments, the system comprises a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a PEG concentration of about 5% to about 8.5% w / v. In some embodiments, the system comprises a reagent composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 6.5% w / v. In some embodiments, the system comprises a reagent composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 7%. In some embodiments, the system comprises a reagent composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 7.4%. In some embodiments, the system comprises a reagent composition that, when added to an isothermal amplification process (e.g., diluted), produces a PEG concentration of about 8.4%. For example, but not as a limitation, when about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the reagent composition from the system of this disclosure is added to an isothermal amplification process, it produces a PEG reaction concentration of about 5% to about 8.5% w / v. For example, but not as a limitation, the systems disclosed herein may include at least one container or reservoir comprising a reagent composition containing about 10% to about 30% w / v PEG, for example, about 15% to about 25% w / v PEG or about 20% w / v PEG.

[0283] In some embodiments, the system may include at least one container or reservoir comprising a reagent composition comprising about 2% to about 8.5% w / v PEG, for example, about 5% to about 8% w / v PEG, about 6% to about 8% w / v PEG, about 7% to about 8% w / v PEG, about 6% to about 7% w / v PEG, about 5% to about 7% w / v PEG, or about 8% to about 8.5% w / v PEG. In some embodiments, the system includes a reagent composition comprising about 5.5% w / v PEG. In some embodiments, the system may include at least one container or reservoir comprising a reagent composition comprising about 6.3% w / v PEG. In some embodiments, the system includes a reagent composition comprising about 6.5% w / v PEG. In some embodiments, the system may include at least one container or reservoir comprising a reagent composition comprising about 7.7% w / v PEG. In some embodiments, the system may include at least one container or reservoir comprising a reagent composition comprising about 7.4% w / v PEG. In some embodiments, the system may include at least one container or reservoir comprising a reagent composition containing about 8.5% w / v PEG. In some embodiments, the PEG has a molecular weight between about 30,000 Daltons and about 40,000 Daltons, for example, about 35,000 Daltons.

[0284] In some embodiments, the system may include at least one container or reservoir containing a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a trehalose concentration of about 1.5% to about 6% w / v. In some embodiments, the system may include at least one container or reservoir containing a reagent composition that, when added to an isothermal amplification method (e.g., diluted), produces a trehalose concentration of about 2% to about 6% w / v. For example, but not as a limitation, when about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the reagent composition from the system of this disclosure is added to an isothermal amplification method, it produces a trehalose concentration of about 2% to about 6% w / v, for example, a trehalose reaction concentration of about 2% to about 4%. For example, but not as a limitation, the systems disclosed herein may include at least one container or reservoir comprising a reagent composition containing about 5% to about 20% w / v trehalose, such as about 5% to about 15% w / v trehalose or about 10% to about 15% w / v trehalose.

[0285] In some embodiments, the system may include at least one container or reservoir containing a reagent composition comprising about 3% to about 5% w / v trehalose, for example about 3.5% to about 4.5% w / v trehalose. In some embodiments, the system includes a reagent composition comprising about 4% w / v trehalose.

[0286] In some embodiments, the system may include at least one container or reservoir comprising a reagent composition containing about 1% to about 2% w / v trehalose, for example, about 1.5% to about 2% w / v trehalose. In some embodiments, the system includes a reagent composition containing about 1.95% w / v trehalose.

[0287] In some embodiments, the system may include at least one container or reservoir comprising a reagent composition containing about 2% to about 3% w / v trehalose, for example, about 2.5% to about 3% w / v trehalose. In some embodiments, the system includes a reagent composition containing about 2.75% w / v trehalose.

[0288] In some embodiments, the system disclosed herein may include at least one container or reservoir containing a composition (e.g., a reagent composition) comprising about 10% to about 25% w / v PEG (e.g., about 10% to about 20% w / v PEG) and about 5% to about 20% w / v trehalose (e.g., about 5% w / v to about 15% w / v trehalose).

[0289] In some embodiments, the system disclosed herein may include at least one container or reservoir containing a composition (e.g., a reagent composition) comprising about 2% to about 8.5% w / v PEG (e.g., about 5% to about 8.5% w / v PEG) and about 2% to about 5% w / v trehalose (e.g., about 1.95% w / v trehalose or about 4% w / v trehalose).

[0290] In some embodiments, the reagent composition in the system disclosed herein may further comprise one or more of the following components (in any combination), and / or the system disclosed herein may comprise one or more additional reagent compositions comprising one or more of the following components (in any combination): at least one recombinase, at least one single-stranded DNA-binding protein, at least one DNA polymerase, dNTPs, buffers, reducing agents, ATP or ATP analogs, at least one recombinase-loading protein, a first primer and optionally a second primer, a probe, reverse transcriptase, creatine kinase, nucleases, and a template nucleic acid molecule, such as a single-stranded (e.g., RNA) or double-stranded nucleic acid. In some embodiments, the composition in the system disclosed herein further comprises dNTPs, buffers, salts, ATP or ATP analogs, and / or congestants.

[0291] In some embodiments, the system of this disclosure may include at least one container or reservoir comprising a composition further comprising dNTPs, buffers, salts, ATP, and / or ATP analogs. In some embodiments, the system of this disclosure may include a reagent composition further comprising non-protein components, including but not limited to dNTPs, buffers, ATP, salts, and creatine phosphate. In some embodiments, the system of this disclosure may include a reagent composition further comprising dNTPs, buffers (e.g., Tris buffer), ATP, salts (e.g., potassium acetate), and creatine phosphate. In some embodiments, the composition of this disclosure further comprises a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP, primers, and template nucleic acid. In some embodiments, the composition of this disclosure does not contain reverse transcriptase. Alternatively, such reagents may be present in a different container than trehalose and PEG. For example, but not as a limitation, the system of this disclosure may include a second composition (e.g., a second reagent composition located in a second container or reservoir of the system), the second composition comprising an enzyme for isothermal reaction, said enzyme including but not limited to: at least one recombinase (e.g., UvsX), at least one single-stranded DNA-binding protein (e.g., gp32), at least one DNA polymerase (e.g., Pol I), at least one recombinase-loading protein (e.g., UvsY), exonuclease (e.g., exonuclease III), creatine kinase, and / or at least one reverse transcriptase (e.g., EIAV-RT). In some embodiments, the second reagent composition does not contain reverse transcriptase. In some embodiments, the system of this disclosure may include a third composition (e.g., a third reagent composition located in a third container or reservoir of the system), said third composition comprising one or more primers (e.g., a first primer and a second primer) and one or more probes. In some embodiments, the system of this disclosure may include a fourth composition (e.g., a fourth reagent composition located in a fourth container or reservoir of the system), said fourth composition comprising an activator, such as MgOAc. In some embodiments, trehalose may also be present in the enzyme-containing reagent composition. In some embodiments, trehalose may also be present in a reagent composition comprising one or more primers (e.g., a first primer and a second primer) and one or more probes.

[0292] In some embodiments, the system disclosed herein may include at least one container or reservoir comprising a reagent composition (e.g., in a first container or reservoir) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.79 or less.

[0293] In some embodiments, the system of this disclosure may include at least one container or reservoir comprising a reagent composition (e.g., in a first container or reservoir) comprising dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition in a wt% ratio of about 0.3 to about 0.79, for example, about 0.3 to about 0.75. In some embodiments, the system of this disclosure may include at least one container or reservoir comprising a reagent composition (e.g., in a first container or reservoir) primarily composed of dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition in a wt% ratio of about 0.3 to about 0.79, for example, about 0.3 to about 0.75. In some embodiments, the system of this disclosure may include at least one container or reservoir comprising a reagent composition (e.g., in a first container or reservoir) comprising: dNTPs, a buffer (e.g., Tris buffer), ATP, a salt (e.g., potassium acetate), creatine phosphate, trehalose, and PEG, wherein trehalose and PEG are present in the composition at a wt% ratio of about 0.3 to about 0.79, for example, about 0.3 to about 0.75. In some embodiments, PEG may be present in the composition at a concentration of about 10% to about 30% w / v, and trehalose may be present in the composition at a concentration of about 5% to about 20% w / v.

[0294] In some embodiments, the reagents and / or compositions of this disclosure present in the system may be lyophilized.

[0295] Examples of suitable containers or reservoirs include, but are not limited to, bottles, test tubes, vials, and microplates. Containers or reservoirs can be made of various materials, such as glass or plastic.

[0296] In some implementations, the system may include other materials required from a commercial and user perspective, including additional buffers and diluents.

[0297] VII. Exemplary Implementation

[0298] A. This disclosure provides an isothermal amplification method for amplifying target nucleic acids, wherein the isothermal amplification method includes:

[0299] (i) A reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less; and

[0300] (ii) Contact the target nucleic acid with the reagent composition to amplify the target nucleic acid.

[0301] B. This disclosure provides an isothermal amplification method for amplifying target nucleic acids, wherein the isothermal amplification method includes:

[0302] (i) Providing a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), and wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less; and

[0303] (ii) Contact the target nucleic acid with the reagent composition to amplify the target nucleic acid.

[0304] B1. The isothermal amplification method according to A or B, wherein the wt% ratio of trehalose to PEG is from about 0.25 to about 0.79.

[0305] B2. The isothermal amplification method according to any one of A-B1, wherein the wt% ratio of trehalose to PEG is about 0.25 to about 0.75.

[0306] B3. The isothermal amplification method according to any one of A-B2, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.70.

[0307] B4. The isothermal amplification method according to any one of A-B3, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.65.

[0308] B5. The isothermal amplification method according to any one of A-B4, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.55.

[0309] B5-1. The isothermal amplification method according to any one of A-B5, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.50.

[0310] B6. The isothermal amplification method according to any one of A-B5-1, wherein PEG is present in the composition in an amount of about 10% to about 30% by weight-volume ratio (w / v).

[0311] B7. The isothermal amplification method according to any one of A-B6, wherein PEG is present in the composition in an amount of about 15% to about 20% by weight-volume ratio (w / v).

[0312] B8. The isothermal amplification method according to any one of A-B7, wherein the reagent composition contains PEG in an amount of about 2% to about 8% by weight / volume ratio (w / v).

[0313] B9. The isothermal amplification method according to any one of A-B8, wherein trehalose is present in the composition in an amount of about 5% to about 20% w / v.

[0314] B10. The isothermal amplification method according to any one of A-B9, wherein trehalose is present in the composition in an amount of about 5% to about 15% w / v.

[0315] B11. The isothermal amplification method according to any one of A-B10, wherein trehalose is present in the composition in an amount of about 5% to about 12% w / v.

[0316] B12. The isothermal amplification method according to any one of A-B11, wherein the reagent composition contains trehalose in an amount of about 3% to about 12% w / v.

[0317] B12-1. The isothermal amplification method according to any one of A-B12, wherein the reagent composition contains trehalose in an amount of about 6% to about 12% w / v.

[0318] B13. The isothermal amplification method according to any one of A-B12, wherein the reagent composition contains trehalose in an amount of about 3% to about 10% w / v.

[0319] B13-1. The isothermal amplification method according to any one of A-B13, wherein the reagent composition contains trehalose in an amount of about 5% to about 10% w / v.

[0320] B14. The isothermal amplification method according to any one of A-B13-1, wherein the composition further comprises ATP, dNTP, creatine phosphate, and one or more of one or more salts.

[0321] B15. The isothermal amplification method according to any one of A-B14, wherein the composition further comprises ATP, dNTPs, creatine phosphate, and one or more salts.

[0322] B16. The isothermal amplification method according to any one of A-B15, wherein the reagent composition further comprises one or more of the following: DNA polymerase, recombinase-loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe and reverse transcriptase.

[0323] C. This disclosure provides an isothermal amplification method for amplifying a target nucleic acid, the method comprising contacting the target nucleic acid with a reagent composition comprising trehalose and polyethylene glycol (PEG) to amplify the target nucleic acid, wherein the weight percentage (wt%) ratio of trehalose and PEG in the reagent composition is about 0.79 or less.

[0324] D. This disclosure provides an isothermal amplification method for amplifying target nucleic acids, wherein the isothermal amplification method is performed in the presence of trehalose and polyethylene glycol (PEG) at a weight percentage (wt%) ratio of trehalose to PEG of about 0.79 or less.

[0325] D1. The isothermal amplification method according to C or D, wherein the wt% ratio of trehalose to PEG is about 0.3 to about 0.79.

[0326] D2. The isothermal amplification method according to any one of C-D1, wherein the wt% ratio of trehalose to PEG is about 0.45 to about 0.75.

[0327] D3. The isothermal amplification method according to any one of C-D2, wherein the wt% ratio of trehalose to PEG is about 0.50 to about 0.70.

[0328] D4. The isothermal amplification method according to any one of C-D3, wherein the wt% ratio of trehalose to PEG is about 0.50 to about 0.60.

[0329] D5. The isothermal amplification method according to any one of C-D4, wherein PEG is present in the reagent composition in an amount of about 2% to about 30% by weight-volume ratio (w / v).

[0330] D6. The isothermal amplification method according to any one of C-D5, wherein the PEG is present in the isothermal amplification method and / or reagent composition in an amount of about 2% to about 20% by weight-volume ratio (w / v).

[0331] D7. The isothermal amplification method according to any one of C-D6, wherein PEG is present in the isothermal amplification method and / or reagent composition in an amount of about 2% to about 8% by weight (w / v).

[0332] D8. The isothermal amplification method according to any one of C-D7, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 20% w / v.

[0333] D9. The isothermal amplification method according to any one of C-D8, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 15% w / v.

[0334] D10. The isothermal amplification method according to any one of C-D9, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 12% w / v.

[0335] D11. The isothermal amplification method according to any one of C-D10, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 10% w / v.

[0336] D12. The isothermal amplification method according to any one of C-D11, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 6% w / v.

[0337] D12-1. This disclosure provides an isothermal amplification method for amplifying a target nucleic acid, the method comprising contacting the target nucleic acid with a reagent composition comprising about 5% to about 15% w / v trehalose and about 2% to about 8% by weight polyethylene glycol (PEG) to amplify the target nucleic acid.

[0338] D13. The isothermal amplification method according to any one of A-D12-1, wherein the PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons.

[0339] D14. The isothermal amplification method according to any one of A-D13, wherein the isothermal amplification method is selected from the group consisting of: rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR).

[0340] D15. The isothermal amplification method according to D14, wherein the isothermal amplification method is RPA.

[0341] D16. The isothermal amplification method according to D14, wherein the isothermal amplification method is NEAR.

[0342] D17. The isothermal amplification method according to any one of A-D16, wherein the target nucleic acid is bacterial, eukaryotic, or viral nucleic acid.

[0343] D18. The isothermal amplification method according to any one of A-D17, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, parvovirus B19, HAV, chlamydia, gonorrhea, WNV, Zika virus, dengue virus, chikungunya virus, influenza, babesi, malaria, Usutu virus, or HEV.

[0344] D18-1. An isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from HIV-1.

[0345] D18-2. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from HIV-2.

[0346] D18-3. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from HBV.

[0347] D18-4. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from HCV.

[0348] D18-5. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from CMV.

[0349] D18-6. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from parvovirus B19.

[0350] D18-7. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from HAV.

[0351] D18-8. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from Chlamydia.

[0352] D18-9. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from gonorrhea.

[0353] D18-10. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from WNV.

[0354] D18-11. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from Zika virus.

[0355] D18-12. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from dengue virus.

[0356] D18-13. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from Chikungunya virus.

[0357] D18-14. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from influenza.

[0358] D18-15. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from the genus Babesia.

[0359] D18-16. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from malaria.

[0360] D18-17. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from Usutu virus.

[0361] D18-18. The isothermal amplification method according to any one of A-D18, wherein the target nucleic acid is derived from HEV.

[0362] D19. The isothermal amplification method according to any one of A-D18-18, wherein the isothermal amplification method, for example, amplifies at least two or at least three target nucleic acids in a multiplex reaction.

[0363] D19-1. The isothermal amplification method according to D19, wherein the target nucleic acid is derived from chikungunya virus and dengue virus.

[0364] D19-2. The isothermal amplification method according to D19, wherein the target nucleic acid is derived from HIV-1, HIV-2, HBV, and HCV.

[0365] D20. The isothermal amplification method according to any one of A-D19-2, wherein the target nucleic acid is present in the sample.

[0366] D21. The isothermal amplification method according to D20, wherein the sample is a tissue sample, and the target nucleic acid is isolated from the tissue sample prior to amplification.

[0367] D22. The isothermal amplification method according to D20, wherein the sample is a biological fluid.

[0368] D23. The isothermal amplification method according to D22, wherein the biological fluid is blood.

[0369] D24. The isothermal amplification method according to D22 or D23, wherein the target nucleic acid is isolated from biological fluids prior to amplification.

[0370] D25. The isothermal amplification method according to any one of C-D24 further includes contacting the target nucleic acid with one or more of the following: DNA polymerase, recombinase-loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe, reverse transcriptase and activator.

[0371] D26. The isothermal amplification method according to any one of C-D24, wherein the reagent composition further comprises one or more of the following: DNA polymerase, recombinase loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe and reverse transcriptase.

[0372] D27. The isothermal amplification method according to any one of A-D26 further includes detection of the target nucleic acid (e.g., if present).

[0373] D28. The isothermal amplification method according to D27, wherein the detection of the target nucleic acid (e.g., if present) includes an optical detection method.

[0374] E. This disclosure provides a composition for performing an isothermal amplification method, the composition comprising trehalose and PEG, wherein the trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less.

[0375] E1. The composition according to E, wherein the wt% ratio of trehalose to PEG is about 0.75 or less.

[0376] E2. The composition according to E or E1, wherein the wt% ratio of trehalose to PEG is from about 0.25 to about 0.79.

[0377] E3. The composition according to any one of E-E2, wherein the wt% ratio of trehalose to PEG is from about 0.30 to about 0.75.

[0378] E4. The composition according to any one of E-E3, wherein the wt% ratio of trehalose to PEG is from about 0.45 to about 0.75.

[0379] E5. The composition according to any one of E-E4, wherein the wt% ratio of trehalose to PEG is about 0.50 to 0.70.

[0380] E6. The composition according to any one of E-E5, wherein the wt% ratio of trehalose to PEG is about 0.50 to about 0.60.

[0381] E7. The composition according to any one of E-E6, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 2% to about 30% by weight (w / v).

[0382] E8. The composition according to any one of E-E7, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 2% to about 25% by weight (w / v).

[0383] E9. The composition according to any one of E-E8, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 5% to about 25% by weight (w / v).

[0384] E10. The composition according to any one of E-E9, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 10% to about 20% by weight (w / v).

[0385] E11. The composition according to any one of E-E10, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 2% to about 8% by weight (w / v).

[0386] E12. The composition according to any one of E-E11, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 3% to about 20% w / v.

[0387] E13. The composition according to any one of E-E12, wherein trehalose is present in the isothermal amplification method and / or the composition in an amount of about 3% to about 15% w / v.

[0388] E14. The composition according to any one of E-E13, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 3% to about 12% w / v.

[0389] E15. The composition according to any one of E-E14, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 3% to about 5% w / v.

[0390] E16. The composition according to E15, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 4% w / v.

[0391] E17. The composition according to any one of E-E16, wherein the PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons.

[0392] E18. The composition according to any one of E-E17 further comprises one or more of the following: DNA polymerase, recombinase, recombinase loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease, nucleic acid probe and reverse transcriptase.

[0393] E19. The composition according to any one of E-E18, further comprising ATP, dNTPs or a mixture of dNTPs and ddNTPs, and one or more reducing agents.

[0394] E20. The composition according to any one of E-E19 further comprises ATP, dNTPs, creatine phosphate, and one or more salts.

[0395] E21. The composition according to any one of E-E20, wherein the isothermal amplification method is selected from the group consisting of: rolling circle amplification (RCA), sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR).

[0396] E22. The composition according to E21, wherein the isothermal amplification method is RPA.

[0397] E23. The composition according to E21, wherein the isothermal amplification method is NEAR.

[0398] F. This disclosure provides a composition for an isothermal amplification method, the composition comprising trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.79 or less.

[0399] G. This disclosure provides a composition for an isothermal amplification method, the composition comprising trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.75 or less.

[0400] H. This disclosure provides a composition for an isothermal amplification method, the composition comprising trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.25 to about 0.79.

[0401] H1. The composition according to F, G or H, wherein the wt% ratio of trehalose to PEG is from about 0.30 to about 0.60.

[0402] I. This disclosure provides a composition for an isothermal amplification method, which mainly comprises: trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.25 to about 0.79.

[0403] I1. The composition according to I, wherein the wt% ratio of trehalose to PEG is from about 0.30 to about 0.60.

[0404] J. This disclosure provides a system for performing the isothermal amplification method as described in any one of A-D28.

[0405] K. This disclosure provides a system for performing an isothermal amplification method, the system comprising a container or reservoir containing the composition described in any one of E-I1.

[0406] K1. The system according to J or K, wherein the system is automated.

[0407] L. This disclosure provides a kit for performing the isothermal amplification method as described in any one of A-D28.

[0408] M. The present invention provides a kit comprising the composition described in any one of E-I1.

[0409] Example

[0410] The subject matter disclosed herein will be better understood by referring to the following embodiments. These embodiments are provided as examples of the subject matter disclosed herein and are not intended to limit it.

[0411] Example 1: The combination of trehalose and PEG improves RPA amplification.

[0412] This embodiment discloses the addition of trehalose and PEG to the RPA reaction. Figure 1 Exemplary results related to the implementation of RPA in the disclosed subject matter, measured by monitoring the change of fluorescence signal (dRn) over time (cycles) using a real-time fluorescence detection system, are illustrated. The RPA reaction was set up as described herein, using the PEG and trehalose concentrations listed in Table 1, and carried out at 40°C.

[0413] The combination of 4% trehalose and polyethylene glycol (PEG) improved HCV detection. Figure 1For example, 4% trehalose and PEG can be added to the final reaction, which is derived from a composition containing the non-protein component (referred to herein as "NPC") used in the RPA reaction. When HCV was present at a level of 30x of the limit of detection (LOD), a signal was detected between 20 and 30 cycles using 4% trehalose, while no signal was detected using the master mixture without trehalose. Figure 1 Beyond specific theories, trehalose promotes the relaxation of target nucleic acids, which can facilitate the binding of enzymes and / or primers to target amplicones. Furthermore, trehalose lowers the melting temperature (Tm) of target DNA / RNA, increases reaction crowding, and improves enzyme thermostability. This improved amplification has also been observed in other HxV and IC targets (data not shown) and on both the NOVA automation (ADBB) and Bio-Rad systems. HxV is a multiplex assay that detects HIV-1, HIV-2, HBV, and HCV nucleic acids.

[0414] Table 1

[0415] NPC = Non-protein component composition

[0416] Figure 2 The exemplary results further illustrate that the addition of 4% wt trehalose improved amplification. The RPA reaction was set up as described herein, using the PEG and trehalose concentrations listed in Table 2, and performed at 40°C. On the Bio-Rad system, the addition of 4% wt trehalose to the final reaction (from NPC) improved the detection of HCV target nucleic acids at near the limit of detection (i.e., 2xLOD). Figure 2 Improved amplification was also observed for other HxV and IC target nucleic acids. Figure 2 (and data not displayed).

[0417] Table 2

[0418]

[0419] Figure 3A and 3B Exemplary results for various trehalose / PEG ratios are illustrated. The RPA reactions were set up as described herein, using the PEG and trehalose concentrations listed in Tables 3A and 3B, and performed at 40°C. The PEG and trehalose concentrations provided in Tables 3A and 3B represent the concentrations of trehalose and PEG in the final reaction, obtained by adding trehalose and PEG from NPC to the final reaction. The addition of trehalose resulted in a final reaction concentration of 4% wt of trehalose obtained from NPC, which improved the RPA amplification signal of chikungunya and dengue target nucleic acids. Figure 3AIncreasing the PEG concentration from 5.5% wt to 6.5% wt improved the RPA amplification signal of chikungunya and dengue target nucleic acids. Figure 3B ).

[0420] Table 3A

[0421]

[0422] Table 3B

[0423]

[0424] Figure 4 Exemplary results related to different trehalose / PEG ratios are illustrated. The RPA reaction was set up as described herein, using the PEG and trehalose concentrations listed in Table 4, and carried out at 40°C. On the NOVA automated system, a trehalose / PEG ratio of 1.04 improved the sensitivity and signal strength of HCV at 2xLOD. Figure 4 However, other HxV targets were not affected by higher concentrations of trehalose (data not shown).

[0425] Table 4

[0426]

[0427] Figures 5A-5C Exemplary results related to different trehalose / PEG ratios are illustrated. The RPA reaction was set up as described herein, using the PEG and trehalose concentrations listed in Table 5, and performed at 40°C. A trehalose / PEG ratio of 0.52 in NPC significantly improved the detection of multiple HxV targets, including HBV, HCV, and an internal control. Figures 5A-5C Improved amplification was observed across multiple targets, with the most significant improvement observed for HCV, which was detected in 30 cycles using a reaction mixture containing trehalose and PEG at a wt% ratio of 0.52. Figure 5B ).

[0428] Table 5

[0429]

[0430] A trehalose / PEG wt% ratio of 0.52 in NPC was found to significantly improve amplification on the NOVA automated system. Specifically, adding trehalose and PEG from NPC to the final reaction, yielding a final reaction composition of 4% wt trehalose and 7.7% wt PEG (corresponding to a trehalose / PEG wt% ratio of 0.52 in NPC), was identified as providing optimal amplification for all HxV targets. This example demonstrates that specific trehalose to PEG ratios and amounts significantly improve the amplification of target nucleic acids.

[0431] Figures 6A-6B Exemplary results related to different trehalose / PEG ratios are illustrated. The RPA reaction was set up as described herein, using the PEG and trehalose concentrations listed in Table 6, and carried out at 40°C. The concentrations provided in Table 6 represent the final trehalose and PEG concentrations obtained by adding trehalose and PEG from NPC to the final reaction.

[0432] Table 6

[0433]

[0434] The 0.26 trehalose / PEG ratio in NPC improved the detection of the DENV target. Figure 6A However, it increased the number of false positives. Figure 6B A trehalose / PEG wt% ratio of 0.31 was found to significantly improve amplification on the NOVA automated system without producing false positives. Specifically, a final concentration of 2% trehalose and 6.5% PEG from NPC (resulting in a trehalose / PEG wt% ratio of 0.31) was identified as providing enhanced amplification of the DENV target without producing false positives. This example demonstrates that specific trehalose to PEG ratios and amounts significantly improve the amplification of target nucleic acids while maintaining assay specificity.

[0435] * * * * * * * *

[0436] Although the disclosed subject matter and its advantages have been described in detail herein, it should be understood that various changes, substitutions, and alterations may be made to this document without departing from the spirit and scope of this disclosure. Furthermore, the scope of this application is not intended to be limited to the specific embodiments of the methods, machines, manufactures, and material compositions, apparatuses, methods, and steps described in the specification. Therefore, the appended claims are intended to cover such methods, machines, manufactures, material compositions, apparatuses, methods, or steps within their scope.

[0437] This application references various patents, patent applications, publications, product specifications and solutions, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Claims

1. An isothermal amplification method for amplifying target nucleic acids, wherein the isothermal amplification method comprises: (i) A reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less; as well as (ii) Contact the target nucleic acid with the reagent composition to amplify the target nucleic acid.

2. An isothermal amplification method for amplifying target nucleic acids, wherein the isothermal amplification method comprises: (i) Provide a reagent composition, wherein the reagent composition is prepared using a composition comprising trehalose and polyethylene glycol (PEG), and wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less; as well as (ii) Contact the target nucleic acid with the reagent composition to amplify the target nucleic acid.

3. The isothermal amplification method according to claim 1 or 2, wherein the wt% ratio of trehalose to PEG is about 0.25 to about 0.

79.

4. The isothermal amplification method according to any one of claims 1-3, wherein the wt% ratio of trehalose to PEG is about 0.25 to about 0.

75.

5. The isothermal amplification method according to any one of claims 1-4, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.

70.

6. The isothermal amplification method according to any one of claims 1-5, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.

60.

7. The isothermal amplification method according to any one of claims 1-6, wherein the wt% ratio of trehalose to PEG is about 0.30 to about 0.

55.

8. The isothermal amplification method according to any one of claims 1-7, wherein PEG is present in the composition in an amount of about 10% to about 30% by weight-volume ratio (w / v).

9. The isothermal amplification method according to any one of claims 1-8, wherein PEG is present in the composition in an amount of about 15% to about 20% by weight-volume ratio (w / v).

10. The isothermal amplification method according to any one of claims 1-9, wherein the reagent composition comprises about 2% to about 8% by weight (w / v) PEG.

11. The isothermal amplification method according to any one of claims 1-10, wherein trehalose is present in the composition in an amount of about 5% to about 20% w / v.

12. The isothermal amplification method according to any one of claims 1-11, wherein trehalose is present in the composition in an amount of about 5% to about 15% w / v.

13. The isothermal amplification method according to any one of claims 1-12, wherein trehalose is present in the composition in an amount of about 5% to about 12% w / v.

14. The isothermal amplification method according to any one of claims 1-13, wherein the reagent composition comprises trehalose in an amount of about 3% to about 12% w / v.

15. The isothermal amplification method according to any one of claims 1-14, wherein the reagent composition comprises trehalose in an amount of about 3% to about 10% w / v.

16. The isothermal amplification method according to any one of claims 1-15, wherein the composition further comprises ATP, dNTPs, creatine phosphate, and one or more salts.

17. The isothermal amplification method according to any one of claims 1-16, wherein the composition further comprises ATP, dNTPs, creatine phosphate, and one or more salts.

18. The isothermal amplification method according to any one of claims 1-17, wherein the reagent composition further comprises one or more of the following: DNA polymerase, recombinase loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe and reverse transcriptase.

19. An isothermal amplification method for amplifying a target nucleic acid, the method comprising contacting the target nucleic acid with a reagent composition comprising trehalose and polyethylene glycol (PEG) to amplify the target nucleic acid, wherein the trehalose and PEG are present in the reagent composition at a weight percentage (wt%) ratio of about 0.79 or less.

20. An isothermal amplification method for amplifying target nucleic acids, wherein the isothermal amplification method is performed in the presence of trehalose and polyethylene glycol (PEG), wherein the weight percentage (wt%) ratio of trehalose to PEG is about 0.79 or less.

21. The isothermal amplification method according to claim 19 or 20, wherein the wt% ratio of trehalose to PEG is from about 0.3 to about 0.

79.

22. The isothermal amplification method according to any one of claims 19-21, wherein the wt% ratio of trehalose to PEG is about 0.45 to about 0.

75.

23. The isothermal amplification method according to any one of claims 19-22, wherein the wt% ratio of trehalose to PEG is about 0.50 to about 0.

70.

24. The isothermal amplification method according to any one of claims 19-23, wherein the wt% ratio of trehalose to PEG is about 0.50 to about 0.

60.

25. The isothermal amplification method according to any one of claims 19-24, wherein PEG is present in the reagent composition in an amount of about 2% to about 30% by weight (w / v).

26. The isothermal amplification method according to any one of claims 19-25, wherein PEG is present in the isothermal amplification method and / or reagent composition in an amount of about 2% to about 20% by weight-volume ratio (w / v).

27. The isothermal amplification method according to any one of claims 19-26, wherein PEG is present in the isothermal amplification method and / or reagent composition in an amount of about 2% to about 8% by weight-volume ratio (w / v).

28. The isothermal amplification method according to any one of claims 19-27, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 20% w / v.

29. The isothermal amplification method according to any one of claims 19-28, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 15% w / v.

30. The isothermal amplification method according to any one of claims 19-29, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 12% w / v.

31. The isothermal amplification method according to any one of claims 19-30, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 10% w / v.

32. The isothermal amplification method according to any one of claims 19-31, wherein trehalose is present in the isothermal amplification method and / or reagent composition in an amount of about 3% to about 6% w / v.

33. The isothermal amplification method according to any one of claims 1-32, wherein the PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons.

34. The isothermal amplification method according to any one of claims 1-33, wherein the isothermal amplification method is selected from the group consisting of: rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR).

35. The isothermal amplification method according to claim 34, wherein the isothermal amplification method is RPA.

36. The isothermal amplification method according to claim 34, wherein the isothermal amplification method is NEAR.

37. The isothermal amplification method according to any one of claims 1-36, wherein the target nucleic acid is bacterial, eukaryotic, or viral nucleic acid.

38. The isothermal amplification method according to any one of claims 1-37, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, parvovirus B19, HAV, chlamydia, gonorrhea, WNV, Zika virus, dengue virus, chikungunya virus, influenza, babesi, malaria, Usutu virus, or HEV.

39. The isothermal amplification method according to any one of claims 1-38, wherein the isothermal amplification method, for example, amplifies at least two or at least three target nucleic acids in a multiplex reaction.

40. The isothermal amplification method according to any one of claims 1-39, wherein the target nucleic acid is present in the sample.

41. The isothermal amplification method according to claim 40, wherein the sample is a tissue sample, and the target nucleic acid is isolated from the tissue sample prior to amplification.

42. The isothermal amplification method according to claim 40, wherein the sample is a biological fluid.

43. The isothermal amplification method according to claim 42, wherein the biological fluid is blood.

44. The isothermal amplification method according to claim 42 or 43, wherein the target nucleic acid is isolated from biological fluids prior to amplification.

45. The isothermal amplification method according to any one of claims 19-44, the method further comprising contacting the target nucleic acid with one or more of the following: DNA polymerase, recombinase loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe, reverse transcriptase and activator.

46. ​​The isothermal amplification method according to any one of claims 19-44, wherein the reagent composition further comprises one or more of the following: DNA polymerase, recombinase loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease (e.g., exonuclease), one or more primers, nucleic acid probe and reverse transcriptase.

47. A composition for performing an isothermal amplification method, the composition comprising trehalose and PEG, wherein the trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.79 or less.

48. The composition according to claim 47, wherein the wt% ratio of trehalose to PEG is about 0.75 or less.

49. The composition according to claim 47 or 48, wherein the wt% ratio of trehalose to PEG is from about 0.25 to about 0.

79.

50. The composition according to any one of claims 47-49, wherein the wt% ratio of trehalose to PEG is from about 0.30 to about 0.

75.

51. The composition according to any one of claims 47-50, wherein the wt% ratio of trehalose to PEG is from about 0.45 to about 0.

75.

52. The composition according to any one of claims 47-51, wherein the wt% ratio of trehalose to PEG is from about 0.50 to about 0.

70.

53. The composition according to any one of claims 47-52, wherein the wt% ratio of trehalose to PEG is about 0.50 to about 0.

60.

54. The composition according to any one of claims 47-53, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 2% to about 30% by weight (w / v).

55. The composition according to any one of claims 47-54, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 2% to about 25% by weight (w / v).

56. The composition according to any one of claims 47-55, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 5% to about 25% by weight (w / v).

57. The composition according to any one of claims 47-56, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 10% to about 20% by weight (w / v).

58. The composition according to any one of claims 47-55, wherein PEG is present in the isothermal amplification method and / or the composition in an amount of about 2% to about 8% by weight (w / v).

59. The composition according to any one of claims 47-58, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 3% to about 20% w / v.

60. The composition according to any one of claims 47-59, wherein trehalose is present in the isothermal amplification method and / or the composition in an amount of about 3% to about 15% w / v.

61. The composition according to any one of claims 47-60, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 3% to about 12% w / v.

62. The composition according to any one of claims 47-61, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 3% to about 5% w / v.

63. The composition according to claim 62, wherein trehalose is present in the isothermal amplification method and / or composition in an amount of about 4% w / v.

64. The composition according to any one of claims 47-63, wherein the PEG has a molecular weight between about 20,000 Daltons and about 50,000 Daltons.

65. The composition according to any one of claims 47-64, further comprising one or more of the following: DNA polymerase, recombinase, recombinase loading protein, single-strand binding protein, ATP, dNTP or a mixture of dNTP and ddNTP, reducing agent, creatine kinase, nuclease, nucleic acid probe and reverse transcriptase.

66. The composition according to any one of claims 47-65, further comprising one or more of the following: ATP, dNTPs or a mixture of dNTPs and ddNTPs, and a reducing agent.

67. The composition according to any one of claims 47-65, further comprising ATP, dNTPs, creatine phosphate, and one or more salts.

68. The composition according to any one of claims 47-67, wherein the isothermal amplification method is selected from the group consisting of: rolling circle amplification (RCA), sequence-based amplification (NASBA), strand substitution amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), and nicking enzyme amplification reaction (NEAR).

69. The composition according to claim 68, wherein the isothermal amplification method is RPA.

70. The composition according to claim 68, wherein the isothermal amplification method is NEAR.

71. A composition for an isothermal amplification method, the composition comprising trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) ratio of about 0.79 or less.

72. A composition for an isothermal amplification method, the composition comprising trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition at a weight percentage (wt%) of about 0.75 or less.

73. A composition for an isothermal amplification method, the composition comprising trehalose, PEG, ATP, dNTP, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.25 to about 0.

79.

74. The composition according to any one of claims 71-73, wherein the wt% ratio of trehalose to PEG is from about 0.30 to about 0.

60.

75. A composition for an isothermal amplification method, the composition comprising primarily trehalose, PEG, ATP, dNTPs, creatine phosphate, and one or more salts, wherein trehalose and PEG are present in the composition in a weight percentage (wt%) ratio of about 0.25 to about 0.

79.

76. The composition according to claim 75, wherein the wt% ratio of trehalose to PEG is from about 0.30 to about 0.

60.

77. A system for performing the isothermal amplification method according to any one of claims 1-46.

78. A system for performing an isothermal amplification method, the system comprising a container or reservoir comprising the composition of any one of claims 47-76.

79. The system according to claim 77 or 78, wherein the system is automated.

80. A kit for performing the isothermal amplification method according to any one of claims 1-46.

81. A kit comprising the composition of any one of claims 47-76.

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