Reducing agent compositions for isothermal amplification of nucleic acids

Abstract

The present disclosure relates to compositions for use in the isothermal amplification of nucleic acids in a sample. In particular, the present disclosure provides compositions that include an activator and reducing agent and methods for amplifying at least one target nucleic acid in a sample using the composition. The present disclosure further provides systems and kits for performing such methods and using such compositions.

Description

[0001] REDUCING AGENT COMPOSITIONS FOR ISOTHERMAL AMPLIFICATION OF NUCLEIC ACIDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The application claims priority to U.S. Provisional Application No. 63 / 612,737, filed December 20, 2023, the contents of which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The subject matter disclosed herein relates to methods for the isothermal amplification of target nucleic acids and compositions, kits and systems for performing such methods.

[0006] BACKGROUND

[0007] The ability to amplify nucleic acids lies at the heart of modern biological and medical research. In particular, amplification of nucleic acids is commonly used in nucleic acid-based diagnostic methods for the detection of an infection, e.g., viral infection or bacterial infection, a disease and / or a genetic mutation in a subject. For example, identification of bacterial or viral nucleic acid in a blood sample can be useful in determining whether the blood is safe for donating. Other examples include the identification of specific genetic mutations for early detection of cancer or for determining the best course of treatment for a cancer.

[0008] Among the various nucleic acid amplification techniques, polymerase chain reaction (PCR) is the most common because of its sensitivity and efficiency at amplifying short nucleic acid sequences. While PCR is of great utility, it also has limitations. In particular, PCR relies on multiple cycles of thermal melting (denaturing) at high temperatures followed by hybridization and elongation at a reduced temperature (“thermal cycling”), which is a time-consuming process and requires specialized equipment. In view of the technical disadvantages of PCR and other amplification techniques that require thermal cycling, methods have been developed which enable isothermal amplification of nucleic acids. Isothermal amplification of nucleic acids includes amplification processes that do not require temperature cycling or rapid heating and cooling for amplification of a target nucleic acid to occur. As isothermal amplification processes become more commonly used for diagnostic applications, there is a need in the art for more stable reagent compositions for performing such processes.

[0009] SUMMARY

[0010] The purpose and advantages of the disclosed subject matter will be set forth in and are apparent from the description that follows, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the devices particularly pointed out in the written description and claims hereof, as well as from the appended drawings.

[0011] To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter provides a composition for performing an isothermal amplification process, where the composition comprises a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, the concentration ratio is about 1 :0.25 to about 1 :3. In certain embodiments, the concentration ratio is about 1 : 1 to about 1 :3. In certain embodiments, the concentration ratio is about 1:2 to about 1 :3. In certain embodiments, the composition includes from about 1 mM to about 200 mM of the reducing agent. In certain embodiments, the composition includes from about 10 mM to about 200 mM of the reducing agent. In certain embodiments, the composition includes from about 50 mM to about 100 mM of the reducing agent. In certain embodiments, the composition includes from about 0.1 mM to about 500 mM of the activator. In certain embodiments, the composition includes from about 1 mM to about 300 mM of the activator. In certain embodiments, the composition includes from about 1 mM to about 250 mM of the activator. In certain embodiments, the composition includes from about 100 mM to about 300 mM of the activator. In certain embodiments, the reducing agent is a trialkylphosphine. In certain embodiments, the reducing agent is tris(hydroxypropyl)phosphine (THPP). In certain embodiments, the activator is magnesium, manganese or a salt form thereof. In certain embodiments, the activator is magnesium or a salt form thereof.

[0012] In certain embodiments, the reducing agent is stable (e.g., does not undergo significant oxidation) in the composition for a time period greater than about 14 days. In certain embodiments, less than about 20% of the reducing agent is oxidized when stored for a time period greater than about 14 days. In certain embodiments, the composition is stored at a temperature from about 2°C to about 40°C. In certain embodiments, the composition is stored at a temperature from about 25°C to about 40°C. In certain embodiments, the composition comprises a Tris buffer. In certain embodiments, the composition has a pH from about 5 to about 11, e.g., a pH from about 6 to about 9.

[0013] In certain embodiments, the composition consists essentially of the reducing agent and the activator. In certain embodiments, the composition consists of the reducing agent and the activator.

[0014] In certain embodiments, the composition is for use in an isothermal amplification process selected from the group consisting of rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), Transcription-Mediated Amplification (TMA), Single Primer Isothermal Amplification (SPIA), Helicase-dependent amplification (HD A), Loop mediated amplification (LAMP), Recombinase-Polymerase Amplification (RPA) and Nicking Enzyme Amplification Reaction (NEAR). In certain embodiments, the isothermal amplification process is RPA. In certain embodiments, the isothermal amplification process is NEAR.

[0015] The present disclosure further provides isothermal amplification processes using the compositions disclosed herein. In certain embodiments, the isothermal amplification process for amplifying a target nucleic acid includes contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, the present disclosure provides an isothermal amplification process for amplifying a target nucleic acid that is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

[0016] In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure includes (a) contacting a solution comprising a plurality of nucleic acids and one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1:5 to generate an activated reaction mixture and (c) incubating the activated reaction mixture to amplify the target nucleic acid if present in the plurality of nucleic acids. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the reducing agent from about 1 mM to about 10 mM. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the activator from about 10 mM to about 20 mM.

[0017] In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure includes (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present in the plurality of nucleic acids. In certain embodiments, the reaction mixture comprises a reaction concentration of the reducing agent from about 1 mM to about 10 mM. In certain embodiments, the reaction mixture comprises a reaction concentration of the activator from about 10 mM to about 20 mM.

[0018] In certain embodiments, the composition for use in the isothermal amplification processes of the present disclosure includes the reducing agent and the activator at a concentration ratio of about 1 :0.25 to about 1 :3. In certain embodiments, the concentration ratio is about 1 : 1 to about 1 :3. In certain embodiments, the concentration ratio is about 1 :2 to about 1 :3. In certain embodiments, the composition includes a concentration ratio of 1 mM to about 200 mM of the reducing agent. In certain embodiments, the composition includes a concentration ratio of 10 mM to about 200 mM of the reducing agent. In certain embodiments, the composition includes from about 50 mM to about 100 mM of the reducing agent. In certain embodiments, the composition includes from about 0.1 mM to about 500 mM of the activator. In certain embodiments, the composition includes from about 1 mM to about 300 mM of the activator. In certain embodiments, the composition includes from about 1 mM to about 250 mM of the activator. In certain embodiments, the composition includes from about 100 mM to about 300 mM of the activator. In certain embodiments, the reducing agent is a trialkylphosphine. In certain embodiments, the reducing agent is THPP. In certain embodiments, the activator is magnesium, manganese or a salt form thereof. In certain embodiments, the activator is magnesium or a salt form thereof. In certain embodiments, the reducing agent is stable (e.g., does not undergo significant oxidation) in the composition for a time period greater than about 14 days. In certain embodiments, less than about 20% of the reducing agent is oxidized when stored for a time period greater than about 14 days. In certain embodiments, the composition is stored at a temperature from about 2°C to about 40°C. In certain embodiments, the composition is stored at a temperature from about 25°C to about 40°C. In certain embodiments, the composition comprises a Tris buffer. In certain embodiments, the composition has a pH from about 5 to about 11, e.g., about 6 to about 9.

[0019] In certain embodiments, the one or more isothermal amplification reagents can include one or more of: a recombinase, a single-stranded DNA binding protein, a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a recombinase loading protein, a first primer, a second primer, a probe, a nuclease, a creatine kinase and a reverse transcriptase.

[0020] In certain embodiments, the isothermal amplification process is selected from the group consisting of rolling circle amplification (RCA), nucleic acid sequencebased amplification (NASBA), strand displacement amplification (SDA), Transcription- Mediated Amplification (TMA), Single Primer Isothermal Amplification (SPIA), Helicasedependent amplification (HDA), Loop mediated amplification (LAMP), Recombinase- Polymerase Amplification (RPA) and Nicking Enzyme Amplification Reaction (NEAR). In certain embodiments, the isothermal amplification process is RPA. In certain embodiments, the isothermal amplification process is NEAR.

[0021] In certain embodiments, the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid. In certain embodiments, the target nucleic acid is derived from SARS- CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Parvovirus Bl 9, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Virus, Influenza, Babesia, Malaria, Usutu Virus or HEV.

[0022] In certain embodiments, the target nucleic acid is derived from HIV-1. In certain embodiments, the target nucleic acid is derived from HIV-2. In certain embodiments, the target nucleic acid is derived from HBV. In certain embodiments, the target nucleic acid is derived from HCV. In certain embodiments, the target nucleic acid is derived from Malaria. In certain embodiments, the target nucleic acid is derived from Babesia. In certain embodiments, the target nucleic acid is derived from Parvovirus Bl 9.

[0023] In certain embodiments, the isothermal amplification process amplifies at least two target nucleic acids or at least three target nucleic acids, e.g., in a multiplex reaction.

[0024] In certain embodiments, the sample is a tissue sample. In certain embodiments, the target nucleic acid is isolated from the tissue sample prior to amplification.

[0025] In certain embodiments, the sample is a biological fluid. In certain embodiments, the biological fluid is blood, e.g., whole blood, lysed whole blood, serum or plasma. In certain embodiments, the target nucleic acid is isolated from the biological fluid prior to amplification.

[0026] In another aspect, the present disclosure provides a system for performing the methods described herein. In certain embodiments, the present disclosure provides a system that includes a container comprising the compositions described herein. In certain embodiments, the system is an automated system.

[0027] The present disclosure provides kits including the compositions described herein. The present disclosure further provides kits for performing the methods described herein.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations and equivalents in form and function, without departing from the scope of this disclosure.

[0030] FIG. 1A provides curves showing the amplification kinetics of exemplary RPA reactions performed using reagents that were stored at 2-8°C or -70°C for 7 days. PC refers to the protein components of the RPA reaction, NPC refers to the non-protein components of the RPA reaction and OC refers to the oligonucleotide components of the RPA reaction. The reducing agent dithiothreitol (DTT) is included in the NPC.

[0031] FIG. IB provides curves showing the amplification kinetics of exemplary RPA reactions performed using reagents that were stored at 2-8°C or -70°C for 15 days, demonstrating decreased performance when the NPC is stored at 2-8°C. The reducing agent DTT is included in the NPC.

[0032] FIG. 2A provides curves showing the amplification kinetics of exemplary RPA reactions performed using reagents that were stored at 25°C, 2-8°C or -70°C for 14 days, demonstrating decreased performance when the NPC is stored at 25°C. The reducing agent DTT is included in the NPC.

[0033] FIG. 2B provides curves showing the amplification kinetics of exemplary RPA reactions performed using reagents that were stored at 25°C, 2-8°C or -70°C for 21 days, demonstrating decreased performance when the NPC is stored at 25°C and 2-8°C. The reducing agent DTT is included in the NPC. FIG. 3A provides curves showing the amplification kinetics of exemplary RPA reactions performed using fresh reagents that were not stored, demonstrating that a reducing agent is necessary for full reaction performance. The reducing agent is DTT.

[0034] FIG. 3B provides curves showing the amplification kinetics of exemplary RPA reactions performed using reagents that were stored at 2-8°C and -70°C for 21 days, demonstrating that a stable reducing agent is necessary for full reaction performance. The reducing agent is DTT.

[0035] FIG. 4A provides curves showing the amplification kinetics of exemplary RPA reactions performed using 5 mM DTT, TCEP or THPP that were stored at 2-8°C for 18 and 31 days in the NPC.

[0036] FIG. 4B provides curves showing the amplification kinetics of exemplary RPA reactions performed using DTT, TCEP or THPP in both NPC and PC that were stored at -20°C or 2-8°C for 21 days.

[0037] FIG. 5A provides curves showing the amplification kinetics of exemplary RPA reactions performed using a solution including THPP and MgOAc that was stored at 37°C for 0 (fresh), 28 and 90 days compared with an NPC with DTT stored at 2-8°C for 0 (fresh), 28 or 90 days. The study demonstrated THPP stored in the MgOAc solution provided reductant activity in the reaction much longer than DTT in NPC.

[0038] FIG. 5B provides the components of the formulations and storage conditions of such formulations used in the RPA reactions shown in FIG. 5 A.

[0039] FIG. 6A provides curves showing the amplification kinetics of exemplary RPA reactions performed using a stock solution including 80 mM THPP and MgOAc in Tris buffer (pH 8.5) that was stored at 2-8°C or 37°C for 232 days.

[0040] FIG. 6B provides the results of an Ellman’s assay of a stock solution including 80 mM THPP (starting concentration) and MgOAc in 10 mM Tris buffer (pH 8.5) that was stored at 2-8°C or 37°C for 232 days. Ellman’s assay measures the number of free sulfhydryl groups available to act as a reducing agent. At 232 days, 37°C and 2-8°C storage left about 40 mM and about 66 mM available THPP in solution, respectively. The minimum starting concentration-equivalent of approximately 8 mM is needed to support the reaction.

[0041] FIG. 7 provides the results of an Ellman’s assay of a stock solution including 62 mM THPP (starting concentration) in 10 mM Tris buffer (pH 8.5) that was stored at 37°C for up to 49 days, demonstrating stability of THPP in the presence of MgOAc. FIG. 8 provides the results of an Ellman’s assay of a stock solution including 62 mM TCEP (starting concentration) in 10 mM Tris buffer (pH 8.5) that was stored at 37°C for up to 18 days, demonstrating that MgOAc did not improve TCEP stability.

[0042] FIG. 9 provides the results of an Ellman’s assay of stock solutions including 80 mM THPP and different concentrations of MgOAc in 10 mM Tris buffer (pH 8.5) that were stored at 37°C for up to 36 days.

[0043] FIG. 10 provides the results of an Ellman’s assay of stock solutions including 80 mM THPP and different concentrations of MgOAc in 10 mM Tris buffer (pH 8.5) that were stored at 45°C or 54°C for 1 week.

[0044] DETAILED DESCRIPTION

[0045] The present disclosure provides improved methods of amplifying and detecting nucleic acids in a sample using isothermal amplification processes, e.g., Recombinase Polymerase Amplification (RPA). The present disclosure further provides compositions, systems and kits for performing isothermal amplification reactions.

[0046] The present disclosure is based, in part, on the observation that a reducing agent, THPP, included in an activator composition has increased stability and can support isothermal amplification processes after long term storage (e.g., at temperatures above 0°C) with stability much beyond the more conventional reducing agent DTT. As shown in Example 1, the presence of a reducing agent, e.g., THPP, in an activator composition minimized the loss of active reducing agent, e.g., THPP, over a long term storage period. Such increased stability supports the use of the presently disclosed compositions in systems (e.g., automated systems) that store bulk solutions of reagents for extended periods of time, where aliquots of the bulk solutions are used to perform isothermal amplification processes on the system.

[0047] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0048] I. Definitions;

[0049] II. Isothermal Amplification Processes;

[0050] III. Nucleic Acid Detection Processes;

[0051] IV. Methods of Use;

[0052] V. Systems;

[0053] VI. Kits; and

[0054] VII. Exemplary Embodiments. I. Definitions

[0055] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0056] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0057] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” For example, but not by way of limitation, reference to “an” or “the” “target nucleic acid” encompasses a single target nucleic acid, as well as a combination and / or mixture of two or more different target nucleic acids.

[0058] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.

[0059] As used herein, the term “activator” refers to an agent that is required to initiate the isothermal amplification reaction. For example, but not by way of limitation, the activator can serve as a cofactor for one or more enzymes, e.g. , DNA polymerase, used in an isothermal amplification process of the present disclosure.

[0060] As used herein, the term “amplified” refers to the process of making multiple copies of the nucleic acid from a single or lower copy number of nucleic acid molecules. The amplified nucleic acid can be referred to as an amplicon.

[0061] The term “amplification process” refers generally to any process where a portion of a nucleic acid is copied or replicated into at least one additional nucleic acid molecule. An amplification process produces amplified nucleic acids. The term “biological fluid,” as used herein, refers to any bodily fluid or bodily fluid derivative in which the analyte can be measured. Non-limiting examples of a biological fluid include dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, tears, or the like. In certain embodiments, the biological fluid is blood.

[0062] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0063] The term “coupled” can refer to the connecting or uniting of two or more components by an interaction, bond, link, force or tie in order to keep two or more components together. In certain embodiments, the term “coupled” encompasses either direct or indirect binding where, for example, a first component is directly bound to a second component, or one or more intermediate molecules are disposed between the first component and the second component. Exemplary bonds comprise covalent bonds, ionic bonds, van der Waals interactions and other bonds identifiable by a skilled person.

[0064] The terms “detect” or “detection,” as used herein, indicates the determination of the existence and / or presence of a target nucleic acid in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and / or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of the presence or absence of a target or signal, without dependence on the quantity or amount of the target or signal beyond its presence or absence.

[0065] The terms “expression” or “expresses,” as used herein, refer to transcription and translation occurring within a cell. The level of expression of a gene and / or nucleic acid in a cell can be determined on the basis of either the amount of corresponding messenger RNA (mRNA) that is present in the cell or the amount of the protein encoded by the gene and / or nucleic acid that is produced by the cell. For example, mRNA transcribed from a gene and / or nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a gene and / or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0066] As used herein, the term “hybridization,” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.

[0067] As used herein, a “label” refers to 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) incorporated by reference herein,32P,14C,125I,3H and131I, fluorogens (such as Rare Earth Chelate or lucifer yellow and its derivatives), Rhodamine (rhodamine) and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial fluorescence plain enzyme) (U.S. Patent number 4,737,456), fluorescein, 2,3-dihydros phthalazine diketone, as well as enzymes producing detectable signals, e.g., horseradish peroxidase (HRP), alkaline phosphorus sour enzyme, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)) and heterocyclic oxidases (such as uricase and xanthine oxidase).

[0068] The term “nucleic acid,” “nucleic acid molecule” or “polynucleotide” as used herein refers to any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically 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. Often, the nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. In certain embodiments, the nucleic acid is isolated. In certain embodiments, the term “isolated nucleic acid” can refer to a nucleic acid removed from a subject or a sample, e.g. , removed from its original environment (e.g. , the natural environment or a host cell if recombinantly expressed).

[0069] The term “oligonucleotide,” as used herein, refers to a short nucleic acid sequence comprising from 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). The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, for example, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).

[0070] Oligonucleotides can be single-stranded or double-stranded or can contain portions of both double-stranded and single-stranded sequences. The oligonucleotide can be DNA, both genomic and complimentary DNA (cDNA), RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Oligonucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0071] Any of the oligonucleotides described herein can be modified in any suitable manner so as to stabilize or enhance the binding affinity of the oligonucleotide for its target. For example, an oligonucleotide sequence as described herein can comprise one or more modified oligonucleotide bases.

[0072] The oligonucleotides described herein can be prepared using any suitable method, a variety of which are known in the art (see, for example, Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; M. A. Innis (Ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, N.Y. (1990); P. Tijssen, Hybridization with Nucleic Acid Probes - Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); M. A. Innis (Ed.), PCR Strategies, Academic Press: New York, N.Y. (1995); and F. M. Ausubel (Ed.), Short Protocols in Molecular Biology, John Wiley & Sons: Secaucus, N.J. (2002); Narang et a\., Melh. 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 also can be designed using a variety of tools, such as the Primer-BLAST tool provided by the National Center of Biotechnology Information (NCBI). Oligonucleotide synthesis can be performed on oligo synthesizers such as those commercially available from Perkin Elmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or Milligen (Bedford, MA). Alternatively, oligonucleotides can be custom made and obtained from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), BioSearch Technologies, Inc. (Novato, CA), and the like. Oligonucleotides can be purified using any suitable method known in the art, such as, for example, native acrylamide gel electrophoresis, anion-exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255: 137-149 (1983), incorporated herein by reference), and reverse phase HPLC (see, e.g., McFarland et al., Nucleic Acids Res., 7: 1067-1080 (1979), incorporated herein by reference).

[0073] The sequence of the oligonucleotides can be verified 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), 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), incorporated herein by reference), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions (Wu et al., Anal. Biochem., 290: 347-352 (2001), incorporated herein by reference), and the like. The term “plurality” refers to a number larger than one. In certain embodiments, the term “plurality of nucleic acids” refers to a number of nucleic acids larger than one. For example, but not by way of limitation, a plurality of target nucleic acids includes at least two target nucleic acids.

[0074] The terms “primer,” “primer sequence,” “primer oligonucleotide,” and “amplification oligonucleotide” as used herein, refer to an oligonucleotide which is capable of acting as a point of initiation of synthesis of an extension product that is a complementary strand of nucleic acid (all types of DNA or RNA) when placed under suitable amplification conditions (e.g. , buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., a DNA-dependent or RNA-dependent polymerase). The amplification oligonucleotides of the present disclosure can be of any suitable size, and desirably comprise, consist essentially of, or consist of about 15 to 50 nucleotides, preferably about 20 to 40 nucleotides. The oligonucleotides of the present disclosure can contain additional nucleotides in addition to those described herein.

[0075] The terms “probe,” “probe sequence,” and “probe oligonucleotide,” refer to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of a target sequence that has been amplified) under appropriate hybridization conditions. In general, a probe sequence is identified as being either “complementary” (i.e., complementary to the coding or sense strand (+)), or “reverse complementary” (i.e., complementary to the anti-sense strand (-)). The probes of the present disclosure can be of any suitable size, and desirably comprise, consist essentially of, or consist of about 10-50 nucleotides, preferably about 12-35 nucleotides.

[0076] As used herein, the terms “set,” “primer set,” “probe set,” and “primer and probe set,” refer to two or more oligonucleotides which together are capable of priming the amplification of a target sequence or target nucleic acid of interest (e.g., a target sequence within an infectious agent) and / or at least one probe which can detect the target sequence or target nucleic acid. In certain embodiments, the term “set” refers to a pair of oligonucleotides including a first oligonucleotide, referred herein as a “forward primer” that hybridizes with the 5 ’-end of the target sequence or target nucleic acid to be amplified and a second oligonucleotide, referred herein as a “reverse primer” that hybridizes with the complement of the target sequence or target nucleic acid to be amplified.

[0077] As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence; for example, as a segment of a full-length protein or protein fragment. A reference sequence can be, for example, a sequence identifiable in a database such as GenBank and UniProt and others identifiable to those skilled in the art.

[0078] As used herein, “sequence identity” or “identity” in the context of two polynucleotide or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule.

[0079] As used herein, “percentage of sequence identity” or “percentage of identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0080] As understood by those skilled in the art, determination of percent identity between any two sequences can be accomplished using certain well-known mathematical algorithms. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, the local homology algorithm of Smith et al.; the homology alignment algorithm of Needleman and Wunsch; the search-for-similarity-method of Pearson and Lipman; the algorithm of Karlin and Altschul, modified as in Karlin and Altschul. Computer implementations of suitable mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, FASTA, among others identifiable by skilled persons. Sequence alignment algorithms also are disclosed in, for example, 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., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(jy. 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(lTy. 3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997), each of which is incorporated herein by reference in its entirety).

[0081] As used herein, the term “reducing agent” refers to an agent that reduces the disulfide bonds of a polypeptide. In certain embodiments, the “reducing agent” can be used to stabilize enzymes and other proteins with free sulfhydryl groups. For example, but not by way of limitation, a reducing agent reduces the disulfide bonds of an enzyme used in an isothermal amplification process of the present disclosure.

[0082] As used herein, the term “subject” or “individual” refers to a vertebrate or an invertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of 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 certain embodiments, the individual or subject is a human.

[0083] The terms “target nucleic acid”, “target sequence”, or “target nucleic acid sequence,” as used herein, refers to a nucleic acid sequence of interest to be amplified using the methods of the present disclosure.

[0084] IL Isothermal Amplification Processes

[0085] The presently disclosed subject matter provides improved and / or more stable reducing agent compositions and provides amplification processes using such compositions. In certain embodiments, the amplification process is an isothermal amplification process. Isothermal amplification processes include amplification processes that do not require temperature cycling or rapid heating and cooling for amplification of a target nucleic acid to occur.

[0086] Non-limiting examples of isothermal amplification processes include rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement 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). Additional non-limiting disclosure regarding isothermal amplification methods is provided in Oliveira et al., Frontiers in Sensors 2:752600 (2021), the contents of which is incorporated herein by reference in its entirety.

[0087] In certain embodiments, the isothermal amplification process is RPA.

[0088] In certain embodiments, the isothermal amplification process is NEAR.

[0089] The present disclosure provides isothermal amplification processes that are performed by contacting a sample (e.g., a sample comprising one or more target nucleic acids) with a composition comprising a reducing agent and an activator, e.g., in the presence of other reagents (e.g., enzymes) for performing the amplification of the one or more target nucleic acids. In certain embodiments, the present disclosure provides isothermal amplification process that are performed by contacting a sample (e.g., a sample comprising one or more target nucleic acids) with a composition consisting essentially of a reducing agent and an activator, e.g, in the presence of other reagents (e.g., enzymes) for performing the amplification of the one or more target nucleic acids. In certain embodiments, the present disclosure provides isothermal amplification process that are performed by contacting a sample (e.g., a sample comprising one or more target nucleic acids) with a composition consisting of a reducing agent and an activator, e.g., in the presence of other reagents (e.g., enzymes) for performing the amplification of the one or more target nucleic acids. For example, but not by way of limitation, an isothermal amplification process of the present disclosure includes initiating amplification of one or more target nucleic acids in a sample by contacting a composition described herein with the sample, e.g., in the presence of other reagents (e.g., enzymes) for performing the amplification of the one or more target nucleic acids.

[0090] In certain embodiments, a composition described herein can be combined with other reagents (e.g., enzymes) to generate a reagent composition that is subsequently contacted with a sample (e.g., a sample comprising one or more target nucleic acids) to amplify the one or more target nucleic acids in the sample. For example, but not by way of limitation, an isothermal amplification process of the present disclosure can include (i) combining a composition disclosed herein (e.g., a portion of a composition disclosed herein) with other reagents (e.g., enzymes) to generate a reagent composition and (ii) contacting a sample with the reagent composition to amplify the one or more target nucleic acids in the sample. In certain embodiments, an isothermal amplification process of the present disclosure can include (i) combining a composition disclosed herein (e.g., a portion of a composition disclosed herein) with other reagents (e.g., enzymes) to generate a reagent composition, (ii) contacting a sample with the reagent composition and (iii) amplifying the one or more target nucleic acids in the sample. In certain embodiments, a method of the present disclosure can include (i) combining a composition disclosed herein (e.g., a portion of a composition disclosed herein) with other reagents (e.g., enzymes) to generate a reagent composition, (ii) contacting a sample with the reagent composition, (iii) amplifying the one or more target nucleic acids in the sample and (iv) detecting the one or more amplified target nucleic acids. In certain embodiments, detecting the one or more amplified target nucleic acids can occur during the amplification of the one or more target nucleic acids.

[0091] In certain embodiments, a percentage of a composition that includes an activator and a reducing agent as described herein can be combined with other reagents (e.g., enzymes) to generate a reagent composition that is subsequently contacted with a sample (e.g., a sample comprising one or more target nucleic acids) to amplify the one or more target nucleic acids in the sample. For example, but not by way of limitation, an isothermal amplification process of the present disclosure can include (i) combining a percentage of a composition that includes an activator and a reducing agent as described herein with other reagents (e.g., enzymes) to generate a reagent composition, (ii) contacting a sample with the reagent composition and (iii) amplifying the one or more target nucleic acids in the sample. In certain embodiments, a method of the present disclosure can include (i) combining a percentage of a composition that includes an activator and a reducing agent as described herein (or a portion of a composition disclosed herein) with other reagents (e.g., enzymes) to generate a reagent composition, (ii) contacting a sample with the reagent composition, (iii) amplifying the one or more target nucleic acids in the sample and (iv) detecting the one or more amplified target nucleic acids. In certain embodiments, detecting the one or more amplified target nucleic acids can occur during the amplification of the one or more target nucleic acids.

[0092] A. Compositions

[0093] The present disclosure provides compositions for performing an isothermal application process. For example, but not by way of limitation, the present disclosure provides compositions comprising one or more reagents, e.g., reagent compositions, for performing an isothermal application process of the present disclosure. In certain embodiments, the present disclosure provides compositions comprising one or more reagents, e.g., reagent compositions, for performing an RPA process.

[0094] In certain embodiments, a composition of the present disclosure includes a reducing agent and an activator. In certain embodiments, the present disclosure provides a composition consisting essentially of a reducing agent and an activator. In certain embodiments, the present disclosure provides a composition consisting of a reducing agent and an activator.

[0095] As shown in Example 1, the presence of an activator in the composition that includes the reducing agent increases the stability of the reducing agent and allows for the long-term storage of the composition, e.g., at temperatures above freezing (e.g., at 2-8°C, at 25°C or at 37°C). For example, but not by way of limitation, the presence of an activator in the composition that includes the reducing agent increases the stability of the reducing agent and allows for the storage of the composition (e.g. , at temperatures above 0°C) for a period greater than about 2 weeks, greater than about 3 weeks, greater than about 1 month, greater than about 2 months, greater than about 3 months, greater than about 4 months, greater than about 5 months, greater than about 6 months, greater than about 7 months, greater than about 8 months or greater than about 9 months (e.g., before being used in an isothermal amplification process). As shown in Example 1, the presence of an activator in a composition that includes the reducing agent increases the stability of the reducing agent and allows storage of the composition for a period greater than about 7 months, e.g, at temperatures above freezing (e.g, at 2-8°C, at 25°C or at 37°C).

[0096] Non-limiting examples of reducing agents for use in the presently disclosed compositions include trialkylphosphines. In certain embodiments, the reducing agent includes tris(3-hydroxypropyl)-phosphine (THPP). In certain embodiments, the reducing agent is THPP. In certain embodiments, the reducing agent is not dithiothreitol (DTT).

[0097] Non-limiting examples of activators for use in the presently disclosed compositions include compounds that initiate the isothermal amplification reaction, e.g., by functioning as a cofactor for the DNA polymerase used in the isothermal amplification reaction. In certain embodiments, the activator is an agent that results in metal ions (e.g., divalent metal ions) in solution. In certain embodiments, the activator can be agent that results in manganese ions (e.g., divalent manganese ions) in solution. In certain embodiments, the activator can be manganese or a salt form thereof. For example, but not by way of limitation, the activator can be manganese chloride (MnCh) and / or manganese acetate. In certain embodiments, the activator can be agent that results in magnesium ions (e.g., divalent magnesium ions) in solution. In certain embodiments, the activator can be magnesium or a salt form thereof. For example, but not by way of limitation, the activator can be magnesium chloride (MgCh) and / or magnesium acetate (MgOAc). In certain embodiments, the activator is MgOAc. In certain embodiments, a composition of the present disclosure includes a trialkylphosphine (e.g., THPP) and a magnesium or a salt thereof. In certain embodiments, the present disclosure provides a composition consisting essentially of a trialkylphosphine (e.g., THPP) and a magnesium or a salt thereof. In certain embodiments, the present disclosure provides a composition consisting of a trialkylphosphine e.g., THPP) and a magnesium or a salt thereof. In certain embodiments, a composition of the present disclosure includes THPP and a magnesium or a salt thereof e.g., MgOAc). In certain embodiments, the present disclosure provides a composition consisting essentially of THPP and a magnesium or a salt thereof e.g., MgOAc). In certain embodiments, the present disclosure provides a composition consisting of THPP and a magnesium or a salt thereof e.g., MgOAc).

[0098] In certain embodiments, a composition of the present disclosure includes a reducing agent and an activator at a concentration ratio of about 1:0.01 to about 1:5. For example, but not by way of limitation, the composition can include a reducing agent and an activator at a concentration ratio of about 1:0.01 to about 1:4.8, about 1:0.01 to about 1:4.6, about 1:0.01 to about 1:4.4, about 1:0.01 to about 1:4.2, about 1:0.01 to about 1:4.0, about 1:0.01 to about 1:3.8, about 1:0.01 to about 1:3.6, about 1:0.01 to about 1:3.4, about 1:0.01 to about 1:3.2, about 1:0.01 to about 1:3.0, about 1:0.01 to about 1:2.8, about 1:0.01 to about 1:2.6, about 1:0.01 to about 1:2.4, about 1:0.01 to about 1:2.2, about 1:0.01 to about 1:2.0, about 1:0.01 to about 1:1.8, about 1:0.01 to about 1:1.6, about 1:0.01 to about 1:1.4, about 1:0.01 to about 1:1.2, about 1:0.01 to about 1:1.0, about 1:0.01 to about 1:0.8, about 1:0.01 to about 1:0.6, about 1:0.01 to about 1:0.4, about 1:0.01 to about 1:0.2, about 1:0.01 to about 1:0.1, about 1:0.01 to about 1:0.05, about 1:0.05 to about 1:5, about 1:0.1 to about 1:5, about 1:0.2 to about 1:5, about 1:0.4 to about 1:5, about 1:0.6 to about 1:5, about 1:0.8 to about 1:5, about 1 : 1 to about 1:5, about 1 : 1.2 to about 1:5, about 1 : 1.4 to about 1:5, about 1 : 1.6 to about 1:5, about 1:1.8 to about 1:5, about 1:2 to about 1:5, about 1:2.2 to about 1:5, about 1:2.4 to about 1:5, about 1:2.6 to about 1:5, about 1:2.8 to about 1:5, about 1:3.0 to about 1:5, about 1:3.2 to about 1:5, about 1:3.4 to about 1:5, about 1:3.6 to about 1:5, about 1:3.8 to about 1:5, about 1 :4 to about 1:5, about 1 :4.2 to about 1:5, about 1 :4.4 to about 1:5, about 1 :4.6 to about 1:5, about 1 :4.8 to about 1:5, about 1 :0.05 to about 1 :4, about 1 : 0.05 to about 1:3, about 1: 0.05 to about 1:2, about 1: 0.05 to about 1:1, about 1:1 to about 1:3 or about 1 : 1 to about 1 :2. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1:0.01 to about 1:4. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.1 to about 1 :3. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.25 to about 1 :3. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.5 to about 1 :3. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :3. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :4. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :2 to about 1 :4. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :2. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.01. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 0.05. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.1. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.25. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :0.5. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :2. In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 :3.

[0099] In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5.

[0100] In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :4.

[0101] In certain embodiments, the composition can include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :3.

[0102] As shown in Example 1, a reducing agent is stabilized when incorporated into a composition that includes an activator (e.g., MgOAc), where the concentration ratio of the reducing agent to the activator is about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, no more than about 5% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). For example, but not by way of limitation, no more than about 6%, no more than about 7%, no more than about 8%, no more than about 9%, no more than about 10%, no more than about 11%, no more than about 12%, no more than about 13%, no more than about 14%, no more than about 15%, no more than about 16%, no more than about 17%, no more than about 18%, no more than about 19%, no more than about 20%, no more than about 21%, no more than about 22%, no more than about 23%, no more than about 24%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, no more than about 50%, no more than about 55%, no more than about 60%, no more than about 65%, no more than about 70%, no more than about 75%, no more than about 80%, no more than about 85%, no more than about 90%, no more than about 91%, no more than about 92%, no more than about 93%, no more than about 94%, no more than about 95%, no more than about 96%, no more than about 97%, no more than about 98% or no more than about 99% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, for a composition comprising about 80 mM of the reducing agent, no more than about 80%, no more than about 81%, no more than about 82%, no more than about 83%, no more than about 84%, no more than about 85%, no more than about 86%, no more than about 87%, no more than about 88%, no more than about 89% or no more than about 90% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, for a composition comprising about 800 mM of the reducing agent, no more than about 90%, no more than about 91%, no more than about 92%, no more than about 93%, no more than about 94%, no more than about 95%, no more than about 96%, no more than about 897%, no more than about 98% or no more than about 99% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, no more than about 20% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3), e.g., as shown in Example 1. In certain embodiments, no more than about 25% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, no more than about 50% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, no more than about 90% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, no more than about 99% of the reducing agent is oxidized when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, such stabilization of the reducing agent is observed over a 2-day period, a 3- day period, a 4-day period, a 5-day period, a 6-day period, a 7-day period, a 10-day period, a 15-day period, a 18-day period, a 25-day period, a 39-day period, a 49-day period, a 50- day period, a 100-day period, a 150-day period, a 200-day period, a 250-day period or a 270 day period. In certain embodiments, such stabilization is observed over a time period greater than about 2 days, greater than about 5 days, greater than about 10 days, greater than about 15 days, greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 50 days, greater than about 100 days, greater than about 150 days, greater than about 200 days or greater than about 250 days.

[0103] In certain embodiments, no more than about 50% of the reducing agent is oxidized when present in a composition that includes an activator and stored over a time period greater than about 14 days.

[0104] In certain embodiments, no more than about 60% of the reducing agent is oxidized when present in a composition that includes an activator and stored over a time period greater than about 14 days. In certain embodiments, no more than about 70% of the reducing agent is oxidized when present in a composition that includes an activator and stored over a time period greater than about 14 days.

[0105] In certain embodiments, no more than about 80% of the reducing agent is oxidized when present in a composition that includes an activator and stored over a time period greater than about 14 days.

[0106] In certain embodiments, no more than about 90% of the reducing agent is oxidized when present in a composition that includes an activator and stored over a time period greater than about 14 days.

[0107] In certain embodiments, the decay of a reducing agent is reduced, decreased and / or minimized when incorporated into a composition that includes an activator (e.g., MgOAc), where the concentration ratio of the reducing agent to the activator is about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). For example, but not by way of limitation, the decay of the reducing agent is reduced or decreased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95% when present in a composition that includes an activator, e.g., where the composition includes the reducing agent and the activator at a concentration ratio of about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, such a reduction in decay is observed over a 2-day period, a 3-day period, a 4-day period, a 5-day period, a 6-day period, a 7-day period, a 10-day period, a 15-day period, a 18-day period, a 25-day period, a 39-day period, a 49-day period, a 50-day period, a 100-day period, a 150-day period, a 200-day period, a 250-day period or a 270 day period. In certain embodiments, such a reduction in decay is observed over a time period greater than about 2 days, greater than about 5 days, greater than about 10 days, greater than about 15 days, greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 50 days, greater than about 100 days, greater than about 150 days, greater than about 200 days or greater than about 250 days.

[0108] In certain embodiments, the composition includes from about 1 mM to about 200 mM, e.g., about 10 mM to about 200 mM of a reducing agent, e.g., THPP. For example, but not by way of limitation, the composition includes from about 5 mM to about 200 mM, about 10 mM to about 200 mM, about 15 mM to about 200 mM, about 20 mM to about 200 mM, about 25 mM to about 200 mM, about 30 mM to about 200 mM, about 35 mM to about 200 mM, about 40 mM to about 200 mM, about 45 mM to about 200 mM, about 50 mM to about 200 mM, about 55 mM to about 200 mM, about 60 mM to about 200 mM, about 65 mM to about 200 mM, about 70 mM to about 200 mM, about 75 mM to about 200 mM, about 10 mM to about 190 mM, about 10 mM to about 180 mM, about 10 mM to about 170 mM, about 10 mM to about 160 mM, about 10 mM to about 150 mM, about 10 mM to about 140 mM, about 10 mM to about 130 mM, about 10 mM to about 120 mM, about 10 mM to about 110 mM, about 10 mM to about 100 mM, about 10 mM to about 90 mM, about 10 mM to about 85 mM, about 20 mM to about 150 mM, about 40 mM to about 120 mM, about 50 mM to about 110 mM, about 60 mM to about 100 mM or about 70 mM to about 90 mM. In certain embodiments, the composition includes from about 1 mM to about 150 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 5 mM to about 150 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 10 mM to about 150 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 15 mM to about 150 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 20 mM to about 150 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 1 mM to about 100 mM of a reducing agent, e.g, THPP. In certain embodiments, the composition includes from about 5 mM to about 150 mM of a reducing agent, e.g, THPP. In certain embodiments, the composition includes from about 10 mM to about 150 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 50 mM to about 100 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 60 mM to about 100 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 70 mM to about 90 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 40 mM to about 120 mM of a reducing agent, e.g., THPP.

[0109] In certain embodiments, the composition includes from about 1 mM to about 100 mM of a reducing agent, e.g., THPP.

[0110] In certain embodiments, the composition includes about 80 mM of a reducing agent, e.g., THPP.

[0111] In certain embodiments, the composition includes from about 0.1 mM to about 500 mM of an activator, e.g., MgOAc. For example, but not by way of limitation, the composition includes from about 0.5 mM to about 500 mM, about 1 mM to about 500 mM, about 5 mM to about 500 mM, 10 mM to about 500 mM, about 20 mM to about 500 mM, about 40 mM to about 500 mM, about 60 mM to about 500 mM, about 80 mM to about 500 mM, about 100 mM to about 500 mM, about 120 mM to about 500 mM, about 140 mM to about 500 mM, about 160 mM to about 500 mM, about 180 mM to about 500 mM, about 200 mM to about 500 mM, about 220 mM to about 500 mM, about 240 mM to about 500 mM, about 260 mM to about 500 mM, about 280 mM to about 500 mM, about 300 mM to about 500 mM, about 320 mM to about 500 mM, about 340 mM to about 500 mM, about 360 mM to about 500 mM, about 380 mM to about 500 mM, about 400 mM to about 500 mM, about 420 mM to about 500 mM, about 440 mM to about 500 mM, about 460 mM to about 500 mM, about 480 mM to about 500 mM, about 0.1 mM to about 500 mM, about 0.1 mM to about 480 mM, about 0.1 mM to about 460 mM, about 0.1 mM to about 440 mM, about 0.1 mM to about 420 mM, about 0.1 mM to about 400 mM, about 0.1 mM to about 380 mM, about 0.1 mM to about 360 mM, about 0.1 mM to about 340 mM, about 0.1 mM to about 320 mM, about 0.1 mM to about 300 mM, about 0.1 mM to about 280 mM, about 0.1 mM to about 260 mM, about 0.1 mM to about 240 mM, about 0.1 mM to about 220 mM, about 0.1 mM to about 200 mM, about 0.1 mM to about 180 mM, about 0.1 mM to about 160 mM, about 0.1 mM to about 140 mM, about 0.1 mM to about 120 mM, about 0.1 mM to about 100 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 60, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 1 mM to about 100 mM, about 5 mM to about 100 mM, about 100 mM to about 300 mM or about 60 mM to about 240 mM. In certain embodiments, the composition includes from about 10 mM to about 300 mM of an activator, e.g. , MgOAc. In certain embodiments, the composition includes from about 15 mM to about 250 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes from about 10 mM to about 100 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes from about 1 mM to about 100 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes from about 1 mM to about 20 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes from about 100 mM to about 300 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes about 1 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes about 20 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes about 40 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes about 80 mM of an activator, e.g., MgOAc. In certain embodiments, the composition includes about 160 mM of an activator, e.g., MgOAc.

[0112] In certain embodiments, the composition includes from about 10 mM to about 250 mM of an activator, e.g., MgOAc.

[0113] In certain embodiments, the composition includes from about 10 mM to about 230 mM of an activator, e.g., MgOAc.

[0114] In certain embodiments, the composition includes from about 100 mM to about 250 mM of an activator, e.g., MgOAc.

[0115] In certain embodiments, the composition includes about 224 mM of an activator, e.g., MgOAc.

[0116] In certain embodiments, a composition of the present disclosure includes THPP and MgOAc at the ratios and amounts described herein. In certain embodiments, the present disclosure provides a composition consisting essentially of THPP and MgOAc at the ratios and amounts described herein. In certain embodiments, the present disclosure provides a composition consisting of THPP and MgOAc at the ratios and amounts described herein.

[0117] In certain embodiments, a composition of the present disclosure includes THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, a composition of the present disclosure includes THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1:3 or about 1 :0.25 to about 1 :3), where THPP is present in the composition at a concentration from about 1 mM to about 200 mM and MgOAc is present in the composition at a concentration from about 0.1 mM to about 500 mM. In certain embodiments, a composition of the present disclosure includes THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3), where THPP is present in the composition at a concentration from about 1 mM to about 100 mM and MgOAc is present in the composition at a concentration from about 10 mM to about 300 mM.

[0118] In certain embodiments, a composition of the present disclosure includes THPP and MgOAc at a ratio from about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM.

[0119] In certain embodiments, a composition of the present disclosure includes THPP and MgOAc at a ratio from about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 1 mM to about 10 mM and MgOAc is present in the composition at a concentration from about 10 mM to about 30 mM.

[0120] In certain embodiments, the present disclosure provides a composition consisting essentially of THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, a composition of the present disclosure consists essentially of THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3), where THPP is present in the composition at a concentration from about 1 mM to about 200 mM and MgOAc is present in the composition at a concentration from about 0.1 mM to about 500 mM. In certain embodiments, a composition of the present disclosure consists essentially of THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3), where THPP is present in the composition at a concentration from about 1 mM to about 100 mM and MgOAc is present in the composition at a concentration from about 10 mM to about 300 mM.

[0121] In certain embodiments, a composition of the present disclosure consists essentially of THPP and MgOAc at a ratio from about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM.

[0122] In certain embodiments, a composition of the present disclosure consists essentially of THPP and MgOAc at a ratio from about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 1 mM to about 10 mM and MgOAc is present in the composition at a concentration from about 10 mM to about 30 mM.

[0123] In certain embodiments, the present disclosure provides a composition consisting of THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3). In certain embodiments, a composition of the present disclosure consists of THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3), where THPP is present in the composition at a concentration from about 1 mM to about 200 mM and MgOAc is present in the composition at a concentration from about 0.1 mM to about 500 mM. In certain embodiments, a composition of the present disclosure consists of THPP and MgOAc at a ratio from about 1 :0.01 to about 1 :5 (e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3), where THPP is present in the composition at a concentration from about 1 mM to about 100 mM and MgOAc is present in the composition at a concentration from about 10 mM to about 300 mM.

[0124] In certain embodiments, a composition of the present disclosure consists of THPP and MgOAc at a ratio from about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM.

[0125] In certain embodiments, a composition of the present disclosure consists of THPP and MgOAc at a ratio from about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 1 mM to about 10 mM and MgOAc is present in the composition at a concentration from about 10 mM to about 30 mM.

[0126] In certain embodiments, the composition further includes a buffer. In certain embodiments, the buffer can be an ionic buffer. In certain embodiments, the buffer comprises a Tris buffer. In certain embodiments, the buffer can be a zwitterionic buffer. In certain embodiments, the buffer comprises a MES buffer. In certain embodiments, the buffer comprises a HEPES buffer. In certain embodiments, the buffer comprises a MES buffer. In certain embodiments, the buffer comprises a MOPS buffer. In certain embodiments, the buffer comprises sodium acetate. In certain embodiments, the buffer comprises N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).

[0127] In certain embodiments, the composition includes a buffer at a concentration of about 1 to about 100 mM, e.g., about 1 to about 75 mM, about 1 to about 50 mM or about 1 to about 25 mM. In certain embodiments, a composition of the present disclosure includes a 10 mM Tris buffer.

[0128] In certain embodiments, the composition has a pH from about 5 to about 11, e.g., from about 5 to about 10, from about 6 to about 11, from about 6 to about 10, from about 6 to about 9, from about 7 to about 9 or from about 8 to about 9. In certain embodiments, the composition has a pH of about 8.3. In certain embodiments, the composition has a pH of about 8.5.

[0129] In certain embodiments, a composition of the present disclosure includes a reducing agent, an activator and a buffer. In certain embodiments, the present disclosure provides a composition consisting essentially of a reducing agent, an activator and a buffer. In certain embodiments, the present disclosure provides a composition consisting of a reducing agent, an activator and a buffer.

[0130] In certain embodiments, a composition of the present disclosure includes a trialkylphosphine (e.g., THPP), a magnesium or a salt thereof and a buffer. In certain embodiments, the present disclosure provides a composition consisting essentially of a trialkylphosphine (e.g., THPP), a magnesium or a salt thereof and a buffer. In certain embodiments, the present disclosure provides a composition consisting of a trialkylphosphine (e.g., THPP), a magnesium or a salt thereof and a buffer.

[0131] In certain embodiments, a composition of the present disclosure includes THPP, MgOAc and a buffer. In certain embodiments, the present disclosure provides a composition consisting essentially of THPP, MgOAc and a buffer. In certain embodiments, the present disclosure provides a composition consisting of THPP, MgOAc and a buffer.

[0132] In certain embodiments, the composition is stored at a temperature from about -20°C to about 40°C. In certain embodiments, the composition is stored at a temperature from about 2°C to about 40°C, e.g., about 20°C to about 40°C. In certain embodiments, the composition is stored at a temperature from about 2°C to about 40°C, e.g., about 20°C to about 40°C.

[0133] In certain embodiments, a composition described herein is stored for a 2-day period, a 3-day period, a 4-day period, a 5-day period, a 6-day period, a 7-day period, a 10- day period, a 15-day period, a 18-day period, a 25-day period, a 39-day period, a 49-day period, a 50-day period, a 100-day period, a 150-day period, a 200-day period, a 250-day period or a 270 day period prior to use in an isothermal amplification process of the present disclosure. In certain embodiments, a composition described herein is stored for a time period greater than about 2 days, greater than about 5 days, greater than about 10 days, greater than about 15 days, greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 50 days, greater than about 100 days, greater than about 150 days, greater than about 200 days or greater than about 250 days prior to use in an isothermal amplification process of the present disclosure.

[0134] In certain embodiments, a composition described herein is stored for a 2-day period, a 3-day period, a 4-day period, a 5-day period, a 6-day period, a 7-day period, a 10- day period, a 15-day period, a 18-day period, a 25-day period, a 39-day period, a 49-day period, a 50-day period, a 100-day period, a 150-day period, a 200-day period, a 250-day period or a 270 day period at a temperature from about 2°C to about 40°C prior to use in an isothermal amplification process of the present disclosure. In certain embodiments, a composition described herein is stored for a time period greater than about 2 days, greater than about 5 days, greater than about 10 days, greater than about 15 days, greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 50 days, greater than about 100 days, greater than about 150 days, greater than about 200 days or greater than about 250 days at a temperature from about 2°C to about 40°C prior to use in an isothermal amplification process of the present disclosure.

[0135] In certain embodiments, the composition is freeze-dried, e.g., lyophilized. In certain embodiments, the composition is resuspended in a buffer (e.g., described herein) prior to use in an isothermal amplification process of the present disclosure.

[0136] In certain embodiments, the present disclosure can provide an additional composition that includes other components useful for performing an isothermal amplification. For example, but not by way of limitation, the present disclosure can provide an additional composition, e.g., a second composition, that includes one or more enzymes for performing an isothermal amplification and / or one or more probes or primers for performing an isothermal amplification. In certain embodiments, the present disclosure can provide an additional composition, e.g., a second composition, that includes one or more of the following (in any combination):

[0137] In certain embodiments, the present disclosure can provide an additional composition, e.g., a second composition, that includes 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 crowding agent, ATP or an ATP analog, at least one recombinase loading protein, a first primer and, optionally, a second primer, a probe, a creatine kinase, a nuclease and / or a reverse transcriptase. In certain embodiments, the second composition of the present disclosure can include a recombinase, a singlestranded binding protein, a polymerase, dNTPs, ATP and / or a primer. In certain embodiments, a composition of the present disclosure, e.g., a second composition, does not include a reverse transcriptase.

[0138] In certain embodiments, an isothermal amplification process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5. In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :3, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM. In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1:5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM. In certain embodiments, the one or more isothermal amplification reagents can be any one of or a combination of any of the reagents disclosed herein, e.g., reagents required for performing an RPA process. Non-limiting examples of such reagents include a recombinase, a single-stranded DNA binding protein, a recombinase loading protein, a creatine kinase, a nuclease, a reverse transcriptase a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a first primer, a second primer and a probe.

[0139] In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :3, where the reducing agent is present in the composition at a concentration from about 50 mM to about 150 mM and the activator is present in the composition at a concentration from about 100 mM to about 300 mM, to obtain a reaction concentration of 1 mM to about 20 mM of the reducing agent and a reaction concentration of about 5 mM to about 25 mM of the activator. In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5, where the reducing agent is present in the composition at a concentration from about 50 mM to about 150 mM and the activator is present in the composition at a concentration from about 100 mM to about 300 mM, to obtain a reaction concentration of 1 mM to about 20 mM of the reducing agent and a reaction concentration of about 5 mM to about 25 mM of the activator. In certain embodiments, the one or more isothermal amplification reagents can be any one of or a combination of any of the reagents disclosed herein, e.g., reagents required for performing an RPA process. Non-limiting examples of such reagents include a recombinase, a single-stranded DNA binding protein, a recombinase loading protein, a creatine kinase, a nuclease, a reverse transcriptase a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a first primer, a second primer and a probe.

[0140] In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising THPP and magnesium or a salt form thereof at a concentration ratio of about 1 : 1 to about 1 :3, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and the magnesium or salt form thereof is present in the composition at a concentration from about 100 mM to about 300 mM, to obtain a reaction concentration of 1 mM to about 20 mM of THPP and a reaction concentration of about 5 mM to about 25 mM of the magnesium or salt form thereof. In certain embodiments, an isothermal amplification process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition disclosed herein, e.g., a composition comprising THPP and a magnesium or a salt form thereof at a concentration ratio of about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and the magnesium or salt form thereof is present in the composition at a concentration from about 100 mM to about 300 mM, to obtain a reaction concentration of 1 mM to about 20 mM of THPP and a reaction concentration of about 5 mM to about 25 mM of the magnesium or salt form thereof. In certain embodiments, the one or more isothermal amplification reagents can be any one of or a combination of any of the reagents disclosed herein, e.g., reagents required for performing an RPA process. Non-limiting examples of such reagents include a recombinase, a single-stranded DNA binding protein, a recombinase loading protein, a creatine kinase, a nuclease, a reverse transcriptase a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a first primer, a second primer and a probe.

[0141] In certain embodiments, a portion of a composition of the present disclosure is added to an isothermal amplification process (e.g., an RPA process) to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent. For example, but not by way of limitation, a portion of a composition of the present disclosure is added to an isothermal amplification process (e.g., an RPA process) to obtain a reaction concentration of about 1 mM to about 15 mM of the reducing agent, about 1 mM to about 10 mM of the reducing agent, about 1 mM to about 9 mM of the reducing agent, about 1 mM to about 8 mM of the reducing agent, about 1 mM to about 7 mM of the reducing agent, about 1 mM to about 6 mM of the reducing agent, about 1 mM to about 5 mM of the reducing agent, about 2 mM to about 10 mM of the reducing agent, about 3 mM to about 10 mM of the reducing agent, about 4 mM to about 10 mM of the reducing agent, about 5 mM to about 10 mM of the reducing agent, about 3 mM to about 9 mM of the reducing agent or about 4 mM to about 5 mM of the reducing agent. In certain embodiments, a portion of a composition of the present disclosure is added to an isothermal amplification process (e.g., an RPA process) to obtain a reaction concentration of about 5 mM or about 8 mM of the reducing agent. In certain embodiments, a portion of a composition of the present disclosure is added to an isothermal amplification process (e.g., an RPA process) to obtain a reaction concentration of about 4 mM or about 8 mM of the reducing agent, e.g., THPP. For example, but not by way of limitation, about 1%, about 2%, 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 a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent, e.g., about 5 mM or about 8 mM of the reducing agent. In certain embodiments, about 2% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent, e.g., about 5 mM or about 8 mM of the reducing agent. In certain embodiments, about 5% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent, e.g., about 5 mM or about 8 mM of the reducing agent. In certain embodiments, about 2% to about 10% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent, e.g., about 5 mM or about 8 mM of the reducing agent. In certain embodiments, about 2% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 4 mM to about 8 mM of the reducing agent. In certain embodiments, about 5% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 4 mM to about 8 mM of the reducing agent. In certain embodiments, about 2% to about 10% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 4 mM to about 8 mM of the reducing agent.

[0142] In certain embodiments, a portion of a composition of the present disclosure is added to a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent. For example, but not by way of limitation, a portion of a composition of the present disclosure is added to a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 1 mM to about 15 mM of the reducing agent, about 1 mM to about 10 mM of the reducing agent, about 1 mM to about 9 mM of the reducing agent, about 1 mM to about 8 mM of the reducing agent, about 1 mM to about 7 mM of the reducing agent, about 1 mM to about 6 mM of the reducing agent, about 1 mM to about 5 mM of the reducing agent, about 2 mM to about 10 mM of the reducing agent, about 3 mM to about 10 mM of the reducing agent, about 4 mM to about 10 mM of the reducing agent, about 5 mM to about 10 mM of the reducing agent, about 3 mM to about 9 mM of the reducing agent or about 4 mM to about 5 mM of the reducing agent. In certain embodiments, a portion of a composition of the present disclosure is added to a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 5 mM or about 8 mM of the reducing agent.

[0143] In certain embodiments, a composition of the present disclosure is contacted with a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 1 mM to about 20 mM of the reducing agent. For example, but not by way of limitation, a composition of the present disclosure is contacted with a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 1 mM to about 15 mM of the reducing agent, about 1 mM to about 10 mM of the reducing agent, about 1 mM to about 9 mM of the reducing agent, about 1 mM to about 8 mM of the reducing agent, about 1 mM to about 7 mM of the reducing agent, about 1 mM to about 6 mM of the reducing agent, about 1 mM to about 5 mM of the reducing agent, about 2 mM to about 10 mM of the reducing agent, about 3 mM to about 10 mM of the reducing agent, about 4 mM to about 10 mM of the reducing agent, about 5 mM to about 10 mM of the reducing agent, about 3 mM to about 9 mM of the reducing agent or about 4 mM to about 5 mM of the reducing agent. In certain embodiments, a composition of the present disclosure is contacted with a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 5 mM or about 8 mM of the reducing agent.

[0144] In certain embodiments, a portion of a composition of the present disclosure is added to an isothermal amplification process (e.g., an RPA process) to obtain a reaction concentration of about 5 mM to about 25 mM of the activator. For example, but not by way of limitation, a portion of a composition of the present disclosure is added to an isothermal amplification process (e.g., an RPA process) to obtain a reaction concentration of about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 11 mM to about 15 mM, about 12 mM to about 15 mM, about 13 mM to about 15 mM or about 12 to about 16 mM of the activator. For example, but not by way of limitation, about 2%, 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 a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 5 mM to about 25 mM of the activator. In certain embodiments, about 2% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 5 mM to about 25 mM of the activator, e.g., about 12 mM or about 14 mM of the activator. In certain embodiments, about 5% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 5 mM to about 25 mM of the activator, e.g., about 12 mM or about 14 mM of the activator. In certain embodiments, about 2% to about 10% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 5 mM to about 25 mM of the activator, e.g., about 12 mM or about 14 mM of the activator. In certain embodiments, about 2% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 10 mM or about 20 mM of the activator. In certain embodiments, about 5% to about 20% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 10 mM or about 20 mM of the activator. In certain embodiments, about 2% to about 10% of a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 10 mM or about 20 mM of the activator.

[0145] In certain embodiments, a portion of a composition of the present disclosure is added to a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 5 mM to about 25 mM of the activator. For example, but not by way of limitation, a portion of a composition of the present disclosure is added to a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 11 mM to about 15 mM, about 12 mM to about 15 mM, about 13 mM to about 15 mM or about 12 to about 16 mM of the activator. For example, but not by way of limitation, about 2%, 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 a composition of the present disclosure is added to an isothermal amplification process to obtain a reaction concentration of about 5 mM to about 25 mM of the activator. In certain embodiments, a portion of a composition of the present disclosure is added to a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 10 mM to about 20 mM of the activator.

[0146] In certain embodiments, a composition of the present disclosure is contacted with a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 5 mM to about 25 mM of the activator. For example, but not by way of limitation, a composition of the present disclosure is contacted with a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 11 mM to about 15 mM, about 12 mM to about 15 mM, about 13 mM to about 15 mM or about 12 to about 16 mM of the activator. For example, but not by way of limitation, a composition of the present disclosure is contacted with an isothermal amplification process to obtain a reaction concentration of about 5 mM to about 25 mM of the activator. In certain embodiments, a composition of the present disclosure is contacted with a reagent composition including one or more other reagents (e.g., enzymes) for performing an isothermal amplification process and / or a sample comprising target nucleic acids to obtain a reaction concentration of about 10 mM to about 20 mM of the activator.

[0147] B. RPA Process

[0148] In certain embodiments, the isothermal amplification process is RPA. RPA relies on the properties of recombinase and related protein components to invade doublestranded nucleic acids with single stranded homologous nucleic acids permitting sequence specific priming of nucleic acid polymerase reactions.

[0149] RPA amplification reactions exploit enzymes known as recombinases, which form complexes with oligonucleotide primers and pair the primers with their homologous sequences in duplex nucleic acids. A single-stranded nucleic acid binding (SSB) protein binds to the displaced nucleic acid strand and stabilizes the resulting loop. Nucleic acid amplification is then initiated from the primer, but only if the target sequence is present. Once initiated, the amplification reaction progresses rapidly, so that starting with just a few target copies of nucleic acid, the highly specific amplification reaches detectable levels within minutes.

[0150] In certain embodiments, in a first step, a first and a second single stranded nucleic acid primer is contacted with a recombinase (e.g., UvsX), a recombinase loading agent (e.g., UvsY) and a single strand DNA binding protein (e.g., gp32) to form a first and a second nucleoprotein primer. The single stranded nucleic acid primers are specific for and are complementary to the target nucleic acid molecule. In the second step, the first nucleoprotein primer is contacted to the double stranded target nucleic acid molecule to create a first D loop structure at a first portion of the double stranded target nucleic acid molecule (Step 2a). Further, the second nucleoprotein primer is contacted to the double stranded target nucleic acid molecule to create a second D loop structure at a second portion of the double stranded target nucleic acid molecule (Step 2b). The D loop structures are formed such that the 3’ ends of the first nucleic acid primer and said second nucleic acid primer 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 Step 2a and Step 2b can be performed in any order or simultaneously.

[0151] In a D loop structure, the primer is hybridized to 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 displaced by the primer. The structure resembles a capital D where the straight part of the D represents the double stranded part of the structure and the curved part of the D represents the single stranded displaced second strand of the target nucleic acid.

[0152] In the third step, the 3’ end of the first and the second nucleoprotein primer is extended with one or more polymerases capable of strand displacement synthesis and dNTPs to generate a first and second double stranded target nucleic acid molecule and a first and second displaced strand of nucleic acid. The first and second double stranded target nucleic acid molecules may serve as target nucleic acid molecules in step two during subsequent rounds of amplification.

[0153] Steps two and step three are repeated until a desired degree of amplification of the target nucleic acid is achieved.

[0154] During the amplification process described above, the first and second displaced strand of nucleic acid may hybridize to each other after Step (c) to form a third double stranded target nucleic acid molecule.

[0155] In certain embodiments, the present disclosure provides an RPA process that includes the use of a composition comprising a reducing agent and an activator at the ratios and amounts described herein. For example, but not by way of limitation, the present disclosure provides an RPA process that is initiated by a composition that includes the reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 :0.01 to about 1 :3 or about 1 :0.25 to about 1 :3. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes the reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1 : 1 to about 1 :5. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes the reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1 : 1 to about 1 :3. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes the reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1 :2 to about 1 :3. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes about 1 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes about 10 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes about 0.01 mM to about 300 mM of an activator. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes about 0.01 mM to about 250 mM of an activator, e.g., about 0.1 mM to about 250 mM of an activator. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes about 10 mM to about 250 mM of an activator.

[0156] In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes the reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1 :1 to about 1:5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM.

[0157] In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes a reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1 : 1 to about 1 :5, where the reducing agent (e.g., THPP) is present in the composition at a concentration from about 50 mM to about 150 mM and the activator (e.g., MgOAc) is present in the composition at a concentration from about 100 mM to about 300 mM, and where the reducing agent (e.g., THPP) is present in the RPA process at a concentration from about 1 mM to about 10 mM and the activator (e.g., MgOAc) is present in the RPA process at a concentration from about 10 mM to about 30 mM.

[0158] In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes THPP and MgOAc at a concentration ratio of about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM, and where THPP is present in the RPA process at a concentration from about 1 mM to about 10 mM and MgOAc is present in the RPA process at a concentration from about 10 mM to about 30 mM. In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes a reducing agent (e.g., THPP) and an activator (e.g., MgOAc) at a concentration ratio of about 1:2 to about 1 :3, where the reducing agent (e.g., THPP) is present in the composition at a concentration from about 50 mM to about 150 mM and the activator e.g., MgOAc) is present in the composition at a concentration from about 100 mM to about 300 mM, and where the reducing agent e.g., THPP) is present in the RPA process at a concentration from about 4 mM to about 7 mM and the activator e.g., MgOAc) is present in the RPA process at a concentration from about 12 mM to about 18 mM.

[0159] In certain embodiments, the present disclosure provides an RPA process that is initiated by a composition that includes THPP and MgOAc at a concentration ratio of about 1 :2 to about 1 :3, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and MgOAc is present in the composition at a concentration from about 100 mM to about 300 mM, and where THPP is present in the RPA process at a concentration from about 4 mM to about 7 mM and MgOAc is present in the RPA process at a concentration from about 12 mM to about 18 mM.

[0160] In certain embodiments, the RPA process further includes the use of a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP, a primer and a template nucleic acid (e.g., target nucleic acid). In certain embodiments, an RPA process can 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, at least one nuclease, at least one creatine kinase, at least one recombinase loading protein, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a first primer and, optionally, a second primer, a probe and a template nucleic acid molecule, e.g., a singlestranded (e.g., RNA) or double stranded target nucleic acid. In certain embodiments, the RPA reaction can contain, e.g., a reverse transcriptase. In certain embodiments, the RPA reaction does not include a reverse transcriptase.

[0161] In certain embodiments, an RPA process of the present disclosure includes combining non-protein components (NPC) (e.g., a reaction buffer, dNTPs, ATP and salts), protein components (PC) that include the enzymes required for the RPA process (e.g., a DNA polymerase, a recombinase, a recombinase loading protein, a single stranded binding protein, creatine kinase, a nuclease (e.g., an exonuclease) and / or a reverse transcriptase) and oligonucleotide components (OC) (e.g., one or more primers and / or one or more probes specific to a target nucleic acid or two or more target nucleic acids e.g. , in a multiplex RPA process)) with the sample containing nucleic acids (e.g., the nucleic acids can include the target nucleic acid(s)). In certain embodiments, these components can be added in any order to the sample, or the NPC, PC and / or OC can be included in a “master mix” that is then added to the sample. In certain embodiments, an activator, e.g., magnesium (e.g., as magnesium acetate (MgOAc)), is then added to the reaction, followed by the incubation of the reaction at a specified temperature, e.g., 40°C, to amplify the target nucleic acid(s). As described herein, a reducing agent, e.g., THPP, is included in the solution containing the activator. For example, but not by way of limitation, the activator solution includes a reducing agent, e.g., THPP, and an activator, e.g., MgOAc, at the ratios and / or amounts described herein.

[0162] Alternatively, in certain embodiments, an RPA process of the present disclosure includes combining non-protein components (NPC) (e.g., a reaction buffer, dNTPs, ATP and salts), protein components (PC) that include the enzymes required for the RPA process (e.g., a DNA polymerase, a recombinase, a recombinase loading protein, a single stranded binding protein, creatine kinase, a nuclease (e.g., an exonuclease) and / or a reverse transcriptase) and oligonucleotide components (OC) (e.g., one or more primers and / or one or more probes specific to a target nucleic acid or two or more target nucleic acids (e.g., in a multiplex RPA process)) with an activator composition of the present disclosure. As described herein, a reducing agent, e.g., THPP, is included in the composition containing the activator. In certain embodiments, these components can be added in any order to the activator composition, or the NPC, PC and / or OC can be included in a “master mix” that is then added to the activator composition. In certain embodiments, a sample containing nucleic acids (e.g., the nucleic acids can include the target nucleic acid(s)) is then added to the reaction, followed by the incubation of the reaction at a specified temperature, e.g., 40°C, to amplify the target nucleic acid(s).

[0163] In certain embodiments, the recombinase (e.g., UvsX), recombinase loading agent (e.g., UvsY) and single strand DNA binding protein (e.g., gp32) can be derived from a myoviridae phage. In certain embodiments, the myoviridae phage can be, for example, T4, T2, T6, Rb69, Aehl, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rbl4, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rbl6, Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3 or phage LZ2. In certain embodiments, the combination of Rb69 UvsX, Rb69 UvsY and Rb69 gp32 can be used. In certain embodiments, the combination of Aehl UvsX, Aehl UvsY and Rb69 gp32 can be used. In certain embodiments, the combination of T4 UvsX, T4 UvsY and Rb69 gp32 can be used. In certain embodiments, the combination of T4 UvsX, Rb69 UvsY and T4 gp32 can be used.

[0164] In certain embodiments, the recombinase (e.g., UvsX), recombinase loading agent (e.g., UvsY) and single strand DNA binding protein (e.g., gp32) can each be native, hybrid or mutant proteins from the same or different myoviridae phage sources. A native protein can be a wildtype or natural variant of a protein. A mutant protein (also called a genetically engineered protein) is a native protein with natural or manmade mutations such as insertions, deletions, substitutions, or a combination thereof, that are at the N terminus, C terminus, or interior (between the N terminus and the C terminus). A hybrid protein (also called a chimeric protein) comprises sequences from at least two different organisms. For example, but not by way of limitation, a hybrid UvsX protein can contain an amino acid from one species (e.g., T4) but a DNA binding loop from another species (e.g., T6). The hybrid protein can contain improved characteristics compared to a native protein. The improved characteristics can be increased or more rapid RPA amplification rate or a decreased or more controllable RPA amplification rate.

[0165] In certain embodiments, the recombinase (e.g., UvsX) can be a mutant UvsX. In certain embodiments, the mutant UvsX is an Rb69 UvsX comprising at least one mutation in the Rb69 UvsX amino acid sequence, wherein the mutation is selected from the group consisting of (a) an amino acid which is not histidine at position 64, a serine at position 64, the addition of one or more glutamic acid residues at the C-terminus, the addition of one or more aspartic acid residues at the C-terminus, and a combination thereof. In certain embodiments, the mutant UvsX is a T6 UvsX having at least one mutation in the T6 UvsX amino acid sequence, wherein the mutation is selected from the group consisting of (a) an amino acid which is not histidine at position 66; (b) a serine at position 66; (c) the addition of one or more glutamic acid residues at the C-terminus; (d) the addition of one or more aspartic acid residues at the C-terminus; and (e) a combination thereof.

[0166] In certain embodiments, the RPA processes of the present disclosure are performed with a polymerase which is a large fragment polymerase. In certain embodiments, the large fragment polymerase can be selected from the group consisting of E. Coli Pol I, Bacillus subtilis Pol I, Staphylococcus aureus Pol I and homologs thereof. In certain embodiments, the RPA processes are performed in the presence of about 0.01 mg / mL to about 0.5 mg / mL of a DNA Polymerase, e.g., about 0.08 mg / mL to about 0.2 mg / mL of a DNA Polymerase. In certain embodiments, the RPA processes are performed in the presence of about 10 units / mL to about 10,000 units / mL of a DNA Polymerase, e.g., about 500 units / mL to about 5,000 units / mL of a DNA Polymerase.

[0167] In certain embodiments, the RPA processes are performed in the presence of heparin. Heparin can serve as an agent to reduce the level of non-specific primer noise, and to increase the ability of E. coli exonuclease III or E. coli exonuclease IV to rapidly polish 3’ blocking groups or terminal residues from recombination intermediates.

[0168] In certain embodiments, the dNTPs for use in the RPA processes of the present disclosure include, for example, dATP, dGTP, dCTP and dTTP. In certain embodiments, the ddNTPs for use in the RPA processes of the present disclosure include, for example, ddATP, ddTTP, ddGTP and ddGTP. In certain embodiments, dNTPs and / or ddNTPs can be used at concentrations of about 1 pM to about 500 pM per each dNTP and / or ddNTP species.

[0169] In certain embodiments, the RPA processes are performed with a blocked primer. A blocked primer is a primer which does not allow elongation with a polymerase. Where a blocked primer is used, an unblocking agent is also used to unblock the primer to allow elongation. The unblocking agent can be an endonuclease or exonuclease which can cleave the blocking group from the primer. In certain embodiments, unblocking agents include E. coli exonuclease III and E. coli endonuclease IV. In certain embodiments, the unblocking agent is E. coli exonuclease III. In certain embodiments, the unblocking agent is E. coli endonuclease IV.

[0170] In certain embodiments, the RPA processes are performed in the presence of two or more primers, e.g., (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 certain embodiments, the RPA processes are performed in the presence of at least three primers. In certain embodiments, the RPA processes are performed in the presence of at least two probes. In certain embodiments, the RPA processes are performed in the presence of about 1 nM to about 1,000 nM of one or more primers and / or probes, e.g., about 10 nM to about 500 nM of one or more primers and / or probes. In certain embodiments, the RPA processes are performed in the presence of about 1 nM to about 1,000 nM of one or more primers, e.g., about 10 nM to about 500 nM of one or more primers. In certain embodiments, the RPA processes are performed in the presence of about 1 nM to about 1,000 nM of a forward primer, e.g., about 10 nM to about 500 nM of a forward primer. In certain embodiments, the RPA processes are performed in the presence of about 1 nM to about 1,000 nM of a reverse primer, e.g., about 10 nM to about 500 nM of a reverse primer. In certain embodiments, the RPA processes are performed in the presence of about 1 nM to about 1,000 nM of one or more probes, e.g., about 10 nM to about 500 nM of one or more probes, e.g., detection probes.

[0171] In certain embodiments, only one of the nucleic acid primers used in the RPA processes of the present disclosure is coated with recombinase / recombinase loading agent / single stranded DNA binding protein. That is, an RPA can be performed with one primer which is uncoated and one primer which is coated with any one or a combination of recombinase, recombinase loading agent and single stranded DNA binding protein.

[0172] In certain embodiments, the recombinase loading agent (e.g., UvsY) is omitted. That is, any of the RPA reactions of this disclosure can be performed in the absence of the recombinase loading agent (e.g., UvsY).

[0173] In certain embodiments, an RPA process of the present disclosure can be employed using RNA as an initial template, e.g., to amplify a target nucleic acid derived from an RNA virus, by using reverse transcriptase to first produce a DNA copy of the RNA template after which the DNA copy can be subjected to RPA-based nucleic acid amplification. Performing RPA with RNA templates is typically referred to in the art as Reverse Transcriptase RPA or RT-RPA. In certain embodiments, the reverse transcriptase used in the methods of the present disclosure can be selected from: OmniScript (Qiagen), Sensi Script (Qiagen), MonsterScript (Epicentre), Transcriptor (Roche), HIV RT (Ambion), Superscript III (Invitrogen), ThermoScript (Invitrogen), Thermo-X (Invitrogen), ImProm II (Promega) and EIAV-RT. In certain embodiments, the reverse transcriptase is EIAV-RT.

[0174] In certain embodiments, the reverse transcriptase can be omitted from the RPA reaction. For example, but not by way of limitation, any of the RPA reactions of the present disclosure can be performed in the absence of a reverse transcriptase. In certain embodiments, an RPA reaction of the present disclosure is performed in the absence of a reverse transcriptase if the target nucleic acid to be analyzed is DNA.

[0175] In certain embodiments, the ATP or analog thereof can be used at a concentration of about 1 and about 10 mM. Non-limiting examples of an ATP analog include ATP-y-S, ATP-P-S and ddATP.

[0176] In certain embodiments, the RPA process is performed in the presence of a crowding agent. The crowding agent can be selected from the group comprising polyethylene glycol (e.g., PEG1450, PEG3000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000, PEG30000, PEG35000, PEG40000 and / or a PEG compound with molecular weight between 15,000 and 20,000 daltons), polyethylene oxide (PEO), polyvinyl alcohol, polystyrene, Ficoll, dextran, PVP, albumin, trehalose and a combination thereof. In certain embodiments, the crowding agent has a molecular weight of less than 200,000 daltons. In certain embodiments, the crowding agent may be present in the reaction in an amount of about 0.5% to about 15% weight to volume (w / v). In certain embodiments, the crowding agent can be present in the reaction in an amount of about 1% to about 10% w / v.

[0177] In certain embodiments, the following reagents, in addition to the reducing agent and the activator, can be employed for performing an RPA process of the present disclosure: Tris-HCl, Potassium Acetate, a crowding agent, dNTPs, ATP, Phosphocreatine, Glycerol, Creatine Kinase, UvsX, UvsY, DNA polymerase, GP32, Exonuclease III, BSA and EIAV. In certain embodiments, additional reagents can be employed, including but not limited to, forward primers, reverse primers, probes and ROX reference dyes.

[0178] In certain embodiments, about 5 mM to about 100 mM Tris-HCl at a pH of about 6.5-9.0, e.g., 8.3, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 50 mM to about 100 mM potassium acetate can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 1 mMto about 5 mM dNTPs can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 1 mM to about 10 mM of ATP, e.g., about 2 mM to about 5 mM ATP, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 20 mM to about 100 mM Phosphocreatine, e.g, about 40 mM to about 100 mM Phosphocreatine, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.01 mg / mL to about 10 mg / mL BSA can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 5% to about 10% Glycerol can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.01 mg / mL to about 0.5 mg / mL Creatine Kinase, e.g., about 0.1 mg / mL to about 0.5 mg / mL Creatine Kinase, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 1% to about 10% w / v of a crowding agent can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.1 mg / mL to about 1.0 mg / mL UvsX, e.g., about 0.3 mg / mL to about 1.0 mg / mL UvsX, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.01 mg / mL to about 0.25 mg / mL UvsY, e.g., about 0.09 mg / mL to about 0.25 mg / mL UvsY, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.01 mg / mL to about 0.5 mg / mL DNA Polymerase, e.g., about 0.08 mg / mL to about 0.2 mg / mL DNA Polymerase, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.1 mg / mL to about 2.0 mg / mL GP32, e.g., about 0.4 mg / mL to about 0.8 mg / mL GP32, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.01 mg / mL to about 0.5 mg / mL Exonuclease III, e.g., about 0.1 mg / mL to about 0.5 mg / mL Exonuclease III, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure. In certain embodiments, about 0.5 pg / mL to about 100.0 pg / mL equine infectious anemia virus reverse transcriptase (EIAV-RT), e.g., about 0.5 pg / mL to about 1.5 pg / mL, about 1.5 pg / mL to about 20 pg / mL or about 20 pg / mL to about 70 pg / mL EIAV-RT, can be employed in an RPA process of the present disclosure or included in a composition (e.g., a second composition) of the present disclosure.

[0179] In certain embodiments, the following reagents, in addition to the reducing agent and the activator, can be employed at the following concentrations (or included in a reagent composition (e.g., a second reagent composition) of the present disclosure) for performing an RPA process of the present disclosure: about 5 mM to about 100 mM Tris- HC1 at pH of about 6.5-9.0, e.g., 8.3; about 5 mM to about 10 mM of a 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 of ATP, e.g., about 2 mM to about 5 mM ATP; about 20 mM to about 100 mM Phosphocreatine, e.g., about 40 mM to about 100 mM Phosphocreatine; about 5 mM to about 40 mM Mg Acetate, e.g., about 10 mM to about 40 mM Mg 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 Creatine Kinase, e.g., about 0.1 mg / mL to about 0.5 mg / mL Creatine Kinase; about 1% to about 10% w / v of a crowding agent (e.g., PEG); about 0.1 mg / mL to about 1.0 mg / mL UvsX, e.g., about 0.3 mg / mL to about 1.0 mg / mL UvsX; about 0.01 mg / mL to about 0.25 mg / mL UvsY, e.g., about 0.09 mg / mL to about 0.25 mg / mL UvsY; about 0.01 mg / mL to about 0.5 mg / mL DNA Polymerase, e.g., about 0.08 mg / mL to about 0.2 mg / mL DNA Polymerase; about 0.1 mg / mL to about 2.0 mg / mL GP32, e.g., about 0.4 mg / mL to about 0.8 mg / mL GP32; about 0.01 mg / mL to about 0.5 mg / mL Exonuclease III, e.g., about 0.1 mg / mL to about 0.5 mg / mL Exonuclease III; and about 0.5 pg / mL to about 100.0 pg / mL equine infectious anemia virus reverse transcriptase (EIAV-RT), e.g., about 0.5 pg / mL to about 1.5 pg / mL EIAV-RT, about 1.5 pg / mL to about 20 pg / mL or about 20 pg / mL to about 70 pg / mL EIAV-RT. In certain embodiments, additional reagents can be employed, including but not limited to, forward primers, reverse primers, probes and ROX reference dyes.

[0180] In certain embodiments, a singleplex RPA reaction for amplifying and detecting a single target nucleic acid, e.g., from a pathogen or infectious agent (e.g., HIV-1, HIV-2, HCV or HBV), can comprise the following reagents, in addition to the reducing agent and the activator, employed at the following concentrations (or included in a reagent composition (e.g., a second reagent composition) of the present disclosure): Tris-HCl IM, pH 8.3 at 50 mM, Potassium Acetate at 100 mM, 20% Polyethylene glycol at 5.5%, 100 mM dNTPs at 1.8 mM, ATP at 2.5 mM, Phosphocreatine at 50 mM, Forward Primer at 420 nM, Reverse Primer at 420 nM, Exo Probe at 120 nM, ROX reference dye at 15 nM, Glycerol at 6.5%, Creatine Kinase at 0.1 mg / ml, UvsX at 0.3 mg / ml, UvsY at 0.09 mg / ml, DNA Polymerase at 0.0798 mg / ml, Gp32 at 0.48 mg / ml, Exonuclease III at 0.1 mg / ml, Bovine Serum Albumin (BSA) at 0.02 mg / ml and EIAV Reverse Transcriptase at 0.0008 mg / ml.

[0181] In certain embodiments, a multiplex RPA reaction for amplifying and detecting at least two target nucleic acids, e.g., for detecting HIV-1 and HBV, can comprise the following reagents, in addition to the reducing agent and the activator, employed at the following concentrations (or included in a reagent composition (e.g., a second reagent composition) of the present disclosure): Tris-HCl IM, pH 8.3 at 50 mM, Potassium Acetate at 100 mM, 20% Polyethylene glycol at 5.5%, 100 mM dNTPs at 2.7 mM, ATP at 3.5 mM, Phosphocreatine at 50 mM, HIV-1 INT Forward Primer at 157.50 nM, HIV-1 INT Reverse Primer at 236.37 nM, HIV-1 INT Exo Probe at 90 nM, HIV-1 LTR Forward Primer at 39.37 nM, HIV-1 LTR Reverse Primer at 39.37 nM, HIV-1 INT Exo Probe at 22.5 nM, HBV Forward Primer at 86.13 nM, HBV Reverse Primer at 86.13 nM, HBV Exo Probe at 90 nM, ROX reference dye at 45 nM, Glycerol at 8.4%, Creatine Kinase at 0.1 mg / ml, UvsX at 0.375 mg / ml, UvsY at 0.0675 mg / ml, DNA Polymerase at 0.1396 mg / ml, Gp32 at 1.2 mg / ml, Exonuclease III at 0.1 mg / ml, Bovine Serum Albumin (BSA) at 0.02 mg / ml and EIAV Reverse Transcriptase at 0.0016 mg / ml.

[0182] In certain embodiments, a multiplex reaction for amplifying and detecting at least two target nucleic acids, e.g., for detecting HIV-1 and HBV, can comprise the following reagents, in addition to the reducing agent and the activator, employed at the following concentrations (or included in a reagent composition (e.g., a second reagent composition) of the present disclosure): Tris-HCl IM, pH 8.3 at 50 mM, Potassium Acetate at 100 mM, 20% Polyethylene glycol at 5.5%, 100 mM dNTPs at 2.7 mM, ATP at 3.5 mM, Phosphocreatine at 50 mM, HIV-1 Forward Primer at 157.50 nM, HIV-1 Reverse Primer at 236.37 nM, HIV-1 Exo Probe at 90 nM, HBV Forward Primer at 86.13 nM, HBV Reverse Primer at 86.13 nM, HBV Exo Probe at 90 nM, ROX reference dye at 45 nM, Glycerol at 8.4%, Creatine Kinase at 0.1 mg / ml, UvsX at 0.375 mg / ml, UvsY at 0.0675 mg / ml, DNA Polymerase at 0.1396 mg / ml, Gp32 at 1.2 mg / ml, Exonuclease III at 0.1 mg / ml, Bovine Serum Albumin (BSA) at 0.02 mg / ml and EIAV Reverse Transcriptase at 0.0016 mg / ml.

[0183] In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

[0184] In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a trialkylphosphine and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a trialkylphosphine and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

[0185] In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and magnesium or a salt form thereof at a concentration ratio of about 1 :0.01 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and magnesium or a salt form thereof at a concentration ratio of about 1 :0.01 to about 1 :5.

[0186] In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5.

[0187] In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a trialkylphosphine and an activator at a concentration ratio of about 1 : 1 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising a trialkylphosphine and an activator at a concentration ratio of about 1 : 1 to about 1 :5. In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and an activator at a concentration ratio of about 1 : 1 to about 1 :5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and an activator at a concentration ratio of about 1 : 1 to about 1 :5.

[0188] In certain embodiments, an RPA process of the present disclosure includes contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and magnesium or a salt form thereof at a concentration ratio of about 1 : 1 to about 1:5, to amplify the target nucleic acid. In certain embodiments, an RPA process for amplifying a target nucleic acid of the present disclosure is initiated by contacting a sample comprising the target nucleic acid and one or more RPA reagents with a composition disclosed herein, e.g., a composition comprising THPP and magnesium or a salt form thereof at a concentration ratio of about 1 : 1 to about 1 :5.

[0189] In certain embodiments, an RPA process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a solution comprising a plurality of nucleic acids and one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 to generate an activated reaction mixture and (c) incubating the activated reaction mixture to amplify the target nucleic acid if present in the plurality of nucleic acids. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0190] In certain embodiments, an RPA process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present. In certain embodiments, the reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0191] In certain embodiments, an RPA process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present. In certain embodiments, the reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the reaction mixture comprises a reaction concentration of the activator, e.g, magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0192] In certain embodiments, an RPA process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :4 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present. In certain embodiments, the reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0193] In certain embodiments, the one or more RPA reagents can include any one of or include a combination of any of the reagents disclosed herein, e.g., reagents required for performing an RPA process. Non-limiting examples of such reagents include a recombinase, a single-stranded DNA binding protein, a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a recombinase loading protein, a first primer, a second primer, a probe, a nuclease, a creatine kinase and a reverse transcriptase.

[0194] In certain embodiments, the reaction volume of an RPA process of the present disclosure can be about 5 pl, about 10 pl, about 20 pl, about 30 pl, about 50 pl, about 75 pl, about 100 pl, about 300 pl, about 1 ml, about 3 ml, about 10 ml, about 30 ml, about 50 ml or about 100 ml. In certain embodiments, the reaction volume of an RPA process of the present disclosure can be from about 50 pl to about 100 pl.

[0195] In certain embodiments, the target nucleic acid can be of any concentration in the RPA reaction. For example, but not by way of limitation, there can be less than about 10,000 copies of the target nucleic acid, less than about 1000 copies of the target nucleic acid, less than about 100 copies of the target nucleic acid, less than about 10 copies of the target nucleic acid or 1 copy of the target nucleic acid in an RPA reaction. In certain embodiments, an isothermal amplification process of the present disclosure can result in a 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.

[0196] In certain embodiments, the reaction temperature of an RPA process of the present disclosure is between about 20°C to about 50°C, about 20°C to about 40°C, about 20°C to about 30°C or about 37°C to about 42°C. In certain embodiments, the reaction temperature is about 40°C.

[0197] In certain embodiments, the reaction time of an RPA process of the present disclosure is about 10 minutes to about 3 hours, about 10 minutes to about 2 hours, about 10 minutes to about 1 hour, is about 10 minutes to about 30 minutes, or in about 10 minutes to about 25 minutes, or about 10 minutes to about 20 minutes, or even about 10 minutes to about 15 minutes from the addition of the reagents sufficient to initiate the RPA process. In certain embodiments, the reaction time of an RPA process of the present disclosure is about 1 minute to about 20 minutes, about 5 minutes to about 20 minutes, about 8 minutes to about 20 minutes, about 1 minute to about 10 minutes or about 5 minutes to about 10 minutes from the addition of the reagents sufficient to initiate RPA amplification. In certain embodiments, the reaction time of an RPA process of the present disclosure is about 1 minute. In certain embodiments, the reaction time of an RPA process of the present disclosure is about 5 minutes. In certain embodiments, the reaction time of an RPA process of the present disclosure is about 20 minutes. In certain embodiments, the RPA reaction time is sufficient to obtain a result, e.g., detection of a target nucleic acid.

[0198] C. NEAR Process

[0199] In certain embodiments, the isothermal amplification process is a NEAR process. In certain embodiments, the present disclosure provides a NEAR process that includes the use of a composition described herein. For example, but not by way of limitation, the present disclosure provides a NEAR process that is performed in the presence of a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 :0.25 to about 1 :3. In certain embodiments, the present disclosure provides a NEAR process that includes the use of a composition that includes a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 :0.25 to about 1 :3. In certain embodiments, the composition includes from about 10 mM to about 200 mM of a reducing agent, e.g., THPP. In certain embodiments, the composition includes from about 0.1 mM to about 500 mM of an activator, e.g., MgOAc.

[0200] In certain embodiments, a NEAR process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a solution comprising a plurality of nucleic acids and one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 to generate an activated reaction mixture and (c) incubating the activated reaction mixture to amplify the target nucleic acid if present in the plurality of nucleic acids. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0201] In certain embodiments, a NEAR process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a solution comprising a plurality of nucleic acids and one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5 to generate an activated reaction mixture and (c) incubating the activated reaction mixture to amplify the target nucleic acid if present in the plurality of nucleic acids. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the activated reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0202] In certain embodiments, a NEAR process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present. In certain embodiments, the reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0203] In certain embodiments, a NEAR process of the present disclosure for amplifying a target nucleic acid includes: (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :4 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present. In certain embodiments, the reaction mixture comprises a reaction concentration of the reducing agent, e.g., THPP, from about 1 mM to about 10 mM. In certain embodiments, the reaction mixture comprises a reaction concentration of the activator, e.g., magnesium or a salt form thereof, from about 10 mM to about 20 mM.

[0204] In NEAR, a target nucleic acid sequence, having a sense and antisense strand, is contacted with a pair of amplification oligonucleotides. The first amplification oligonucleotide comprises a nucleic acid sequence comprising a recognition region at the 3’ end that is complementary to the 3’ end of the target sequence antisense strand, a nicking enzyme site upstream of said recognition region, and a stabilizing region upstream of said nicking enzyme site (see, e.g., U.S. Patent Nos 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 amplification oligonucleotide comprises a nucleotide sequence comprising a recognition region at the 3’ end that is complementary to the 3’ end of the target sequence sense strand, a nicking enzyme site upstream of said recognition region, and a stabilizing region upstream of said nicking enzyme site. Two nicking enzymes are provided. One nicking enzyme is capable of nicking at the nicking enzyme site of the first amplification oligonucleotide but incapable of nicking within said target sequence. The other nicking enzyme is capable of nicking at the nicking enzyme site of the second amplification oligonucleotide but incapable of nicking within said target sequence. A DNA polymerase is employed under conditions for amplification which involves multiple cycles of extension of the amplification oligonucleotides thereby producing a double-stranded nicking enzyme site which are nicked by the nicking enzymes to produce the amplification product. For example, see 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.

[0205] In certain embodiments, reactions use only two templates to prime, one or two nicking enzymes and a polymerase, under isothermal conditions. In exemplary nonlimiting embodiments, the polymerase and the nicking enzyme are thermophilic, and the reaction temperature is significantly above the melting temperature of the hybridized target region. The nicking enzyme nicks only one strand in a double-stranded duplex, so that incorporation of modified nucleotides is not necessary as it is in strand displacement. In certain embodiments, the method is able to amplify RNA without a separate reverse transcription step, although conversion of RNA to DNA by reverse transcription may be used if desired.

[0206] In certain embodiments, the method comprises contacting a target DNA molecule comprising a double-stranded target sequence having a sense strand and an antisense strand, with a forward template and a reverse template, wherein said forward template comprises a nucleic acid sequence comprising a recognition region at the 3’ end that is complementary to the 3’ end of the target sequence antisense strand; a nicking enzyme site upstream of said recognition region, and a stabilizing region upstream of said nicking enzyme site; the reverse template comprises a nucleotide sequence comprising a recognition region at the 3' end that is complementary to the 3’ end of the target sequence sense strand, a nicking enzyme site upstream of the recognition region, and a stabilizing region upstream of the nicking enzyme site; providing a first nicking enzyme that is capable of nicking at the nicking enzyme site of the forward template, and does not nick within the target sequence; providing a second nicking enzyme that is capable of nicking at the nicking enzyme site of the reverse template and does not nick within the target sequence; and providing a DNA polymerase; under conditions wherein amplification is performed by multiple cycles of the polymerase extending the forward and reverse templates along the target sequence producing a double-stranded nicking enzyme site, and the nicking enzymes nicking at the nicking enzyme sites, producing an amplification product.

[0207] In certain embodiments, the DNA polymerase is a thermophilic polymerase. In other examples, the polymerase and said nicking enzymes are stable at temperatures up to 37°C, 42°C, 60°C, 65°C, 70°C, 75°C, 80°C or 85°C. In certain embodiments, the polymerase is stable up to 60°C. In certain embodiments, the polymerase can, for example, be selected from the group consisting of Bst (large fragment), 9° N, VentR® (exo-) DNA Polymerase, THERMINATOR, and THERMINATOR II (New England Biolabs). In certain embodiments, the nicking enzyme can, for example, nick upstream of the nicking enzyme binding site, or the nicking enzyme may nick downstream of the nicking enzyme binding site. In certain embodiments, the forward and reverse templates comprise nicking enzyme sites recognized by the same nicking enzyme and the first and the second nicking enzyme are the same. In certain embodiments, the nicking enzyme can, for example, be selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.BpulOI and Nt.BpulOI.

[0208] In certain embodiments, the target sequence includes from 1 to 5 nucleotides more than the sum of the nucleotides of said forward template recognition region and said reverse template recognition region.

[0209] In certain embodiments, the forward template is provided at the same concentration as the reverse template. In certain embodiments, the forward template is provided at a ratio to the reverse template at the range of ratios of 1 : 100 to 100: 1.

[0210] In certain embodiments, the NEAR reaction time can be about 10 minutes to about 3 hours, about 10 minutes to about 2 hours, about 10 minutes to about 1 hour, about 10 minutes to about 30 minutes, or about 8 minutes to about 25 minutes, or about 8 minutes to about 20 minutes, or even about 8 minutes to about 15 minutes from the addition of the reagents sufficient to initiate NEAR amplification. In certain embodiments, the NEAR reaction time is about 1 minute to about 20 minutes, about 5 minutes to about 20 minutes, about 8 minutes to about 20 minutes, about 1 minute to about 10 minutes or about 5 minutes to about 10 minutes from the addition of the reagents sufficient to initiate NEAR amplification.

[0211] D. Samples

[0212] In certain embodiments, the target nucleic acids that can be amplified using the disclosed methods can be isolated from a sample, e.g., a sample from a subject. Target nucleic acids can be isolated from a 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 by way of limitation, nucleic acids can be isolated from a sample by the use of magnetic microparticles (e.g., copper titanium microparticles), e.g., as shown in FIGS. 4, 9 and 10 of PCT / US2022 / 027067.

[0213] In certain embodiments, the sample can be a tissue sample. In certain embodiments, the sample can be obtained from preserved tissue, e.g., fixed tissue, from frozen tissue or from fresh tissue, e.g., fresh tissue samples. Non-limiting examples of tissues include eye, muscle, skin, tendon, vein, artery, heart, spleen, lymph node, bone, bone marrow, lung, bronchi, trachea, gut, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testicle, ovary, uterus, fallopian tube, thymus, pituitary, thyroid, adrenal or parathyroid tissue. In certain embodiments, the tissue can be cancerous tissue, e.g., tumor tissue.

[0214] In certain embodiments, the sample is a biological fluid sample. In certain embodiments, the biological fluid sample is a bodily secretion. 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, lymphatic fluid, cerebrospinal fluid, interstitial fluid, bronchoalveolar lavage fluid, nasopharyngeal fluid or any other sample suitable for analysis using the methods and techniques described herein. In certain embodiments, the sample can be a nasal swab (e.g., a nasal swab (e.g., at least partially coated with a target nucleic acid) in a buffer) and / or a sample obtained using a nasal swab (e.g., a nasopharyngeal fluid sample).

[0215] In certain embodiments, the biological fluid sample is whole blood. As used herein, “whole blood” refers to blood that has not had any components removed (blood that contains both the fluid and solid components). Transfusion of whole blood, or the red blood cell (RBC) component of whole blood, can increase a patient’s oxygen-carrying capacity by effectively increasing the patient’s RBC count to thereby increase the amount of available oxygen-carrying hemoglobin. In addition to its oxygen-carrying capacity, whole blood transfusions can be a source of platelets, which aid in blood clotting. In certain embodiments, the clinical use, transfusion of platelets can be used to treat thrombocytopenia, certain cancers, aplastic anemia as well as marrow transplants.

[0216] In certain embodiments, biological fluid sample is lysed whole blood. As used herein, “lysed whole blood” refers to blood that has not had any components removed (blood that contains both the fluid and solid components), but where the RBCs have been lysed by exposure to, e.g., a buffer comprising ammonium chloride, potassium carbonate and EDTA. Ammonium chloride, which lyses RBCs, has minimal effect on lymphocytes.

[0217] In certain embodiments, the biological fluid sample is plasma. Plasma is the aqueous portion of blood that remains after centrifugation to remove the cellular components of blood. Plasma can, in certain embodiments, include albumin, coagulation factors, fibrinolytic proteins, immunoglobulin and other proteins. Products derived from plasma donation can, in certain embodiments, be used to treat bleeding disorders and / or lifethreatening trauma / hemorrhages.

[0218] In certain embodiments, the biological fluid sample is serum. As used herein, “serum” is the clear portion of plasma that does not contain fibrinogen, cells or any solid elements.

[0219] In certain embodiments, the sample is obtained from a subject. In certain embodiments, the subject is a vertebrate or an invertebrate, such as a human or non-human animal, for example, a mammal. In certain 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 certain embodiments, the subject is a human.

[0220] III. Nucleic Acid Detection Processes

[0221] Nucleic acid detection as employed herein is used to determine presence (e.g., presence or absence) of a target nucleic acid or a plurality of different target nucleic acids in a sample. In certain embodiments, nucleic acid detection is employed to quantify the amount of a nucleic acid or a plurality of different nucleic acids in a sample. As embodied herein, the nucleic acid detection methods of the present disclosure can be configured to detect a target nucleic acid or plurality of different target nucleic acids using any of a variety or combination of suitable detection techniques.

[0222] In certain embodiments, following amplification of one or a plurality of target nucleic acids present in the sample, the methods of the present disclosure can be configured to detect the amplified nucleic acid(s) via hybridization. For example, but not limitation, such detection can comprise hybridizing a probe oligonucleotide sufficiently complementary to an amplified target nucleic acid to facilitate detection of the target nucleic acid. In certain embodiments, following hybridization of the probe oligonucleotide to the target nucleic acid, the method comprises detecting hybridization of the probe oligonucleotide to the target nucleic acid. For example, but not limitation, such detection can be achieved by observing a signal from a detectable label, whereby (i) the presence of one or more signals indicates hybridization of the probe oligonucleotide to the target nucleic acid and is indicative of 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. Detection of a signal from the probe oligonucleotide can be performed using a variety of suitable methodologies, depending on the type of detectable label.

[0223] In certain embodiments, nucleic acid amplification, e.g., using an isothermal amplification process as described herein, and nucleic acid detection can occur simultaneously, e.g., during an amplification and detection process. In certain embodiments, an amplification and detection process as disclosed herein includes the simultaneous amplification and detection of nucleic acids in a sample, e.g., an eluate. In certain embodiments, the amplification and detection process begins with the incubation of an eluate with the reagents sufficient to initiate amplification of a target nucleic acid in the sample using the isothermal amplification methods and compositions of the present disclosure, if present, and ends with the determination of a result in the sample, e.g., the detection of the target nucleic acid in the eluate or the lack of detection of the target nucleic acid in the sample.

[0224] In certain embodiments, detection of amplified nucleic acids can employ optical detection, digital detection and / or other detection methods known in the art.

[0225] A. Optical Detection

[0226] In certain embodiments, detection of amplified nucleic acids can be performed using optical detection. For example, but not by way of limitation, the detection of the amplified target nucleic acid is mediated by the binding of a labeled probe or by incorporation of a label into amplified copies of the target nucleic acid.

[0227] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, detection is mediated by observation of a fluorescent label (such as fluorescein (e.g., 5 -fluorescein, 6-carboxyfluorescein (e.g., FAM), 3'6- carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6- tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), Fluor Orange 560 fluorophore, Quasar 670 fluorophore and Quasar 705 fluorophore. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties).

[0228] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, optical detection is performed using fluorescence, chemiluminescence, or other means of generating a signal in response to the presence of an analyte. Many assays are performed by measuring the intensity of a light signal generated in the total volume of a reaction mixture. The light signal generated can be measured by an optical means, wherein the light signal generated is emitted by a large number of molecules. Typically, as described herein, assays can involve combining a sample suspected of containing a target nucleic acid, e.g. , target nucleic acids amplified as described herein, with a reagent comprising a labeled probe capable of hybridizing with the target nucleic acid to form a reaction mixture. The signal attributable to the label is then measured after unbound probe is removed from the reaction mixture, typically by performing a wash step. In certain embodiments, the presence of a detectable signal is sufficient to confirm the presence of the target nucleic acid in the sample. In certain embodiments, the signal that is derived from the total volume of the reaction mixture is measured and then compared to a calibration curve to establish the concentration of target nucleic acid present in the sample.

[0229] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, the optical detection strategy comprises the use of probes labeled with both a detectable label and a “quencher molecule” where the quencher molecule is capable of interacting with a detectable label to reduce or eliminate the signal emitted by the detectable label. For example, but not by way of limitation, a detection probe employed in the methods of the present disclosure can have a fluorescent moiety that is covalently linked, e.g., to the 5’ end of the probe, and has a quencher molecule, e.g., at the 3’ end of the probe. In the absence of target sequences, the probe adopts a conformation that brings the quencher close enough to the excited fluorophore to absorb its energy before it can be fluorescently emitted. When the probe binds to its complementary sequence in the target, the fluorophore and the quencher are positioned at a sufficient distance apart to allow fluorescent emission and detection. In certain embodiments, the quencher can be selected from any suitable quencher known in the art, such as, for example, BLACK HOLE QUENCHER® 1 (BHQ-1®), BLACK HOLE QUENCHER® 2 (BHQ-2®), BLACK HOLE QUENCHER®- 1-dT (BHQ-1 dT®), BLACK HOLE QUENCHER® -2-dT (BHQ-2dT®), IOWA BLACK® FQ, and IO WA BLACK® RQ. For example, but not by way of limitation, an oligonucleotide probe used in the methods of the present disclosure can comprise a FAM fluorophore and a BHQ-1 dT® quencher or a BHQ-2dT® quencher. In certain embodiments, an oligonucleotide probe used in the methods of the present disclosure can include a Quasar 670 fluorophore and a BHQ-1® quencher or a BHQ-2® quencher. In certain embodiments, an oligonucleotide probe used in the methods of the present disclosure can include a Quasar 670 fluorophore and a BHQ-1 dT® quencher or a BHQ-2dT® quencher. Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, specific probes, e.g., probes for specific target nucleic acids and / or internal controls, are each labeled with a different fluorophore, thus allowing for simultaneous detection of a plurality of amplified products.

[0230] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, light intensity can be measured using light emitting diodes (LEDs) and / or lasers for excitation and any suitable detector for detection emissions. Fluorescence- optical detection “scanners” can be used which scan the surface of the chip using a focused laser beam, allowing for detection of the emitted fluorescence light. Exemplary fluorescence scanners are described in, e.g., U.S. Pat. Nos. 5,837,475 and 5,945,679. Scanners in which a confocal excitation and detection system has been integrated into an epifluorescence microscope are also known. The systems used in scanners for detecting the emitted fluorescence light are usually “one-channel systems”, z.e., for example, individual photocells or secondary electron multipliers (photomultipliers). Two-dimensional detection systems such as, for example, charged-coupled device (CCD) cameras, also are used for detecting fluorescence or chemiluminescent light of a sample. Commercially available systems have either an optical imaging system which projects the binding surface provided with chemiluminescent markers or fluorescent markers on a CCD sensor by using lens optics, or a combination of image intensifier and CCD camera.

[0231] B. Digital Detection

[0232] In certain embodiments, detection of amplified nucleic acids can be performed using digital detection methods. Because every single target nucleic acid, as an end-point entity, can be detected in principle in the context of digital detection, the components and methods associated with digital detection can significantly increase detection sensitivity for sample analysis compared to systems using analog optical detection. As such, digital detection can be performed using a lower concentration of analyte, e.g., target nucleic acids, which can allow for decreased time to process the sample for detection. Additionally, or alternatively, detection can be performed using a smaller sample volume, less reagent material, less conjugate material, fewer microparticles, or any combination of these, which can reduce costs to perform each assay. As such, and as described herein, sample preparation time can be improved due at least in part to less sample manipulation involved (e.g., faster washing times) and / or improved kinetics of reactions achieved using a lower sample volume, less reagent or conjugate material, and / or fewer particles or beads to obtain an analyte concentration suitable for detection. Assays using less sample volume and / or reagent material can be performed using smaller equipment, which can reduce the footprint of the laboratory system for performing the assays as discussed further herein. In addition, or as a further alternative, increased detection sensitivity can provide additional benefits when used with multiplexing. For example, and without limitation, when multiple analytes and corresponding signals are combined into a single, multiplexed assay, a noise level associated with the detection of each analyte signal can be multiplied to obtain a total noise level of the multiplexed system. By increasing the detection sensitivity of each signal being detected, the improved sensitivity can be multiplied to further reduce the total noise level of the multiplexed system.

[0233] Digital detection can provide increased sensitivity due at least in part to a reduction of noise during detection relative to the signal being measured, for example, producing a higher signal -to-noise ratio. Such improved signal-to-noise ratios are possible by coupling the analyte of interest, e.g., a particular target nucleic acid, to an independently detectable end-point entity. For example, but not limitation, amplified target nucleic acids can be immobilized to microparticles and labeled with detectable conjugates, where the conjugate is a detectable end-point entity in that it can emit an independently detectable signal, either directly or via the conversion of a substrate.

[0234] In certain embodiments, the detection operation employs a digital nanowell detection process. In certain embodiments, a support medium, such as, but not limited to, microparticles, beads, or other labels, can be mixed with the sample in order to perform the digital detection process after amplification. In certain embodiments, reagents including antibodies and coated microparticles can be combined.

[0235] For example, but not by way of limitation, digital nanowell detection processes incorporating microparticles can employ anti-Digoxin microparticles. In certain embodiments, digital nanowell detection incorporating microparticles can be performed in a formulation comprising: Tris-HCl, NaCl, BSA, Tergitol 15-S-40, sodium azide and 0.02 % anti-Digoxin pP (microparticles). For example, but not by way of limitation, digital nanowell detection incorporating microparticles can be performed in the following context: about 50 mM Tris-HCl at a pH of about 8.0; about 150 mM NaCl; about 0.2 % BSA; about 0.5 % Tergitol 15-S-40; about 0.08 % Sodium azide; and about 0.02 % anti-Digoxin pP (microparticles). The solution can be washed, for example to remove excess reagents and / or unbound analyte. Any suitable number of washes can be performed for each washing step, including one, two, or three or more washes, and each wash can be performed in a single chamber or location or among different chambers or locations. For example, and not limitation, as embodied herein, three washes can be performed.

[0236] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, a conjugate can be added to bind with an analyte of interest in the sample. In certain embodiments, a conjugate, e.g., Alkaline Phosphatase- SA, can be added to the sample. In certain embodiments, additional reagents including, but not limited to, Tris-HCl, NaCl, MgCh, ZnCh, fish gelatin, Rabbit IgG, Saponin, calf serum, Goat IgG and Sodium azide, can be added to the sample. For example, and not limitation, the conjugate can include one or more reagents or enzymes selected or configured to react with the analyte of interest to produce a signal for detection by the detection component. In certain embodiments, the digital nanowell detection process will employ conjugates in the following context: about 3000 pM Alkaline Phosphatase- SA; about 100 mM Tris-HCl, at a pH of about 7.5; about 500 mM NaCl; about 1 mM MgCh; about 0.1 ZnCh; about 8.9 g / L fish gelatin; about 30 ug / mL of Rabbit IgG; about 0.1% Saponin; about 10% calf serum; about 5 mg / mL Goat IgG; and about 0.1% Sodium azide. The solution can be washed, for example to remove excess conjugate unbound to the analyte of interest. Any suitable number of washes can be performed for each washing step, including one, two, or three or more washes, and each wash can be performed in a single chamber or location or among different chambers or locations.

[0237] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, detection of the microparticles bound with analytes and conjugates can be performed in a single chamber or location or among different chambers or locations. For purpose of illustration and not limitation, the detection chamber or location can include a surface and a detection region. The microparticles can be added to the detection chamber or location using any suitable technique, including but not limited to pipetting, magnetic force or dielectrophoresis. In certain embodiments, the digital nanowell detection process will employ a detection substrate, e.g., AJ Phos. In certain embodiments, additional reagents including, but not limited to, DEA, MgCh and Tween 20, can be added in combination with the detection substrate. For example, but not by way of limitation, the digital nanowell detection process will employ a detection substrate in the following context: about 200 pM AJ Phos; about 1 M DEA; about 1 mM MgCh; and about 0.05 % Tween 20. As embodied herein, the detection region can include one or more nanowells. The microparticles can be moved to the detection region, for example and as embodied herein, an array of nanowells. The microparticles can be moved to the nanowells using any suitable technique, including but not limited to pipetting, magnetic force or dielectrophoresis. In certain embodiments, oil, e.g., 3 mM Guaiazulene in FC-40 oil, is added to seal the nanowells. In certain embodiments, a dye can be added to increase contrast or otherwise improve optical conditions for detection of the analyte of interest in the nanowells. In certain embodiments, the digital nanowell detection process incorporating microparticles will employ a dye in the following context: about 0.1 % Tween 20; about 10 mM PBS; and about 50 mM Nigrosine. In certain embodiments, one or more images of the microparticles are taken and analyzed to determine the presence or absence of the analyte of interest and / or a concentration of the analyte of interest in the sample.

[0238] IV. Methods of Use

[0239] In certain embodiments, the methods of the present disclosure can be used to detect the presence or absence of a target nucleic acid in a sample. In certain embodiments, the methods of the present disclosure can be used to quantify the amount of a target nucleic acid in a sample. For example, but not by way of limitation, a method of the present disclosure can be used to determine the presence, absence and / or quantity of one or more target nucleic acids in a sample comprising a plurality of nucleic acids.

[0240] In certain embodiments, the methods of the present disclosure can be used to amplify and detect and / or quantify target nucleic acids that are associated with a disease or disorder, e.g., to determine if a subject has the disease or disorder. In certain embodiments, the methods of the present disclosure can be used to amplify and detect and / or quantify target nucleic acids that are markers for a disease or disorder. For example, but not by way of limitation, the present disclosure can be used to determine if a subject has a disease or disorder or is at risk of developing the disease or disorder, e.g., cancer. In certain embodiments, the methods of the present disclosure can be used to determine if a subject has a genetic disorder, e.g., by determining if the subject has a genetic mutation associated with the genetic disorder. In certain embodiments, the methods of the present disclosure can be used to amplify and detect and / or quantify target nucleic acids that are derived from a pathogen, e.g., to determine if a subject is infected with the pathogen. In certain embodiments, the present disclosure can be used to determine the blood type and / or blood group of a subject. In certain embodiments, the present disclosure can be used to quantify target nucleic acids, e.g., to determine a viral titer and / or bacterial load.

[0241] In certain embodiments, the methods of the present disclosure can be used for the screening of blood samples. In certain embodiments, the methods of the present disclosure can be used for the screening of samples derived from a single individual as well as from a plurality of individuals. In certain embodiments, the screening of blood samples can find use in connection with donations of a material, e.g., plasma, platelets, red cells and whole blood. In certain embodiments, the blood sample screened is a whole blood sample. In certain embodiments, the blood sample screened is a lysed whole blood sample. In certain embodiments, the blood sample screened is a serum sample. In certain embodiments, the blood sample screened is a plasma sample.

[0242] In certain embodiments, the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid. In certain embodiments, the target nucleic acid is a bacterial nucleic acid. In certain embodiments, the target nucleic acid is a eukaryotic nucleic acid. In certain embodiments, the target nucleic acid is a viral nucleic acid.

[0243] In certain embodiments, the target nucleic acid is a nucleic acid derived from SARS-CoV-2 (COVID-19), coronaviruses, HIV (e.g., HIV-1 and / or HIV-2), Hepatitis B (HBV), Hepatitis C (HCV), Hepatitis A (HAV), Hepatitis E (HEV), Cytomegalovirus (CMV), Parvovirus Bl 9, Creutzfeldt-Jakob disease (vCJD), Chlamydia, Gonorrhea, West Nile virus (WNV), Zika virus (ZIKV), Dengue, Chikungunya, Influenza (e.g., Influenza A virus, Influenza B virus, or Influenza C virus), Babesia, Malaria, Rubella, Varicella-zoster, Herpes Simplex, Polio, syphilis, Smallpox, Vaccinia, Rabies, human T-lymphotropic virus (HTLV), Usutu Virus or Epstein Barr Virus. In certain embodiments, the target nucleic acid is selected from the group consisting of Hepatitis B (HBV), Hepatitis C (HCV), Hepatitis A (HAV), Hepatitis E (HEV) and a combination thereof. In certain embodiments, the target nucleic acid is a nucleic acid derived from HCV. In certain embodiments, the target nucleic acid is a nucleic acid derived from HIV, e.g., HIV-1 and / or HIV-2. In certain embodiments, the target nucleic acid is a nucleic acid derived from HBV. In certain embodiments, the target nucleic acid is a nucleic acid derived from Dengue. In certain embodiments, the target nucleic acid is a nucleic acid derived from Chikungunya.

[0244] In certain embodiments, the target nucleic acid is a nucleic acid derived from HIV-1.

[0245] In certain embodiments, the target nucleic acid is a nucleic acid derived from HIV-2.

[0246] In certain embodiments, the target nucleic acid is a nucleic acid derived from HCV.

[0247] In certain embodiments, the target nucleic acid is a nucleic acid derived from HBV. In certain embodiments, the target nucleic acid is a nucleic acid derived from

[0248] Babesia.

[0249] In certain embodiments, the target nucleic acid is a nucleic acid derived from Malaria.

[0250] In certain embodiments, the target nucleic acid is a nucleic acid derived from one or more new or emerging pathogens, viruses and / or agents.

[0251] In certain embodiments, if the virus is an RNA-based virus, e.g., HIV-1 and HCV, the nucleic acids to be detected will be RNA. If the virus is a DNA-based virus, e.g., HBV, the nucleic acids to be detected will be DNA. In certain embodiments, the methods can detect ribosomal RNA of the parasite Babesia.

[0252] In certain embodiments, the methods of the present disclosure can be used for the amplification, detection (e.g., presence or absence) or the quantification of two or more target nucleic acids in a sample, e.g., a plurality of target nucleic acids, by multiplexing. In certain embodiments, higher orders of multiplex amplifications can be employed in connection with the methods of the present disclosure, such that the presence of 3, 4, 5, 6, 7, 8, 9, 10 or more target nucleic acids can be detected in a single sample. In certain embodiments, each target nucleic acid is derived from a different pathogen, infectious agent, gene or mRNA. As used herein, “multiplex analysis” refers to concurrent screening for two or more target nucleic acids, e.g. , where each target nucleic acid is derived from a pathogen or infectious agent. As used herein, “multiplex analysis” encompasses concurrent screening of two or more target nucleic acids in a single reaction vessel, e.g, an amplification vessel, as well as screening in separate reaction vessels of two or more target nucleic acids, e.g, where a sample eluate has been split into two more separate reaction vessels, e.g., amplification vessels.

[0253] In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of HIV-1, HIV-2, HCV and / or HBV. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of HIV-1 and HIV-2. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of HIV- 1, HIV-2 and HCV. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of HIV-1, HIV-2 and HBV. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of HCV and HBV. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of HIV- 1, HIV-2, HCV and HBV. In certain embodiments, methods of the present disclosure can be used for the multiplex analysis of HIV-1, HIV-2, HCV and / or HBV, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the multiplex analysis of HIV-1, HIV-2, HCV and / or HBV, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g, about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0254] In certain embodiments, methods of the present disclosure can be used for the analysis of HIV-1, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of HIV-1, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0255] In certain embodiments, methods of the present disclosure can be used for the analysis of HIV-2, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of HIV-2, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0256] In certain embodiments, methods of the present disclosure can be used for the analysis of HCV, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of HCV, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0257] In certain embodiments, methods of the present disclosure can be used for the analysis of HBV, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of HBV, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM. In certain embodiments, methods of the present disclosure can be used for the analysis of Babesia, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of Babesia, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g, about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0258] In certain embodiments, methods of the present disclosure can be used for the analysis of Malaria, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of Malaria, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0259] In certain embodiments, methods of the present disclosure can be used for the analysis of Parvovirus Bl 9, where the methods include the use of a composition comprising a reducing agent and an activator at the ratios and / or amounts described herein. For example, but not by way of limitation, the methods of the present disclosure can be used for the analysis of Parvovirus B19, where the methods include the use of a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5, e.g., about 1 : 1 to about 1 :5, about 1 :0.01 to about 1 :3, about 1 :0.25 to about 1 :3 or about 1 : 1 to about 1 :3. In certain embodiments, the composition can include about 5 mM to about 100 mM of a reducing agent, e.g., about 50 mM to about 90 mM of a reducing agent, and about 10 mM to about 300 mM of an activator, e.g., about 100 mM to about 300 mM of an activator. In certain embodiments, the reaction concentration of the reducing agent can be from about 1 mM to about 10 mM, e.g., about 3 mM to about 8 mM, and the reaction concentration of the activator can be from about 5 mM to about 20 mM, e.g., about 10 mM to about 20 mM.

[0260] In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Zika Virus, WNV, Chikungunya Virus and / or Dengue Virus. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Zika Virus and WNV. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Chikungunya Virus and Dengue Virus. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Chikungunya Virus and WNV. In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Zika Virus and Dengue.

[0261] In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Babesia and Malaria.

[0262] In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Parvovirus B19 and HAV.

[0263] V. Systems

[0264] The present disclosure further provides systems for performing the isothermal amplification methods of the present disclosure. In certain embodiments, the system is an automatic system. In certain embodiments, the automatic system that can be used for performing the isothermal amplification methods of the present disclosure can include a sample preparation area (e.g., an area for isolating nucleic acids from a sample), a nucleic acid amplification area and a nucleic acid detection area. In certain embodiments, the nucleic acid amplification area and the nucleic acid detection area are the same. An automated system for performing the methods of the present disclosure is provided in International Patent Application PCT / US2022 / 027067, which is incorporated herein by reference in its entirety. For example, but not by way of limitation, the system of FIGS. 68A-68D of PCT / US2022 / 027067 can be used to perform the methods of the present disclosure. In certain embodiments, a system of the present disclosure includes containers and / or reservoirs that includes one or more compositions disclosed herein for performing an isothermal amplification reaction (e.g., an RPA process). In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a composition (e.g., a reagent composition) that includes a reducing agent and an activator at the ratios and amounts described herein. For example, but not by way of limitation, a system of the present disclosure can include at least one container or reservoir that includes a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5. In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :4. In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :3. In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a reducing agent and an activator at a concentration ratio of about 1 :2 to about 1 :3. In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a reducing agent and an activator at a concentration ratio of about 1 :2 to about 1 :4.

[0265] In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a composition (e.g., reagent composition) that includes from about 10 mM to about of 200 mM of a reducing agent, e.g., THPP, and about 0.1 mM to about of 500 mM of an activator, e.g., MgOAc.

[0266] In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a composition (e.g., a reagent composition) that comprises (or consists essentially of or consists of) a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5, where the reducing agent is present in the composition at a concentration from about 50 mM to about 150 mM and the activator is present in the composition at a concentration from about 100 mM to about 300 mM.

[0267] In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a composition (e.g., a reagent composition) that comprises (or consists essentially of or consists of) a trialkylphosphine and an activator at a concentration ratio of about 1 : 1 to about 1 :5, where the trialkylphosphine is present in the composition at a concentration from about 50 mM to about 150 mM and the activator is present in the composition at a concentration from about 100 mM to about 300 mM.

[0268] In certain embodiments, a system of the present disclosure can include at least one container or reservoir that includes a composition (e.g., a reagent composition) that comprises (or consists essentially of or consists of) THPP and a magnesium or salt form thereof at a concentration ratio of about 1 : 1 to about 1 :5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and the magnesium or salt form thereof is present in the composition at a concentration from about 100 mM to about 300 mM.

[0269] VI. Kits

[0270] The present disclosure further provides kits for performing the methods of the present disclosure. In certain embodiments, the present disclosure provides kits containing materials and / or components for performing a method of the present disclosure. In certain embodiments, a kit of the present disclosure includes a container containing reagents, e.g., a reagent composition, for performing a method of the present disclosure that comprises a reducing agent and an activator, e.g., at the ratios and amounts described herein. In certain embodiments, the kit contains a reagent composition that include a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. In certain embodiments, the kit contains a reagent composition that include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5. In certain embodiments, the kit contains a reagent composition that include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :4. In certain embodiments, the kit contains a reagent composition that include a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :3. In certain embodiments, the kit contains a reagent composition that include a reducing agent and an activator at a concentration ratio of about 1 :2 to about 1 :4. In certain embodiments, the kit contains a reagent composition that includes from about 10 mM to about of 200 mM of a reducing agent, e.g., THPP, and about 0.1 mM to about of 500 mM of an activator, e.g., MgOAc.

[0271] In certain embodiments, a kit of the present disclosure includes a container containing reagents, e.g, a reagent composition, that comprises (or consists essentially of or consists of) a reducing agent and an activator at a concentration ratio of about 1 : 1 to about 1 :5, where the reducing agent is present in the composition at a concentration from about 50 mM to about 150 mM and the activator is present in the composition at a concentration from about 100 mM to about 300 mM.

[0272] In certain embodiments, a kit of the present disclosure includes a container containing reagents, e.g., a reagent composition, that comprises (or consists essentially of or consists of) a trialkylphosphine and an activator at a concentration ratio of about 1 : 1 to about 1 :5, where the trialkylphosphine is present in the composition at a concentration from about 50 mM to about 150 mM and the activator is present in the composition at a concentration from about 100 mM to about 300 mM.

[0273] In certain embodiments, a kit of the present disclosure includes a container containing reagents, e.g., a reagent composition, that comprises (or consists essentially of or consists of) THPP and a magnesium or salt form thereof at a concentration ratio of about 1 : 1 to about 1:5, where THPP is present in the composition at a concentration from about 50 mM to about 150 mM and the magnesium or salt form thereof is present in the composition at a concentration from about 100 mM to about 300 mM.

[0274] In certain embodiments, a kit of the present disclosure can further include one or more additional reagent compositions (e.g., a second and / or third reagent composition) that includes 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, ATP or an ATP analog, at least one recombinase loading protein, a crowding agent, a first primer and, optionally, a second primer, a probe, a reverse transcriptase and a template nucleic acid molecule, e.g., a single-stranded (e.g., RNA) or double stranded nucleic acid. In certain embodiments, a kit of the present disclosure further includes an additional reagent composition (e.g., a second reagent composition) that includes the protein components (PC) ( / . e. , enzymes) required for the RPA process (e.g., a DNA polymerase, a recombinase, a recombinase loading protein, a single stranded binding protein, creatine kinase, a nuclease (e.g., an exonuclease) and / or a reverse transcriptase). In certain embodiments, a kit of the present disclosure further includes an additional reagent composition (e.g., a second reagent composition) that includes the oligonucleotide components (OC) required for the RPA process (e.g., one or more primers and / or one or more probes specific to a target nucleic acid or two or more target nucleic acids). In certain embodiments, a kit of the present disclosure further includes an additional reagent composition (e.g., a second reagent composition) that includes the non-protein components (NPC) required for the RPA process (e.g, dNTPs, a buffer, salts, ATP or an ATP analog and / or a crowding agent). In certain embodiments, the one or more compositions of a kit disclosed herein can be provided in different containers.

[0275] In certain embodiments, the one or more compositions of a kit disclosed herein can be lyophilized.

[0276] Suitable containers include, but are not limited to, bottles, test tubes, vials and microtiter plates. The containers can be formed from a variety of materials such as glass or plastic.

[0277] In certain embodiments, the kit further includes a package insert that provides instructions for using the components provided in the kit. For example, a kit of the present disclosure can include a package insert that provides instructions for performing methods of the present disclosure.

[0278] In certain embodiments, the kit can include other materials desirable from a commercial and user standpoint, including other buffers and diluents.

[0279] VII. Exemplary Embodiments

[0280] A. The present disclosure provides a composition for performing an isothermal amplification process, wherein the composition comprises a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

[0281] Al. The composition of A, wherein the concentration ratio is about 1 :0.25 to about 1 :3.

[0282] Al-1. The composition of A or Al, wherein the concentration ratio is about 1 : 1 to about 1 :4.

[0283] Al-2. The composition of A or Al-1, wherein the concentration ratio is about 1 : 1 to about 1 :3.

[0284] Al -3. The composition of any one of A-Al-2, wherein the concentration ratio is about 1 :2 to about 1 :3.

[0285] Al-4. The composition of any one of A-Al-3, wherein the composition comprises from about 1 mM to about 200 mM of the reducing agent.

[0286] A2. The composition of any one of A-Al-4, wherein the composition comprises from about 10 mM to about 200 mM of the reducing agent.

[0287] A3. The composition of any one of A-A2, wherein the composition comprises from about 50 mM to about 100 mM of the reducing agent.

[0288] A4. The composition of any one of A-A3, wherein the composition comprises from about 0.1 mM to about 500 mM of the activator. A5. The composition of any one of A-A4, wherein the composition comprises from about 1 mM to about 300 mM of the activator.

[0289] A5-1. The composition of any one of A-A5, wherein the composition comprises from about 100 mM to about 250 mM of the activator.

[0290] A5-2. The composition of any one of A-A5-1, wherein the reducing agent is a tri alkylphosphine.

[0291] A6. The composition of any one of A-A5-2, wherein the reducing agent is tris(hydroxypropyl)phosphine (THPP).

[0292] A7. The composition of any one of A-A6, wherein the activator is magnesium, manganese or a salt form thereof.

[0293] A8. The composition of any one of A-A7, wherein the isothermal amplification process is selected from the group consisting of rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement 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).

[0294] A9. The composition of A8, wherein the isothermal amplification process is RPA.

[0295] A10. The composition of A8, wherein the isothermal amplification process is NEAR.

[0296] Al l. The composition of any one of A-A10, wherein the reducing agent is stable in the composition for a time period greater than about 14 days.

[0297] A12. The composition of any one of A-Al 1, wherein less than about 20% of the reducing agent is oxidized when stored for a time period greater than about 14 days.

[0298] A13. The composition of any one of A-A12, wherein the composition is stored at a temperature from about 2°C to about 40°C.

[0299] A13-1. The composition of any one of A-A13, wherein the composition is stored at a temperature from about 25°C to about 40°C.

[0300] A14. The composition of any one of A-A13-1, wherein the composition comprises a Tris buffer.

[0301] A15. The composition of any one of A-A14, wherein the composition has a pH from about 5 to about 11. Al 6. The composition of any one of A-A15, wherein the composition consists essentially of the reducing agent and the activator.

[0302] Al 7. The composition of any one of A-A16, wherein the composition consists of the reducing agent and the activator.

[0303] A18. The composition of any one of A-A17, wherein the composition is for use in detecting a target nucleic acid in a sample.

[0304] Al 9. The composition of Al 8, wherein the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid.

[0305] A20. The composition of Al 8 or Al 9, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Parvovirus Bl 9, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Virus, Influenza, Babesia, Malaria, Usutu Virus or HEV.

[0306] A21. The composition of any one of A-A20, wherein the target nucleic acid is derived from HIV-1.

[0307] A22. The composition of any one of A-A20, wherein the target nucleic acid is derived from HIV-2.

[0308] A23. The composition of any one of A-A20, wherein the target nucleic acid is derived from HBV.

[0309] A24. The composition of any one of A-A20, wherein the target nucleic acid is derived from HCV.

[0310] A25. The composition of any one of A-A20, wherein the target nucleic acid is derived from Malaria.

[0311] A26. The composition of any one of A-A20, wherein the target nucleic acid is derived from Babesia.

[0312] B. The present disclosure provides an isothermal amplification process for amplifying a target nucleic acid comprising contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

[0313] Bl. The present disclosure provides an isothermal amplification process for amplifying a target nucleic acid, wherein the isothermal amplification process is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5. B2. The present disclosure provides an isothermal amplification process for amplifying a target nucleic acid comprising: (a) contacting a solution comprising a plurality of nucleic acids and one or more isothermal amplification reagents to generate a reaction mixture; (b) contacting the reaction mixture with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 to generate an activated reaction mixture; and (c) incubating the activated reaction mixture to amplify the target nucleic acid if present in the plurality of nucleic acids.

[0314] B2-1. The present disclosure provides an isothermal amplification process for amplifying a target nucleic acid comprising: (a) contacting a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 with one or more isothermal amplification reagents to generate a reaction mixture, (b) contacting the reaction mixture with a solution comprising a plurality of nucleic acids and (c) incubating the reaction mixture comprising the plurality of nucleic acids to amplify the target nucleic acid if present in the plurality of nucleic acids.

[0315] B2-2. The method of isothermal amplification process of any one of B-B2-1 further comprising: detecting the amplified target nucleic acid.

[0316] B3. The isothermal amplification process of any one of B-B2-2, wherein the concentration ratio is about 1 :0.25 to about 1 :3.

[0317] B3-1. The isothermal amplification process of any one of B-B3, wherein the concentration ratio is about 1 :1 to about 1 :4.

[0318] B3-2. The isothermal amplification process of any one of B-B3-1, wherein the concentration ratio is about 1 :1 to about 1 :3.

[0319] B3-3. The isothermal amplification process of any one of B-B3-2, wherein the concentration ratio is about 1 :2 to about 1 :3.

[0320] B4. The isothermal amplification process of any one of B-B3-3, wherein the composition comprises from about 1 mM to about 200 mM of the reducing agent.

[0321] B4-1. The isothermal amplification process of any one of B-B4, wherein the composition comprises from about 10 mM to about 200 mM of the reducing agent.

[0322] B5. The isothermal amplification process of any one of B-B4-1, wherein the composition comprises from about 50 mM to about 100 mM of the reducing agent.

[0323] B6. The isothermal amplification process of any one of B-B5, wherein the composition comprises from about 0.1 mM to about 500 mM of the activator.

[0324] B6-1. The isothermal amplification process of any one of B-B6, wherein the composition comprises from about 1 mM to about 300 mM of the activator. B6-2. The isothermal amplification process of any one of B-B6-1, wherein the composition comprises from about 1 mM to about 250 mM of the activator.

[0325] B6-3. The isothermal amplification process of any one of B-B6-2, wherein the composition comprises from about 10 mM to about 250 mM of the activator.

[0326] B7. The isothermal amplification process of any one of B-B6-3, wherein the reducing agent is a trialkylphosphine.

[0327] B8. The isothermal amplification process of any one of B-B7, wherein the reducing agent is tris(hydroxypropyl)phosphine (THPP).

[0328] B9. The isothermal amplification process of any one of B-B8, wherein the activator is magnesium, manganese or a salt form thereof.

[0329] B9-1. The isothermal amplification process of any one of B-B9, wherein the activator is magnesium or a salt form thereof.

[0330] BIO. The isothermal amplification process of any one of B-B9-1, wherein the reducing agent is stable in the composition for a time period greater than about 14 days.

[0331] Bl 1. The isothermal amplification process of any one of B-B10, wherein less than about 20% of the reducing agent is oxidized when stored for a time period greater than about 14 days.

[0332] B12. The isothermal amplification process of any one of B-Bl l, wherein the composition is stored at a temperature from about 2°C to about 40°C.

[0333] B12-1. The isothermal amplification process of any one of B-B12, wherein the composition is stored at a temperature from about 25°C to about 40°C.

[0334] B13. The isothermal amplification process of any one of B-B12-1, wherein the composition comprises a Tris buffer.

[0335] B14. The isothermal amplification process of any one of B-B13, wherein the composition has a pH from about 5 to about 11.

[0336] B15. The isothermal amplification process of any one of B-B14, wherein the isothermal amplification process is selected from the group consisting of rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement 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).

[0337] Bl 6. The isothermal amplification process of Bl 5, wherein the isothermal amplification process is RPA. Bl 7. The isothermal amplification process of Bl 5, wherein the isothermal amplification process is NEAR.

[0338] Bl 8. The isothermal amplification process of any one of B-B17, wherein the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid.

[0339] Bl 9. The isothermal amplification process of any one of B-B18, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Parvovirus Bl 9, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Virus, Influenza, Babesia, Malaria, Usutu Virus or HEV.

[0340] B20. The isothermal amplification process of any one of B-B19, wherein the isothermal amplification process amplifies at least two target nucleic acids or at least three target nucleic acids, e.g., in a multiplex reaction.

[0341] B21. The isothermal amplification process of any one of B-B20, wherein the sample is a tissue sample.

[0342] B22. The isothermal amplification process of B21, wherein the target nucleic acid is isolated from the tissue sample prior to amplification.

[0343] B23. The isothermal amplification process of any one of B-B22, wherein the sample is a biological fluid.

[0344] B24. The isothermal amplification process of B-B23, wherein the biological fluid is blood.

[0345] B25. The isothermal amplification process of B-B24, wherein the target nucleic acid is isolated from the biological fluid prior to amplification.

[0346] B26. The isothermal amplification process of any one of B-B25, wherein the one or more isothermal amplification reagents comprise one or more of a recombinase, a singlestranded DNA binding protein, a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a recombinase loading protein, a first primer, a second primer, a probe, a nuclease, a creatine kinase and a reverse transcriptase.

[0347] B27. The isothermal amplification process of any one of B-B26, wherein the target nucleic acid is derived from HIV-1.

[0348] B28. The isothermal amplification process of any one of B-B26, wherein the target nucleic acid is derived from HIV-2.

[0349] B29. The isothermal amplification process of any one of B-B26, wherein the target nucleic acid is derived from HBV.

[0350] B30. The isothermal amplification process of any one of B-B26, wherein the target nucleic acid is derived from HCV. B31. The isothermal amplification process of any one of B-B26, wherein the target nucleic acid is derived from Malaria.

[0351] B32. The isothermal amplification process of any one of B-B26, wherein the target nucleic acid is derived from Babesia.

[0352] B33. The isothermal amplification process of any one of B-B32, wherein the composition consists essentially of the reducing agent and the activator.

[0353] B34. The isothermal amplification process of any one of B-B33, wherein the composition consists of the reducing agent and the activator.

[0354] B35. The isothermal amplification process of any one of B-B34, wherein the reaction mixture comprises a reaction concentration of the reducing agent from about 1 mM to about 10 mM.

[0355] B36. The isothermal amplification process of any one of B-B35, wherein the reaction mixture comprises a reaction concentration of the activator from about 10 mM to about 20 mM.

[0356] C. The present disclosure provides a system for performing an isothermal amplification process, wherein the system comprises a container comprising the composition of any one of A-A26.

[0357] Cl. The present disclosure provides a system for performing the isothermal amplification process of any one of B-B36.

[0358] C2. The system of C or Cl, wherein the system is automated.

[0359] D. The present disclosure provides a kit comprising the composition of any one of A-A26.

[0360] DI. The present disclosure provides a kit for performing the isothermal amplification process of any one of B-B36.

[0361] EXAMPLES

[0362] The presently disclosed subject matter will be better understood by reference to the following example, which is provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0363] Example 1: Use of compositions including a reducing agent and an activator for performing an RPA reaction.

[0364] This example discloses the addition of a reducing agent in the activator solution for performing an RPA reaction. A reducing agent, such as DTT, is required during an RPA reaction to reduce disulfide bonds in the RPA enzymes for full protein functionality after storage of the enzymes. The reducing agent must retain stability at the intended storage conditions for an extended period time to ensure useability of the reducing agent. In a typical RPA reaction, reducing agent DTT is included in the reagent composition that includes the non-protein components (also reference to herein as “NPC”) that are used to perform the reaction. The final concentration of DTT in the RPA reaction is 5 mM.

[0365] Initial studies were performed to analyze the stability of DTT in the NPC at various storage temperatures and time periods by analyzing the performance of RPA reactions that use the NPCs stored at the various conditions. As shown in FIG. 1A, the stability of DTT at day 7 when stored at 2-8°C was similar to when stored at -70°C for performing an RPA reaction of HIV-2 nucleic acids. However, as shown in FIG. IB, the presence of DTT in the NPC was not sufficiently stable for RPA reactions of HIV-2 after two or more weeks of storage at 2-8°C compared to when stored at -70°C. In particular, as shown in FIG. IB, after 15 days of being stored at 2-8°C, DTT was not sufficiently stable to support an RPA reaction compared to when stored at -70°C. In addition, the storage temperature of the RPA enzymes at -70°C (referred to as the protein component (“PC”)) did not affect the RPA reaction when the NPC was stored at 2-8°C (FIG. IB) confirming that the storage conditions of the reducing agent are important. Similar results were shown for an RPA reaction for Babesia. Although, longer DTT stability was observed during an RPA reaction for Babesia (FIG. 2A), decay of DTT was observed by day 21 (FIG. 2B). In addition, decay was accelerated at higher temperatures such as 25°C when compared to storage at 2-8°C (FIG. 2B). Similar results were shown for amplification of HBV, HCV, WNV, Parvo / HAV, Zika and an internal control (IC).

[0366] The importance of the storage conditions of the reducing agent was confirmed by performing spike-in experiments. As shown in FIG. 3 A, NPC without DTT does not support a strong RPA reaction, even when NPC is freshly thawed. As shown in FIG. 3B, NPC containing DTT stored at 2-8°C for 21 days performed poorly. However, spiking fresh DTT into an RPA reaction that uses an NPC composition stored at 2-8°C for 21 days recovered RPA signal to the level of the frozen control, showing that DTT had significantly decayed in the NPC during 2-8°C storage (FIG. 3B).

[0367] To achieve a greater storage time of the reducing agent that is needed for an RPA reaction, alternative reducing agents and storage locations were analyzed. Alternative reducing agents including THPP and TCEP were analyzed. When included in the NPC and tested at the final reaction concentration of 5 mM, THPP was stable for greater than 31 days (FIG. 4A) in an RPA reaction for Parvo. In contrast, the RPA reactions for Parvo at 18 days using DTT and 31 days using DTT or TCEP were similar to RPA curves that indicate degraded reducing agent (weak amplitude and low efficiency) (FIG. 4A). When included in the NPC and PC (at 15 mM and ~0.5 mM final reaction concentration, respectively) in an RPA reaction for Parvo and HAV, THPP showed greater stability at day 21 than TCEP and DTT when stored at 2-8°C, and TCEP showed greater stability at day 21 than DTT when stored at 2-8°C (FIG. 4B). As shown in FIG. 4B, THPP when included in both the PC and NPC and stored at 2-8°C for 21 days slightly shifted the RPA curve when compared to the reagents stored at -20°C from frozen control. The use of TCEP shifted the curve more for HAV and appeared significantly decayed for Parvo. The presence of DTT in the PC and NPC resulted in curves similar to the “No Reducing Agent” (No RA) control by day 21, indicating DTT is nearly completely decayed (FIG. 4B). These data show that the THPP has greater stability that DTT and TCEP in the NPC and PC compositions when stored for long durations.

[0368] Next, the location of the reducing agent was analyzed to determine if a greater increase in stability of the reducing agent could be achieved. The reducing agent was added to the solution containing the activator magnesium acetate (MgOAc). The activator solution is added to RPA reaction to initiate amplification. A stability study was performed for RPA reactions for Parvo that included NPC, PC, and OC compositions stored at 2-8°C with a solution including THPP and MgOAc stored at 37°C. The storage temperatures and locations are shown in FIG. 5B. As shown in FIG. 5 A, the THPP stored in the MgOAc solution was significantly more stable than the DTT of the previous formulation. Addition of THPP and MgOAc fully rescued the RPA signal to be comparable to the signal obtained when the reagents were stored at -20°C; whereas, almost no signal was obtained after 1 month using the previous DTT formulation (FIG. 5A). After three months of storage, the use of NPC and PC components that were stored at 2-8°C with a THPP and Mg solution stored at 37°C showed no degradation compared to control at -20°C (FIG. 5A).

[0369] A stability study over a longer time frame was performed and the effect of storage temperatures of the activator solution containing THPP was analyzed. In particular, the stability of a MgOAc solution containing 80 mM THPP in 10 mM Tris (pH 8.5) was analyzed at 2-8°C and 37°C. Each timepoint in the assay was tested with fresh NPC and PC components. As shown in FIG. 6A, the performance of an RPA reaction using a solution containing THPP and MgOAc that was stored at 37°C for 232 days (approximately 7 months) was similar to the performance of an RPA reaction that used a THPP / MgOAc solution that was stored at 2-8°C for 232 days. Similar results were observed after storage of the THPP / MgOAc solution at 2-8°C and 37°C for 9 months. An Ellman’s assay was performed to determine the oxidation of the reducing agent over time. Oxidation of the reducing agent blocks its ability to reduce the disulfide bonds, thereby making the reducing agent inactive. The Ellman’s assay showed that THPP is slowly oxidizing over time, with about 50% loss of active THPP by approximately 7.5 months when stored at 37°C (FIG. 6B), leaving approximately 40 mM of available THPP in solution. A minimum starting concentration of approximately 8 mM THPP in the THPP / MgOAc stock solution is needed to support the reaction. Thus, the amount of active THPP remaining after 7.5 months is sufficient to support an RPA reaction (z.e., 40 mM remaining > 8 mM). When stored at 2- 8°C, a loss of about 14 mM active THPP by approximately 7.5 months was observed, leaving about 66 mM of active THPP (FIG. 6B).

[0370] The stability of THPP in the presence or absence of MgOAc was further analyzed using the Ellman’s assay. It was found that THPP (62 mM) in the presence of MgOAc is much more stable than THPP, TCEP or DTT alone in buffer (FIG. 7). At 37°C, THPP was stabilized by MgOAc (224 mM) and showed a loss of active THPP of no more than 20%. The stabilizing effect of MgOAc on THPP was seen at all temperatures tested between 4°C and 54°C. In addition, THPP was tested in many buffer / pH conditions, but none were nearly as stable as with addition of MgOAc. A Tris (pH 8.3-8.5) buffer was found to be an optimal buffer to be used for the THPP / MgOAc solution, and such a buffer is compatible with RPA. As shown in FIG. 8, TCEP stability was not improved when stored with MgOAc compared with TCEP-only control. Additional buffer and pH conditions were analyzed but only marginal improvement was observed when using extreme pHs that are not compatible with RPA reactions.

[0371] Lower MgOAc concentrations than the nominal MgOAc concentration were also analyzed to determine if they could effectively stabilize THPP. A MgOAc concentration of 20 mM, which is less than 10% of the nominal concentration of MgOAc (z.e., 224 mM), was enough to limit THPP decay by day 36 of 45°C accelerated storage (FIG. 9). As shown in FIG. 9, a solution with 80 mM THPP and 20 mM MgOAc (having an approximate ratio of 1 :0.25) had equivalent stability to a solution with 80 mM THPP and 224 mM MgOAc (having an approximate ratio of 1 :3), as demonstrated by Ellman’s assay results. A MgOAc concentration of 1 mM was also able to stabilize 80 mM THPP in solution when stored for one week at the high temperatures of 45°C and 54°C as determined by Ellman’s assay (FIG. 10). These data show that THPP in the MgOAc activator solution is more stable than when THPP is stored with other components used in RPA reactions. Without being limited to a potential theory, while RPA needs MgOAc to activate the reaction, the small amount of MgOAc required to stabilize THPP can potentially allow this stabilization method of THPP to be used in other reactions besides RPA, where stable reducing agents may be needed but low activator concentrations are preferred.

[0372] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. Accordingly, the appended claims are intended to comprise within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

[0373] Various patents, patent applications, publications, product descriptions and protocols are cited throughout this application, the disclosure of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A composition for performing an isothermal amplification process, wherein the composition comprises a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

2. The composition of claim 1, wherein the concentration ratio is about 1 :0.25 to about 1 :3.

3. The composition of claim 1 or 2, wherein the composition comprises from about 1 mM to about 200 mM of the reducing agent.

4. The composition of any one of claims 1-3, wherein the composition comprises from about 50 mM to about 100 mM of the reducing agent.

5. The composition of any one of claims 1-4, wherein the composition comprises from about 0.1 mM to about 500 mM of the activator.

6. The composition of any one of claims 1-5, wherein the composition comprises from about 1 mM to about 250 mM of the activator.

7. The composition of any one of claims 1-6, wherein the reducing agent is tris(hydroxypropyl)phosphine (THPP).

8. The composition of any one of claims 1-7, wherein the activator is magnesium, manganese or a salt form thereof.

9. The composition of any one of claims 1-8, wherein the isothermal amplification process is selected from the group consisting of rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement 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).

10. The composition of claim 9, wherein the isothermal amplification process is RPA.

11. The composition of claim 9, wherein the isothermal amplification process is NEAR.

12. The composition of any one of claims 1-11, wherein the reducing agent is stable in the composition for a time period greater than about 14 days.

13. The composition of any one of claims 1-12, wherein less than about 20% of the reducing agent is oxidized when stored for a time period greater than about 14 days.

14. The composition of any one of claims 1-13, wherein the composition is stored at a temperature from about 2°C to about 40°C.

15. The composition of any one of claims 1-14, wherein the composition comprises a Tris buffer.

16. The composition of any one of claims 1-15, wherein the composition has a pH from about 5 to about 11.

17. An isothermal amplification process for amplifying a target nucleic acid comprising contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

18. An isothermal amplification process for amplifying a target nucleic acid, wherein the isothermal amplification process is initiated by contacting a sample comprising the target nucleic acid and one or more isothermal amplification reagents with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5.

19. An isothermal amplification process for amplifying a target nucleic acid comprising: a) contacting a solution comprising a plurality of nucleic acids and one or more isothermal amplification reagents to generate a reaction mixture; b) contacting the reaction mixture with a composition comprising a reducing agent and an activator at a concentration ratio of about 1 :0.01 to about 1 :5 to generate an activated reaction mixture; and c) incubating the activated reaction mixture to amplify the target nucleic acid if present in the plurality of nucleic acids.

20. The isothermal amplification process of any one of claims 17-19, wherein the concentration ratio is about 1 :0.25 to about 1 :3.

21. The isothermal amplification process of any one of claims 17-20, wherein the composition comprises from about 1 mM to about 200 mM of the reducing agent.

22. The isothermal amplification process of any one of claims 17-21, wherein the composition comprises from about 50 mM to about 100 mM of the reducing agent.

23. The isothermal amplification process of any one of claims 17-22, wherein the composition comprises from about 0.1 mM to about 500 mM of the activator.

24. The isothermal amplification process of any one of claims 17-23, wherein the composition comprises from about 1 mM to about 250 mM of the activator.

25. The isothermal amplification process of any one of claims 17-24, wherein the reducing agent is tris(hydroxypropyl)phosphine (THPP).

26. The isothermal amplification process of any one of claims 17-25, wherein the activator is magnesium, manganese or a salt form thereof.

27. The isothermal amplification process of any one of claims 17-26, wherein the reducing agent is stable in the composition for a time period greater than about 14 days.

28. The isothermal amplification process of any one of claims 17-27, wherein less than about 20% of the reducing agent is oxidized when stored for a time period greater than about14 days.

29. The isothermal amplification process of any one of claims 17-28, wherein the composition is stored at a temperature from about 2°C to about 40°C.

30. The isothermal amplification process of any one of claims 17-29, wherein the composition comprises a Tris buffer.

31. The isothermal amplification process of any one of claims 17-30, wherein the composition has a pH from about 5 to about 11.

32. The isothermal amplification process of any one of claims 17-31, wherein the isothermal amplification process is selected from the group consisting of rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), strand displacement 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).

33. The isothermal amplification process of claim 32, wherein the isothermal amplification process is RPA.

34. The isothermal amplification process of claim 32, wherein the isothermal amplification process is NEAR.

35. The isothermal amplification process of any one of claims 17-34, wherein the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid.

36. The isothermal amplification process of any one of claims 17-35, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Parvovirus Bl 9, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Virus, Influenza, Babesia, Malaria, Usutu Virus or HEV.

37. The isothermal amplification process of any one of claims 17-36, wherein the isothermal amplification process amplifies at least two target nucleic acids or at least three target nucleic acids, e.g., in a multiplex reaction.

38. The isothermal amplification process of any one of claims 17-37, wherein the sample is a tissue sample.

39. The isothermal amplification process of claim 38, wherein the target nucleic acid is isolated from the tissue sample prior to amplification.

40. The isothermal amplification process of any one of claims 17-39, wherein the sample is a biological fluid.

41. The isothermal amplification process of claim 40, wherein the biological fluid is blood.

42. The isothermal amplification process of claim 40 or 41, wherein the target nucleic acid is isolated from the biological fluid prior to amplification.

43. The isothermal amplification process of any one of claims 17-42, wherein the one or more isothermal amplification reagents comprise one or more of a recombinase, a singlestranded DNA binding protein, a DNA polymerase, dNTPs, a buffer, a crowding agent, ATP or an ATP analog, a recombinase loading protein, a first primer, a second primer, a probe, a nuclease, a creatine kinase and a reverse transcriptase.

44. The isothermal amplification process of any one of claims 19-43, wherein the activated reaction mixture comprises a reaction concentration of the reducing agent from about 1 mM to about 10 mM.

45. The isothermal amplification process of any one of claims 19-44, wherein the activated reaction mixture comprises a reaction concentration of the activator from about 10 mM to about 20 mM.

46. A system for performing an isothermal amplification process, wherein the system comprises a container comprising the composition of any one of claims 1-16.

47. A system for performing the isothermal amplification process of any one of claims 17-45.

48. The system of claim 46 or 47, wherein the system is automated.

49. A kit comprising the composition of any one of claims 1-16.

50. A kit for performing the isothermal amplification process of any one of claims 17-45.

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