Methods of detecting variant nucleic acids

Abstract

The present disclosure relates to methods for detecting target nucleic acids, e.g., variants of target nucleic acids, in a sample, using two different detection probes for the same target, in two different concentrations. The present disclosure further provides compositions, systems and kits for performing such methods.

Description

[0001] METHODS OF DETECTING VARIANT NUCLEIC ACIDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The application claims priority to U.S. Provisional Application No. 63 / 580,287, filed September 1, 2023, the contents of which is incorporated herein by reference in its entirety.

[0004] FIELD

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

[0006] BACKGROUND

[0007] The ability to detect nucleic acids lies at the heart of modern biological and medical research. In particular, detection of nucleic acids is commonly used in nucleic acidbased diagnostic methods for confirming the presence of an infection, e.g., viral infection or bacterial infection, a disease or a genetic mutation in a subject. For example, identification of bacterial or viral nucleic acid, e.g., HIV-1, in a blood sample can be useful in determining whether donated blood is safe for transfusion.

[0008] However, an issue that can occur with nucleic acid detection assays is the underquantification of minority variants of certain pathogens that may be present in a sample. For example, certain subgroups of viruses such as HIV-1 subgroups are commonly underquantified. HIV-1 is classified according to the degree of genetic similarity between isolates. There are currently four groups of HIV- 1 isolates (types): M, N, O and P, and each group includes several subtypes. It has been shown that many nucleic acid detection assays underquantify the minority variants of HIV- 1 such as the O and N group isolates. Therefore, there is a need in the art for more sensitive assays that can detect variants of target nucleic acids.

[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. 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 includes methods for detecting one or more target nucleic acids in a sample. In certain embodiments, the method includes performing an isothermal amplification process and detecting the one or more target nucleic acids with a first probe and a second probe, where the concentration of the second probe is less than the concentration of the first probe. In certain embodiments, the method includes (a) performing an isothermal amplification process that includes contacting a sample with a reagent composition comprising a first probe and a second probe, where the concentration of the second probe in the reagent composition is less than the concentration of the first probe and amplifying the one or more target nucleic acids; and (b) detecting the one or more target nucleic acids with the first probe and the second probe.

[0011] In certain embodiments, the present disclosure provides a method of detecting one or more variants of a target nucleic acid in a sample. In certain embodiments, the method includes performing an isothermal amplification process and detecting the one or more variants of the target nucleic acid with a first probe and a second probe, where the concentration of the second probe is less than the concentration of the first probe. In certain embodiments, the method includes (a) performing an isothermal amplification process that includes contacting a sample with a reagent composition comprising a first probe and a second probe, where the concentration of the second probe in the reagent composition is less than the concentration of the first probe and amplifying the one or more variants of the target nucleic acid; and (b) detecting the one or more variants of the target nucleic acid with the first probe and the second probe.

[0012] In certain embodiments, the concentration of the second probe is about 5 nM to about 200 nM. In certain embodiments, the concentration of the second probe used in a method of the present disclosure is about 15 nM to about 80 nM. In certain embodiments, the concentration of the second probe is about 25 nM or about 65 nM.

[0013] In certain embodiments, the concentration of the first probe is about 20 nM to about 300 nM. In certain embodiments, the concentration of the first probe is about 60 nM to about 170 nM.

[0014] In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 8. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 7. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 5.

[0015] In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe of about 90% or greater. In certain embodiments, the second probe comprises a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe comprises a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe comprises a nucleotide sequence that is identical to or differs by no more than 2 nucleotides from the nucleotide sequence of the first probe.

[0016] In certain embodiments, the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 100 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe. In certain embodiments, the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 50 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe. In certain embodiments, the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 10 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe.

[0017] In certain embodiments, the first probe used in a method of the present disclosure is labeled with a first fluorophore. In certain embodiments, the second probe used in a method of the present disclosure is labeled with a second fluorophore. In certain embodiments, the first fluorophore and the second fluorophore are different. In certain embodiments, the first fluorophore and the second fluorophore are the same.

[0018] In certain embodiments, the isothermal amplification process for use in a method of the present disclosure 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). In certain embodiments, the isothermal amplification process is RPA. In certain embodiments, the isothermal amplification process is NEAR.

[0019] In certain embodiments, the target nucleic acid detected by the methods of the present disclosure 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. 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 HEV.

[0020] In certain embodiments, the target nucleic acid detected by the methods of the present disclosure is a variant of the target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a minority variant of the target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a minority variant of a HIV-1 target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a group N variant of a HIV-1 target nucleic acid, e.g., of the INT gene. In certain embodiments, the variant of the target nucleic acid is a variant of a HIV-2 target nucleic acid, e.g., of the POL gene. In certain embodiments, the variant of the target nucleic acid is a subtype A variant of a HIV-2 target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a minority variant of a HEV target nucleic acid, e.g., of ORF1 or the 5’ UTR (e.g., within the 5’ UTR of ORF 1). In certain embodiments, the variant of the target nucleic acid is a genotype GT3 variant of a HEV target nucleic acid.

[0021] In certain embodiments, the sample being analyzed by a method of the present disclosure is a tissue sample. In certain embodiments, the target nucleic acid is isolated from the tissue sample prior to amplification. In certain embodiments, the sample is a biological fluid. In certain embodiments, the biological fluid is blood. In certain embodiments, the target nucleic acid is isolated from the biological fluid prior to amplification.

[0022] In certain embodiments, the reagent composition for use in the disclosed methods further comprises one or more of the following: a DNA polymerase; a recombinase; a recombinase loading protein; a single stranded binding protein; dNTPs or a mixture of dNTPs and ddNTPs; a reducing agent; creatine kinase; a nuclease; a crowding agent; at least one primer; and a reverse transcriptase.

[0023] The present disclosure further provides a composition for detecting one or more target nucleic acids. In certain embodiments, the composition includes a first probe and a second probe, where the concentration of the second probe is less than the concentration of the first probe. The present disclosure provides a composition for detecting one or more variants of a target nucleic acid that includes a first probe and a second probe, where the concentration of the second probe is less than the concentration of the first probe.

[0024] In certain embodiments, the concentration of the second probe in a composition of the present disclosure is about 5 nM to about 200 nM, e.g., about 15 nM to about 80 nM. In certain embodiments, the concentration of the second probe in a composition of the present disclosure is about 5 nM to about 200 nM, e.g., about 15 nM to about 75 nM. In certain embodiments, the concentration of the second probe is about 25 nM or about 65 nM. In certain embodiments, the concentration of the first probe in a composition of the present disclosure is about 20 nM to about 300 nM. In certain embodiments, the concentration of the first probe in a composition of the present disclosure is about 60 nM to about 170 nM. In certain embodiments, the concentration of the first probe in a composition of the present disclosure is about 80 nM to about 300 nM. In certain embodiments, the concentration of the first probe in a composition of the present disclosure is about 80 nM to about 200 nM. In certain embodiments, the concentration of the first probe in a composition of the present disclosure is about 80 nM to about 180 nM. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in a composition of the present disclosure is about 1.5 to about 20. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in a composition of the present disclosure is about 1.5 to about 10. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in a composition of the present disclosure is about 1.5 to about 8. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in a composition of the present disclosure is about 1.5 to about 5. In certain embodiments, the second probe in the composition comprises a nucleotide sequence having an identity to the first probe of about 85% or greater. In certain embodiments, the second probe in the composition comprises a nucleotide sequence having an identity to the first probe of about 90% or greater. In certain embodiments, the second probe in the composition comprises a nucleotide sequence having an identity to the first probe of about 95% or greater. In certain embodiments, the second probe in the composition comprises a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe in the composition comprises a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe in the composition comprises a nucleotide sequence that is identical to or differs by no more than 2 nucleotides from the nucleotide sequence of the first probe.

[0025] In certain embodiments, the first probe in a composition of the present disclosure is labeled with a first fluorophore. In certain embodiments, the second probe in a composition of the present disclosure is labeled with a second fluorophore. In certain embodiments, the first fluorophore and the second fluorophore are different. In certain embodiments, the first fluorophore and the second fluorophore are the same.

[0026] In certain embodiments, the second probe in a composition of the present disclosure hybridizes to a region of the target nucleic acid that is located at a distance of about 100 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe. In certain embodiments, the second probe in a composition of the present disclosure hybridizes to a region of the target nucleic acid that is located at a distance of about 50 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe. In certain embodiments, the second probe in a composition of the present disclosure hybridizes to a region of the target nucleic acid that is located at a distance of about 10 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe.

[0027] In certain embodiments, the composition further comprises one or more reagents for performing an isothermal amplification process. In certain embodiments, 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). In certain embodiments, the isothermal amplification process is RPA. In certain embodiments, the isothermal amplification process is NEAR.

[0028] In certain embodiments, the composition further comprises one or more of the following: a DNA polymerase; a recombinase; a recombinase loading protein; a single stranded binding protein; dNTPs or a mixture of dNTPs and ddNTPs; a reducing agent; creatine kinase; a nuclease; a crowding agent; at least one primer; and a reverse transcriptase.

[0029] In certain embodiments, the target nucleic acid detected using the compositions of the present disclosure is a variant of the target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a minority variant of the target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a minority variant of a HIV-1 target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a group N variant of a HIV-1 target nucleic acid, e.g., of the INT gene. In certain embodiments, the variant of the target nucleic acid is a variant of a HIV-2 target nucleic acid, e.g., of the POL gene. In certain embodiments, the variant of the target nucleic acid is a subtype A variant of a HIV-2 target nucleic acid. In certain embodiments, the variant of the target nucleic acid is a minority variant of a HEV target nucleic acid, e.g., of 0RF1 or the 5’ UTR (e.g., within the 5’ UTR of ORF 1). In certain embodiments, the variant of the target nucleic acid is a genotype GT3 variant of a HEV target nucleic acid.

[0030] In another aspect, the present disclosure provides a system for performing the methods described herein. In certain embodiments, the system of the present disclosure includes one or more containers or reservoirs comprising a composition described herein. In certain embodiments, the system is an automated system.

[0031] The present disclosure further provides kits for performing the methods described herein.

[0032] The present disclosure provides kits including the compositions described herein.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] FIGS. 1A-1B provide exemplary results of an RPA process that includes the use of a secondary probe at a concentration of 25 nM.

[0036] FIGS. 2A-2C provide diagrams that illustrates exemplary results of an RPA process that includes the detection of HIV-2A and HIV-2B subtypes associated with the addition of 25 nM of a secondary probe.

[0037] FIG. 3A provides exemplary results of an RPA process for detecting an HEV variant that includes the use of a secondary probe at a concentration of 15 nM.

[0038] FIG. 3B provides the design of the primary and secondary probes for detecting amplicons relating to an HEV variant nucleic acid. DETAILED DESCRIPTION

[0039] The present disclosure provides improved methods of amplifying and detecting nucleic acids in a sample using isothermal amplification and detection processes, e.g., Recombinase Polymerase Amplification (RPA). The present disclosure further provides compositions, systems and kits for performing such methods.

[0040] The present disclosure is based, in part, on the observation that the presence of a second probe in an RPA reaction significantly improved the detection of nucleic acid variants. As shown in Example 1, the addition of a second probe resulted in a significant increase in the detection of HIV-1 group N nucleic acids and subtypes of HIV-2, and as shown in Example 2, the addition of a second probe resulted in a significant increase in the detection of HEV genotype GT3 nucleic acids. In particular, as shown in FIGS. 1A-1B, FIGS. 2A-2C and FIG. 3A, the addition of the second probe at a low concentration increased the detection of target nucleic acid variants compared to RPA reactions that did not include the second probe or only include the second probe.

[0041] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0042] I. Definitions;

[0043] II. Nucleic Acid Analysis;

[0044] III. Compositions;

[0045] IV. Methods of Use;

[0046] V. Kits;

[0047] VI. Systems; and

[0048] VII. Exemplary Embodiments.

[0049] I. Definitions

[0050] 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.

[0051] 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. 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.

[0052] 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, i.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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] As used herein, the term “clade” or “subtype” refers to a related human immunodeficiency virus (HIV) classified according to the degree of genetic similarity. There are currently four groups of HIV-1 isolates (types): M, N, O and P. Group M (“major” group) consists of at least 12 clades or subgroups, A-L. Group O (“outlier” group) can include a similar number of clades as Group M. Group N (“non-M, non-O” group) is an HIV-1 isolate that does not belong to any category of group M or group O. Group P is a newly identified group.

[0057] 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.

[0058] 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.

[0059] The term “derived” or “derive” is used herein to mean to obtain from a specified source.

[0060] 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.

[0061] As used herein the term “donor” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which donates a biological sample. For example, but not by way of limitation, the biological sample can be blood, serum, or plasma, e.g., for use in transfusions.

[0062] As used herein “donor blood” refers to blood obtained from a donor, e.g., whole blood, lysed whole blood, serum, or plasma, as well as products derived from such blood, e.g., platelets, packed red blood cells, and plasma-derived products such as, but not limited to: (1) coagulation factors, e.g., factor VIII, von Willebrand factor, and fibrinogen; (2) protease inhibitors, e.g., alphal -antitrypsin and Cl -esterase inhibitor; (3) albumin; and (4) immunoglobulin G (IgG). 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 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).

[0063] 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.

[0064] 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).

[0065] 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).

[0066] 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).

[0067] 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. 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.

[0068] Any of the oligonucleotide sequences described herein can comprise, consist essentially of, or consist of a complement of any of the sequences disclosed herein. The terms “complement” or “complementary sequence,” as used herein, refer to a nucleic acid sequence that forms a stable duplex with an oligonucleotide described herein via Watson-Crick base pairing rules, and typically shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% greater identity with the disclosed oligonucleotide.

[0069] 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 al., Meth. Enzymol., 68: 90-98 (1979); Brown et al., Meth. Enzymol., 68: 109-151 (1979); and Belousov et al., Nucleic Acids Res., 25: 3440-3444 (1997), each of which is incorporated herein by reference in its entirety). Oligonucleotide pairs 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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, e.g., about 12-35 nucleotides. In certain embodiments, the probes (e.g., the first probe and / or the second probe) of the present disclosure comprise, consist essentially of, or consist of about 30-50 nucleotides. 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.

[0074] 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 or a gene sequence or fragment of a gene sequence. 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.

[0075] A “result,” as used herein, refers to the detection of the presence of one or more target nucleic acids. In certain embodiments, a result obtained using the methods of the present disclosure can include determining the absence or presence of one or more target nucleic acids. In certain embodiments, a result obtained using the methods of the present disclosure can include the quantification of one or more target nucleic acids.

[0076] As used interchangeably 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.

[0077] 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.

[0078] 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(1): 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(11): 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).

[0079] 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.

[0080] 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.

[0081] A “variant” or “nucleic acid variant,” as used herein, refers to a nucleic acid that comprise modifications, e.g., variations, compared to a wild type nucleic acid. Non-limiting modifications include nucleotide substitutions (e.g., single nucleotide substitutions), translocations, mutations, deletions and insertions. In certain embodiments, a wild type nucleic acid is the most common form of the nucleic acid occurring in nature. In certain embodiments, the nucleic acid variant is a minority variant. In certain embodiments, a minority variant is a low-frequency variant. In certain embodiments, a nucleic acid variant can differ from the wild type nucleic acid by at least one nucleotide modification, e.g., from about one to about ten nucleotide modifications. In certain embodiments, the sequence of a nucleic acid variant has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity to a wild type nucleic acid sequence. In certain embodiments, a nucleic acid variant can differ from a different variant of the nucleic acid by at least one nucleotide modification, e.g., from about one to about ten nucleotide modifications. In certain embodiments, the sequence of a nucleic acid variant has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity to a different variant of the nucleic acid. For example, but not by way of limitation, different subtypes of a virus, e.g., HIV-2A and HIV-2B, are considered variants and the sequences of a gene in each of these subtypes are considered nucleic acid variants.

[0082] IL Nucleic Acid Analysis

[0083] The presently disclosed subject matter provides methods of detecting target nucleic acids in a sample. In certain embodiments, the present disclosure provides improved methods for detecting variants of target nucleic acids in a sample by using at least two probes, e.g., a first probe and a second probe. As shown in Examples 1 and 2 and FIGS. 1A-1B, FIGS. 2A-2C and FIG. 3A, the use of two probes significantly improved detection of variants of a target nucleic acid compared to the use of a single probe.

[0084] In certain embodiments, a nucleic acid analysis of the present disclosure includes amplification of one or more target nucleic acids, e.g, one or more variants of a target nucleic acid, and detection of the one or more variants using two or more probes, e.g, a first probe and a second probe, as described herein. In certain embodiments, a nucleic acid analysis of the present disclosure includes amplification of one or more target nucleic acids using an isothermal amplification process.

[0085] A. Isothermal Amplification Processes

[0086] In certain embodiments, the amplification process for amplifying the target nucleic acids, e.g., target nucleic acid variants, 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. 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. i. RPA Process

[0087] In certain embodiments, the isothermal amplification process for use in a nucleic acid analysis of the present disclosure is RPA. RPA relies on the properties of recombinase and related protein components to invade double-stranded nucleic acids with single stranded homologous nucleic acids permitting sequence specific priming of nucleic acid polymerase reactions.

[0088] 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.

[0089] 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 (c.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. 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.

[0090] 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.

[0091] Steps two and step three are repeated until a desired degree of amplification of the target nucleic acid is achieved.

[0092] 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.

[0093] In certain embodiments, during the amplification process, the amplified target nucleic acids or amplicons are detected using the first and second probes described herein. For example, but not by way of limitation, the target nucleic acid molecules are amplified and detected simultaneously. In certain embodiments, the first and second probes are detected using optical detection as described herein.

[0094] In certain embodiments, the RPA process 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 singlestranded DNA binding protein, at least one DNA polymerase, dNTPs, a buffer, a reducing agent, ATP or an ATP analog, at least one recombinase loading protein, a first primer and, optionally, a second primer, a reverse transcriptase and a template nucleic acid molecule, e.g., a single-stranded (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. In certain embodiments, the RPA process includes the use of two probes (e.g., the first and second probe) as described herein, e.g., for detection of the amplicons generated by RPA. In certain embodiments, an RPA process of the present disclosure includes combining a reaction buffer, dNTPs and primers (e.g., as a first step). In certain embodiments, the probes (e.g., the first and second probe as described herein), crowding agent, the single strand DNA binding protein, the recombinase, the recombinase loading agent, polymerase and the reverse transcriptase is added to the reaction (e.g., as a second step). Alternatively, the reaction buffer, dNTPs, primers, the probes (e.g., the first and second probe as described herein), the crowding agent, the single strand DNA binding protein, the recombinase, the recombinase loading agent, polymerase and reverse transcriptase is provided as a single reaction solution (e.g., a combination of the first and second steps above). The isolated nucleic acids and an activator, e.g, magnesium, are then added to the reaction (e.g, as a third step), followed by the incubation of the reaction at a specified temperature, e.g. , 40°C (e.g. , as a fourth step). In certain embodiments, the reaction solution can further include one or more nonprotein components (e.g., for extending the primers (e.g., dNTPs), for use as an energy source (e.g., ATP and phosphocreatine), for stabilizing the proteins and reaction (e.g., reaction buffer (e.g., Tris and salts)) and for use as a crowding agent (e.g., polyethylene glycol)).

[0095] In certain embodiments, an RPA process of the present disclosure includes combining non-protein components (NPC) (e.g., a reaction buffer, dNTPs, ATP and / or 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 (e.g., two or more probes as described herein) specific to a target nucleic acid (or variants thereof) or two or more target nucleic acids (e.g., in a multiplex RPA process)) with the sample containing the target nucleic acid(s). In certain embodiments, these components can be added in any order to the sample, or the components 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).

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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 some 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.

[0102] 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.

[0103] 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. 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 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 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.

[0104] 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. coll endonuclease IV. In certain embodiments, the unblocking agent is E. coli exonuclease III. In certain embodiments, the unblocking agent is E. coli endonuclease IV. In certain embodiments, an exonuclease is added to the reaction to cleave one or more probes present in the reaction.

[0105] 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.

[0106] 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.

[0107] In certain embodiments, the RPA processes are performed in the presence of at least two or more probes as described further below.

[0108] In certain embodiments, the RPA processes are performed in the presence of about 1 mM to about 25 mM divalent manganese ions, e.g., about 1 mM to about 20 mM, about 1 mM to about 10 mM or about 1 mM to about 3 mM divalent manganese ions. In certain embodiments, the manganese ions replace the magnesium ions and the reaction can be performed with or without magnesium.

[0109] 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).

[0110] 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), SensiScript (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.

[0111] 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.

[0112] 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.

[0113] In certain embodiments, the following reagents can be employed for performing an RPA process of the present disclosure: Tris-HCl, a reducing agent (e.g., DTT), Potassium Acetate, a crowding agent, dNTPs, ATP, Phosphocreatine, Glycerol, Creatine Kinase, UvsX, UvsY, DNA polymerase, GP32, Exonuclease III, BSA, an activator (e.g., magnesium, e.g., Mg Acetate (MgOAc)), forward primers, reverse primers, at least two probes (e.g., a first probe and a second probe) and EIAV. In certain embodiments, ROX reference dyes can be employed in an RPA process of the present disclosure.

[0114] 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 of the present disclosure.

[0115] In certain embodiments, about 5 mM to about 10 mM of a reducing agent (e.g., DTT) can be employed in an RPA process of the present disclosure or included in a composition of the present disclosure.

[0116] 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 of the present disclosure.

[0117] In certain embodiments, about 1 mM to about 5 mM dNTPs can be employed in an RPA process of the present disclosure or included in a 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 of the present disclosure.

[0118] 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 of the present disclosure.

[0119] In certain embodiments, about 5 mM to about 40 mM Mg Acetate, e.g., about 10 mM to about 40 mM Mg Acetate, can be employed in an RPA process of the present disclosure or included in a composition of the present disclosure.

[0120] 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 of the present disclosure.

[0121] 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 of the present disclosure.

[0122] 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 of the present disclosure.

[0123] 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 of the present disclosure.

[0124] 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 of the present disclosure.

[0125] 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 of the present disclosure.

[0126] 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 of the present disclosure.

[0127] 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 of the present disclosure. In certain embodiments, about 0.5 pg / mL to about 100.0 pg / mL of a reverse transcriptase (e.g., 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 of the present disclosure.

[0128] In certain embodiments, the following reagents can be employed at the following concentrations for performing an RPA process of the present disclosure: about 5 mM to about 100 mM Tris-HCl 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 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; about 0.5 pg / mL to about 80.0 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 100 nM to about 200 nM of a first probe, e.g., about 100 nM to about 150 nM; and about 5 nM to about 80 nM of a second probe, e.g., about 10 nM to about 50 nM. In certain embodiments, additional reagents can be employed, including but not limited to, forward primers, reverse primers and ROX reference dyes.

[0129] 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.

[0130] 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, at least one variant of the target nucleic acid is present in the RPA reaction. In certain embodiments, at least two variants of a target nucleic acid are present in the RPA reaction. In certain embodiments, one of the two variants is present at a lower concentration than the other variant in the RPA reaction. In certain embodiments, there can be less than about 10,000 copies of the variant of the target nucleic acid, less than about 1,000 copies of the variant of the target nucleic acid, less than about 100 copies of the variant of the target nucleic acid, less than about 10 copies of the variant of the target nucleic acid or 1 copy of the variant of the target nucleic acid in an RPA reaction. In certain embodiments, the target nucleic acid variant occurs at a frequency of less than 10% in the sample, e.g., nucleic acid sample. In certain embodiments, the target nucleic acid variant occurs at a frequency of less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% in the sample, e.g., nucleic acid sample. In certain embodiments, the target nucleic acid variant occurs at a frequency of less than 5% in the sample, e.g., nucleic acid sample. In certain embodiments, the target nucleic acid variant occurs at a frequency of less than 1% in the sample, e.g., nucleic acid sample.

[0131] 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.

[0132] 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.

[0133] 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 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. ii. NEAR Process

[0134] In certain embodiments, the isothermal amplification process for use in a nucleic acid analysis of the present disclosure is NEAR.

[0135] 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.

[0136] In certain embodiments, reactions use only two templates to prime, one or two nicking enzymes and a polymerase, under isothermal conditions. In exemplary non-limiting 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.

[0137] 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.

[0138] 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).

[0139] 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. 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.

[0140] 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.

[0141] In certain embodiments, the NEAR processes are performed in the presence of at least two or more probes as described further below.

[0142] 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.

[0143] B. Nucleic Acid Detection

[0144] The presently disclosed subject matter provides a nucleic acid analysis that includes the detection of target nucleic acids amplified by an isothermal amplification process, as described herein. For example, but not by way of limitation, the presently disclosed subject matter provides a nucleic acid analysis that includes the detection of target nucleic acids amplified by an RPA process, as described herein. In certain embodiments, the presently disclosed subject matter provides a nucleic acid analysis that includes the detection of target nucleic acids amplified by a NEAR process, as described herein.

[0145] Nucleic acid detection as employed herein is used to determine the presence (e.g., presence or absence) of a target nucleic acid (or variants 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, variants of a target nucleic acid and / or a plurality of different target nucleic acids using any of a variety or combination of suitable detection techniques. For example, but not by way of limitation, the nucleic acid detection methods of the present disclosure can be configured to detect one or more variants of a target nucleic acid. Following amplification of one or a plurality of target nucleic acids present in the sample, e.g., using an isothermal amplification process, the methods of the present disclosure can be configured to detect the amplified nucleic acid(s) via hybridization. Such detection includes hybridizing a probe oligonucleotide (referred to herein as a “probe”) 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.

[0146] In certain embodiments, nucleic acid amplification and nucleic acid detection can occur simultaneously, e.g., in a system of the present disclosure. In certain embodiments, a method of the present disclosure includes the simultaneous amplification and detection of nucleic acids in a sample, e.g., an eluate. In certain embodiments, a method of the present disclosure has a duration of about 1 minute to about 60 minutes, about 5 minutes to about 60 minutes, about 8 minutes to about 60 minutes, about 8 minutes to about 50 minutes, about 8 minutes to about 40 minutes, about 8 minutes to about 35 minutes, about 8 minutes to about 30 minutes, about 8 minutes to about 25 minutes, about 8 minutes to about 20 minutes, about 1 minute to about 22 minutes, about 5 minutes to about 22 minutes, about 8 minutes to about 22 minutes, about 1 minute to about 20 minutes, about 5 minutes to about 20 minutes, about 8 minutes to about 20 minutes or about 8 minutes to about 15 minutes.

[0147] As disclosed herein, the present disclosure provides the use of two probes to increase the sensitivity of a nucleic analysis disclosed herein. As shown in Examples 1 and 2, the use of two probes significantly improved detection of variants of a target nucleic acid derived from a single pathogen, e.g., a virus. In particular, the addition of a second probe at a concentration less than the first probe resulted in a significant increase in the sensitivity of a nucleic acid analysis compared to a nucleic acid analysis performed in the absence of the second probe. For example, but not by way of limitation, the addition of a second probe at a low concentration, e.g. , a concentration of about 15 nM or about 25 nM, resulted in a significant increase in the sensitivity of a nucleic acid analysis compared to a nucleic acid analysis performed in the absence of the second probe. In certain embodiments, the first and second probes are included in the reagents used to perform the isothermal amplification process, as described above in Section ILA. In certain embodiments, the first and second probes are provided in the reagent composition that comprises the oligonucleotide components (e.g., used for the isothermal amplification process). In certain embodiments, the first and second probes are provided in the reagent composition that consists of the oligonucleotide components. In certain embodiments, the first and second probes are provided in the reagent composition that consists essentially of the oligonucleotide components.

[0148] In certain embodiments, a nucleic acid analysis of the present disclosure can include performing an amplification and detection process in the presence of a first probe and a second probe, where the first probe is present in the process at a concentration from about 20 nM to about 300 nM. For example, but not by way of limitation, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration from about 25 nM to about 300 nM, about 30 nM to about 300 nM, about 35 nM to about 300 nM, about 40 nM to about 300 nM, about 45 nM to about 300 nM, about 50 nM to about 300 nM, about 55 nM to about 300 nM, about 60 nM to about 300 nM, about 65 nM to about 300 nM, about 70 nM to about 300 nM, about 75 nM to about 300 nM, about 80 nM to about 300 nM, about 85 nM to about 300 nM, about 90 nM to about 300 nM, about 95 nM to about 300 nM, about 100 nM to about 300 nM, about 105 nM to about 300 nM, about 110 nM to about 300 nM, about 115 nM to about 300 nM, about 120 nM to about 300 nM, about 125 nM to about 300 nM, about 130 nM to about 300 nM, about 135 nM to about 300 nM, about 140 nM to about 300 nM, about 150 nM to about 300 nM, about 160 nM to about 300 nM, about 170 nM to about 300 nM, about 180 nM to about 300 nM, about 190 nM to about 300 nM, about 200 nM to about 300 nM, about 210 nM to about 300 nM, about 220 nM to about 300 nM, about 230 nM to about 300 nM, about 240 nM to about 300 nM, about 250 nM to about 300 nM, about 260 nM to about 300 nM, about 270 nM to about 300 nM, about 280 nM to about 300 nM, about 290 nM to about 300 nM, about 20 nM to about 290 nM, about 20 nM to about 280 nM, about 20 nM to about 270 nM, about 20 nM to about 260 nM, about 20 nM to about 250 nM, about 20 nM to about 240 nM, about 20 nM to about 230 nM, about 20 nM to about 220 nM, about 20 nM to about 210 nM, about 20 nM to about 200 nM, about 20 nM to about 195 nM, about 20 nM to about 190 nM, about 20 nM to about 185 nM, about 20 nM to about 180 nM, about 20 nM to about 175 nM, about 20 nM to about 170 nM, about 20 nM to about 165 nM, about 20 nM to about 160 nM, about 20 nM to about 155 nM, about 20 nM to about 150 nM, about 20 nM to about 145 nM, about 20 nM to about 140 nM, about 20 nM to about 135 nM, about 20 nM to about 130 nM, about 20 nM to about 125 nM, about 20 nM to about 120 nM, about 20 nM to about 110 nM, about 20 nM to about 100 nM, about 20 nM to about 95 nM, about 20 nM to about 90 nM, about 20 nM to about 85 nM, about 20 nM to about 80 nM, about 20 nM to about 75 nM, about 20 nM to about 70 nM, about 20 nM to about 65 nM, about 50 nM to about 110 nM, about 60 nM to about 110 nM, about 100 nM to about 135 nM, about 100 nM to about 130 nM or about 100 nM to about 125 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 50 nM to about 200 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 50 nM to about 170 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 50 nM to about 150 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 50 nM to about 120 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 60 nM to about 110 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 80 nM to about 175 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 65 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 105 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 120 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a first probe at a concentration of about 169 nM.

[0149] In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 5 nM to about 200 nM. For example, but not by way of limitation, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 6 nM to about 200 nM, about 7 nM to about 200 nM, about 8 nM to about 200 nM, about 9 nM to about 200 nM, about 10 nM to about 200 nM, about 11 nM to about 200 nM, about 12 nM to about

[0150] 200 nM, about 13 nM to about 200 nM, about 14 nM to about 200 nM, about 15 nM to about

[0151] 200 nM, about 16 nM to about 200 nM, about 17 nM to about 200 nM, about 18 nM to about

[0152] 200 nM, about 19 nM to about 200 nM, about 20 nM to about 200 nM, about 21 nM to about

[0153] 200 nM, about 22 nM to about 200 nM, about 23 nM to about 200 nM, about 24 nM to about 200 nM, about 25 nM to about 200 nM, about 30 nM to about 200 nM, about 40 nM to about 200 nM, about 45 nM to about 200 nM, about 50 nM to about 200 nM, about 55 nM to about 200 nM, about 60 nM to about 200 nM, about 65 nM to about 200 nM, about 70 nM to about 200 nM, about 75 nM to about 200 nM, about 80 nM to about 200 nM, about 85 nM to about 200 nM, about 90 nM to about 200 nM, about 95 nM to about 200 nM, about 100 nM to about 200 nM, about 105 nM to about 200 nM, about 110 nM to about 200 nM, about 115 nM to about 200 nM, about 120 nM to about 200 nM, about 125 nM to about 200 nM, about 130 nM to about 200 nM, about 135 nM to about 200 nM, about 140 nM to about 200 nM, about 145 nM to about 200 nM, about 150 nM to about 200 nM, about 155 nM to about 200 nM, about 160 nM to about 200 nM, about 165 nM to about 200 nM, about 170 nM to about 200 nM, about 175 nM to about 200 nM, about 180 nM to about 200 nM, about 185 nM to about 200 nM, about 190 nM to about 200 nM, about 195 nM to about 200 nM, about 5 nM to about 195 nM, about 5 nM to about 190 nM, about 5 nM to about 185 nM, about 5 nM to about 180 nM, about 5 nM to about 175 nM, about 5 nM to about 170 nM, about 5 nM to about 165 nM, about 5 nM to about 160 nM, about 5 nM to about 155 nM, about 5 nM to about 150 nM, about 5 nM to about 145 nM, about 5 nM to about 140 nM, about 5 nM to about 135 nM, about 5 nM to about 130 nM, about 5 nM to about 125 nM, about 5 nM to about 120 nM, about 5 nM to about 110 nM, about 5 nM to about 100 nM, about 5 nM to about 95 nM, about 5 nM to about 90 nM, about 5 nM to about 85 nM, about 5 nM to about 80 nM, about 5 nM to about 75 nM, about 5 nM to about 70 nM, about 5 nM to about 65 nM, about 5 nM to about 60 nM, about 5 nM to about 55 nM, about 5 nM to about 50 nM, about 5 nM to about 49 nM, about 5 nM to about 48 nM, about 5 nM to about 47 nM, about 5 nM to about 46 nM, about 5 nM to about 45 nM, about 5 nM to about 44 nM, about 5 nM to about 43 nM, about 5 nM to about 42 nM, about 5 nM to about 41 nM, about 5 nM to about 40 nM, about 5 nM to about 39 nM, about 5 nM to about 38 nM, about 5 nM to about 37 nM, about 5 nM to about 36 nM, about 5 nM to about 35 nM, about 5 nM to about 34 nM, about 5 nM to about 33 nM, about 5 nM to about 32 nM, about 5 nM to about 31 nM, about 5 nM to about 30 nM, about 5 nM to about 29 nM, about 5 nM to about 28 nM, about 5 nM to about 27 nM, about 5 nM to about 26 nM, about 5 nM to about 25 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 15 nM to about 35 nM, about 15 nM to about 35 nM, about 20 nM to about 80 nM, about 20 nM to about 70 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM or about 20 nM to about 30 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 10 nM to about 70 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 10 nM to about 60 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 10 nM to about 50 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 10 nM to about 40 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 15 nM to about 70 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 15 nM to about 75 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 15 nM to about 65 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 15 nM to about 35 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 20 nM to about 30 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration from about 20 nM to about 70 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration of about 15 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration of about 25 nM. In certain embodiments, a nucleic acid analysis of the present disclosure can be performed in the presence of a second probe at a concentration of about 65 nM.

[0154] In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 20. For example, but not by way of limitation, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 19, about 1.5 to about 18, about 1.5 to about 17, about 1.5 to about 16, about 1.5 to about 15, about 1.5 to about 14, about 1.5 to about 13, about 1.5 to about 12, about 1.5 to about 11, about 1 to about 10, about 1.5 to about 9, about 1.5 to about 8, about 1.5 to about 7, about 1.5 to about 6, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 1.5 to about 2, about 2 to about 20, about 2 to about 19, about 2 to about 18, about 2 to about 17, about 2 to about 16, about 2 to about 15, about 2 to about 14, about 2 to about 13, about 2 to about 12, about 2 to about 11, about 2 to about 10, about 2 to about 9, about 2 to about 8, about 2 to about 7, about

[0155] 2 to about 6, about 2 to about 5, about 2 to about 4, about 4 to about 20, about 5 to about 20, about 4 to about 6, about 4 to about 5 or about 3 to about 5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 10. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 9. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 8. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 7. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 4. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 3. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 2 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 2 to about 5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 2 to about 4. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 3 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about

[0156] 3 to about 5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 4 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 4 to about 5.

[0157] In certain embodiments, the second probe is used in a method of the present disclosure at a concentration that is at least less than about 80% of the concentration of the first probe. In certain embodiments, the second probe is used in a method of the present disclosure at a concentration that is at least less than about 75% of the concentration of the first probe. In certain embodiments, the second probe is used in a method of the present disclosure at a concentration that is at least less than about 70% of the concentration of the first probe. For example, but not by way of limitation, the second probe is used at a concentration that is at least less than about 69%, at least less than about 68%, at least less than about 67%, at least less than about 66%, at least less than about 65%, at least less than about 64%, at least less than about 63%, at least less than about 62%, at least less than about 61%, at least less than about 60%, at least less than about 59%, at least less than about 58%, at least less than about 57%, at least less than about 56%, at least less than about 55%, at least less than about 54%, at least less than about 53%, at least less than about 52%, at least less than about 51%, at least less than about 50%, at least less than about 49%, at least less than about 48%, at least less than about 47%, at least less than about 46%, at least less than about 45%, at least less than about 44%, at least less than about 43%, at least less than about 42%, at least less than about 41%, at least less than about 40%, at least less than about 39%, at least less than about 38%, at least less than about 37%, at least less than about 36%, at least less than about 35%, at least less than about 34%, at least less than about 33%, at least less than about 32%, at least less than about 31%, at least less than about 30%, at least less than about 29%, at least less than about 28%, at least less than about 27%, at least less than about 26%, at least less than about 25%, at least less than about 24%, at least less than about 23%, at least less than about 22%, at least less than about 21% or at least less than about 20% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 65% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 60% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 50% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 40% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 30% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 20% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is at least less than about 15% of the concentration of the first probe.

[0158] In certain embodiments, the second probe is used in a method of the present disclosure at a concentration that is no more than about 80% of the concentration of the first probe. In certain embodiments, the second probe is used in a method of the present disclosure at a concentration that is no more than about 75% of the concentration of the first probe. In certain embodiments, the second probe is used in a method of the present disclosure at a concentration that is no more than about 70% of the concentration of the first probe. For example, but not by way of limitation, the second probe is used at a concentration that is no more than about 69%, no more than about 68%, no more than about 67%, no more than about 66%, no more than about 65%, no more than about 64%, no more than about 63%, no more than about 62%, no more than about 61%, no more than about 60%, no more than about 59%, no more than about 58%, no more than about 57%, no more than about 56%, no more than about 55%, no more than about 54%, no more than about 53%, no more than about 52%, no more than about 51%, no more than about 50%, no more than about 49%, no more than about 48%, no more than about 47%, no more than about 46%, no more than about 45%, no more than about 44%, no more than about 43%, no more than about 42%, no more than about 41%, no more than about 40%, no more than about 39%, no more than about 38%, no more than about 37%, no more than about 36%, no more than about 35%, no more than about 34%, no more than about 33%, no more than about 32%, no more than about 31%, no more than about 30%, no more than about 29%, no more than about 28%, no more than about 27%, no more than about 26%, no more than about 25%, no more than about 24%, no more than about 23%, no more than about 22%, no more than about 21% or no more than about 20% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 65% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 60% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 50% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 40% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 30% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 20% of the concentration of the first probe. In certain embodiments, the second probe is used at a concentration that is no more than about 15% of the concentration of the first probe.

[0159] In certain embodiments, the first probe is used at a concentration that is at least 1.1 times greater than the concentration of the second probe. For example, but not by way of limitation, the first probe is used at a concentration that is at least about 1.1 times greater, at least about 1.2 times greater, at least about 1.3 times greater, at least about 1.4 times greater, at least about 1.5 times greater, at least about 1.6 times greater, at least about 1.7 times greater, at least about 1.8 times greater, at least about 1.9 times greater, at least about 2 times greater, at least about 2.1 times greater, at least about 2.2 times greater, at least about 2.3 times greater, at least about 2.4 times greater, at least about 2.5 times greater, at least about 2.6 times greater, at least about 2.7 times greater, at least about 2.8 times greater, at least about 2.9 times greater, at least about 3 times greater, at least about 3.1 times greater, at least about 3.2 times greater, at least about 3.3 times greater, at least about 3.4 times greater, at least about 3.5 times greater, at least about 3.6 times greater, at least about 3.7 times greater, at least about 3.8 times greater, at least about 3.9 times greater, at least about 4 times greater, at least about 4.1 times greater, at least about 4.2 times greater, at least about 4.3 times greater, at least about 4.4 times greater, at least about 4.5 times greater, at least about 4.6 times greater, at least about 4.7 times greater, at least about 4.8 times greater, at least about 4.9 times greater, at least about 5 times greater, at least about 5.1 times greater, at least about 5.2 times greater, at least about 5.3 times greater, at least about 5.4 times greater, at least about 5.5 times greater, at least about 5.6 times greater, at least about 5.7 times greater, at least about 5.8 times greater, at least about 5.9 times greater, at least about 6 times greater, at least about 6.1 times greater, at least about 6.2 times greater, at least about 6.3 times greater, at least about 6.4 times greater, at least about 6.5 times greater, at least about 6.6 times greater, at least about 6.7 times greater, at least about 6.8 times greater, at least about 6.9 times greater, at least about 7 times greater, at least about 7.1 times greater, at least about 7.2 times greater, at least about 7.3 times greater, at least about 7.4 times greater, at least about 7.5 times greater, at least about 7.6 times greater, at least about 7.7 times greater, at least about 7.8 times greater, at least about 7.9 times greater or at least about 8 times greater, than the concentration of the second probe. In certain embodiments, the first probe is used at a concentration that is at least about 1.5 times greater than the concentration of the second probe. In certain embodiments, the first probe is used at a concentration that is at least about 2.5 times greater than the concentration of the second probe. In certain embodiments, the first probe is used at a concentration that is at least about 3 times greater than the concentration of the second probe. In certain embodiments, the first probe is used at a concentration that is at least about 4 times greater than the concentration of the second probe. In certain embodiments, the first probe is used at a concentration that is at least about 4.5 times greater than the concentration of the second probe. In certain embodiments, the first probe is used at a concentration that is at least about 7 times greater than the concentration of the second probe.

[0160] In certain embodiments, the first and second probe target nucleotide sequences that are variant. For example, but not by way of limitation, the first probe targets a nucleotide sequence of a genomic region (e.g., a gene), and the second probe targets a variant of the nucleotide sequence of the genomic region (e.g., the gene). In certain embodiments, the variant nucleotide sequence includes one or more (e.g., two or more, three or more, four or more or five or more) nucleotide substitutions compared to the nucleotide sequence targeted (e.g., bound by) by the first probe. In certain embodiments, the sequence of a nucleotide variant targeted by (e.g., bound by) the second probe has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity the nucleotide sequence targeted by the first probe. As shown in Examples 1 and 2, the use of a first probe and a second probe targeting variants of a gene sequence resulted in a significant improvement in the detection of a minority gene variant compared to the detection of the minority gene variant using the first probe or second probe alone. In certain embodiments, the first probe targets a nucleotide sequence of a genomic region (e.g., a gene), and the second probe targets a minority variant of the nucleotide sequence of the genomic region (e.g., the gene). In certain embodiments, the first probe targets a wild type nucleotide sequence of a genomic region (e.g., a gene), and the second probe targets a minority variant of the nucleotide sequence of the genomic region (e.g., the gene).

[0161] In certain embodiments, the second probe hybridizes to a variant sequence of a target nucleic acid (or an amplicon of the variant of the target nucleic acid). In certain embodiments, the second probe hybridizes to a target nucleic acid comprising a variant sequence (or an amplicon of the target nucleic acid comprising the variant sequence). In certain embodiments, the second probe is at least about 90% homologous or complementary to a variant sequence of a target nucleic acid (or an amplicon of the variant of the target nucleic acid). For example, but not by way of limitation, the second probe is at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homologous or complementary to a variant sequence of a target nucleic acid (or an amplicon of the variant of the target nucleic acid). In certain embodiments, the second probe is 100% homologous or complementary to a variant sequence of a target nucleic acid (or an amplicon of the variant of the target nucleic acid). In certain embodiments, the variant sequence of the target nucleic acid is a minority variant of the target nucleic acid. In certain embodiments, the second probe is at least about 90% homologous or complementary to a minority variant sequence of a target nucleic acid (or an amplicon of the minority variant sequence of the target nucleic acid). For example, but not by way of limitation, the second probe is at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homologous or complementary to a minority variant sequence of a target nucleic acid (or an amplicon of the target nucleic acid). In certain embodiments, the second probe is 100% homologous or complementary to a minority variant sequence of a target nucleic acid (or an amplicon of the minority variant sequence of the target nucleic acid). In certain embodiments, the variant sequence (e.g., minority variant sequence) of the target nucleic acid comprises at least one modification, e.g., at least two modifications or at least three modifications, compared to the wild type sequence of the target nucleic acid. In certain embodiments, the variant sequence (e.g., minority variant sequence) of the target nucleic acid comprises a single nucleotide modification compared to the wild type sequence of the target nucleic acid. Non-limiting examples of variants to be detected by methods of the present disclosure are provided in Section IV.

[0162] In certain embodiments, the first probe hybridizes to a wild type sequence of a target nucleic acid (or an amplicon of the wild type sequence the target nucleic acid). In certain embodiments, the first probe hybridizes to a target nucleic acid comprising a wild type sequence (or an amplicon of the target nucleic acid comprising the wild type sequence). In certain embodiments, the first probe is at least about 90% homologous or complementary to a wild type sequence of a target nucleic acid (or an amplicon of the wild type sequence the target nucleic acid). For example, but not by way of limitation, the first probe is at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homologous or complementary to a wild type sequence of a target nucleic acid (or an amplicon of the wild type sequence the target nucleic acid). In certain embodiments, the first probe is 100% homologous or complementary to a wild type sequence of a target nucleic acid (or an amplicon of the wild type sequence the target nucleic acid).

[0163] In certain embodiments, the second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 90% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 91% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 92% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 93% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 94% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 95% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 96% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 97% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 98% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.1% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.2% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.3% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.4% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.5% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.6% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.7% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.8% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 99.9% or greater to the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 80% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 81% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 82% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 83% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 84% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 85% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 86% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 87% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 88% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 89% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 90% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 91% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 92% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 93% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 94% to about 99% to the sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 95% to about 99% to the sequence of the first probe.

[0164] In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 9 nucleotides, differs by no more than 8 nucleotides, differs by no more than 7 nucleotides, differs by no more than 6 nucleotides, differs by no more than 5 nucleotides, differs by no more than 4 nucleotides, differs by no more than 3 nucleotides, differs by no more than 2 nucleotides or differs by no more than 1 nucleotide from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 9 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 8 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 7 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 6 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 4 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 2 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 1 nucleotide from the nucleotide sequence of the first probe.

[0165] In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that differs by about 1 to about 10 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that differs by about 2 to about 10 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that differs by about 1 to about 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, a second probe for use in the present disclosure can include a nucleotide sequence that differs by about 2 to about 5 nucleotides from the nucleotide sequence of the first probe.

[0166] In certain embodiments, the first probe and the second probe hybridize to the same amplicon produced by an isothermal amplification reaction. In certain embodiments, the second probe hybridizes to a region (e.g., second region) of the amplicon that is located at a distance of about 500 nucleotides or less from the region of the amplicon (e.g., first region) hybridized to by the first probe. For example, but not by way of limitation, the second probe binds to a region of the amplicon that is located at a distance of about 400 nucleotides or less, about 350 nucleotides or less, about 300 nucleotides or less, about 250 nucleotides or less, about 200 nucleotides or less, about 150 nucleotides or less, about 100 nucleotides or less, about 95 nucleotides or less, about 90 nucleotides or less, about 85 nucleotides or less, about

[0167] 80 nucleotides or less, about 75 nucleotides or less, about 70 nucleotides or less, about 65 nucleotides or less, about 60 nucleotides or less, about 55 nucleotides or less, about 50 nucleotides or less, about 45 nucleotides or less, about 40 nucleotides or less, about 35 nucleotides or less, about 30 nucleotides or less, about 25 nucleotides or less, about 20 nucleotides or less, about 15 nucleotides or less or about 10 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 300 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 200 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 150 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 125 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 100 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 75 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 50 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 40 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 30 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 25 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 20 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 15 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 10 nucleotides or less from the region of the amplicon bound by the first probe. In certain embodiments, the second probe binds to a region of the amplicon that is located at a distance of about 5 nucleotides or less from the region of the amplicon bound by the first probe.

[0168] In certain embodiments, the probes for use in the present disclosure are singlestranded and have a length of 100 nucleotides or less. For example, but not by way of limitation, a probe for use in the present disclosure, e.g., the first and / or second probe, has a length of about 90 nucleotides or less, about 80 nucleotides or less, about 70 nucleotides or less, about 60 nucleotides or less, about 50 nucleotides or less, about 40 nucleotides or less, about 30 nucleotides or less or about 20 nucleotides. In certain embodiments, a probe for use in the present disclosure, e.g., the first and / or second probe, has a length of about 50 nucleotides or less. In certain embodiments, a probe for use in the present disclosure, e.g., the first and / or second probe, has a length from about 10 to about 50 nucleotides. In certain embodiments, a probe for use in the present disclosure, e.g., the first and / or second probe, has a length from about 20 to about 50 nucleotides. In certain embodiments, a probe for use in the present disclosure, e.g., the first and / or second probe, has a length from about 30 to about 50 nucleotides.

[0169] In certain embodiments, a nucleic acid analysis of the present disclosure includes amplification (e.g., isothermal amplification) of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more target nucleic acids (e.g., the one or more variants of the target nucleic acid), using two or more probes, e.g., a first probe and a second probe, as described herein. In certain embodiments, a nucleic acid analysis of the present disclosure includes amplification (e.g., isothermal amplification) of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more amplicons generated by the amplification process using two or more probes, e.g., a first probe and a second probe, as described herein.

[0170] In certain embodiments, a nucleic acid analysis of the present disclosure includes an isothermal amplification process of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more target nucleic acids (e.g., the one or more variants of the target nucleic acid) using two or more probes, e.g., a first probe and a second probe, as described herein. In certain embodiments, the second probe is present in the nucleic acid analysis at a concentration of about 5 nM to about 200 nM, e.g., about 10 nM to about 100 nM or about 10 nM to about 70 nM, e.g., about 25 nM or about 65 nM. In certain embodiments, the second probe is present in the nucleic acid analysis at a concentration of about 10 nM to about 70 nM. In certain embodiments, the first probe is present in the nucleic acid analysis at a concentration of about 20 nM to about 300 nM, e.g., about 50 nM to about 150 nM, e.g., about 67.5 nM, about 105 nM or about 120 nM. In certain embodiments, the first probe is present in the nucleic acid analysis at a concentration of about 50 nM to about 170 nM. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20, e.g., about 1.5 to about 6, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 5 or about 3 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater, e.g., about 90% or greater. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe from about 90% to about 99%. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that differs by about 1 to about 10 nucleotides, e.g., about 1 to about 5 nucleotides, from the nucleotide sequence of the first probe.

[0171] In certain embodiments, a nucleic acid analysis of the present disclosure includes an isothermal amplification process of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more variants using two or more probes, e.g., a first probe and a second probe, where the second probe is used in the nucleic acid analysis at a concentration of about 10 nM to about 70 nM, the first probe is used in the nucleic acid analysis at a concentration of about 50 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10. In certain embodiments, the second probe includes a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe.

[0172] In certain embodiments, a nucleic acid analysis of the present disclosure includes an RPA process of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more target nucleic acids (e.g., the one or more variants of the target nucleic acid) using two or more probes, e.g., a first probe and a second probe, as described herein. In certain embodiments, the second probe is present in the nucleic acid analysis at a concentration of about 5 nM to about 200 nM, e.g., about 10 nM to about 100 nM or about 10 nM to about 70 nM, e.g., about 25 nM or about 65 nM. In certain embodiments, the second probe is present in the nucleic acid analysis at a concentration of about 10 nM to about 70 nM. In certain embodiments, the first probe is present in the nucleic acid analysis at a concentration of about 20 nM to about 300 nM, e.g., about 50 nM to about 150 nM, e.g., about 67.5 nM, about 105 nM or about 120 nM. In certain embodiments, the first probe is present in the nucleic acid analysis at a concentration of about 50 nM to about 170 nM. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20, e.g., about 1.5 to about 6, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 5 or about 3 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater, e.g., about 90% or greater. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe from about 90% to about 99%. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that differs by about 1 to about 10 nucleotides, e.g., about 1 to about 5 nucleotides, from the nucleotide sequence of the first probe.

[0173] In certain embodiments, a nucleic acid analysis of the present disclosure includes an RPA process of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more target nucleic acids (e.g., the one or more variants of the target nucleic acid) using two or more probes, e.g., a first probe and a second probe, where the second probe is used in the nucleic acid analysis at a concentration of about 10 nM to about 70 nM, the first probe is used in the nucleic acid analysis at a concentration of about 50 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10.

[0174] In certain embodiments, a nucleic acid analysis of the present disclosure includes a NEAR process of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more target nucleic acids (e.g., the one or more variants of the target nucleic acid) using two or more probes, e.g., a first probe and a second probe, as described herein. In certain embodiments, the second probe is present in the nucleic acid analysis at a concentration of about 5 nM to about 200 nM, e.g., about 10 nM to about 100 nM or about 10 nM to about 70 nM, e.g., about 25 nM or about 65 nM. In certain embodiments, the first probe is present in the nucleic acid analysis at a concentration of about 20 nM to about 300 nM, e.g., about 50 nM to about 150 nM, e.g., about 67.5 nM, about 105 nM or about 120 nM. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20, e.g., about 1.5 to about 6, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 5 or about 3 to about 6. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater, e.g., about 90% or greater. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe from about 90% to about 99%. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that differs by about 1 to about 10 nucleotides, e.g., about 1 to about 5 nucleotides, from the nucleotide sequence of the first probe.

[0175] In certain embodiments, a nucleic acid analysis of the present disclosure includes a NEAR process of one or more target nucleic acids, e.g., one or more variants of a target nucleic acid, and detection of the one or more target nucleic acids (e.g., the one or more variants of the target nucleic acid) using two or more probes, e.g., a first probe and a second probe, where the second probe is used in the nucleic acid analysis at a concentration of about 10 nM to about 70 nM, the first probe is used in the nucleic acid analysis at a concentration of about 50 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10.

[0176] 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 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. In certain embodiments, the probes (e.g., the first and second probes) are included with the reagents of the isothermal amplification process, as described herein.

[0177] In certain embodiments, detection of amplified nucleic acids can employ optical detection, digital detection and / or other detection methods known in the art. In certain embodiments, the detection operation detects the presence or absence of the target nucleic acid, e.g., variants of the target nucleic acid. In certain embodiments, the detection operation quantifies the amount of target nucleic acid, e.g. , variants of the target nucleic acid, in a sample. i. Optical Detection

[0178] 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 detecting the binding of a labeled probe, e.g., a labeled first probe and a labeled second probe, to the amplified target nucleic acid (e.g., amplicon).

[0179] In certain embodiments, 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).

[0180] 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 amplified target nucleic acid. 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.

[0181] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, the optical detection strategy comprises the use of probes, e.g., first and second probes as described herein, 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 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 certain embodiments, a probe employed in the methods of the present disclosure can have a fluorescent moiety that is covalently linked to a nucleotide present towards the 5’ end of the probe and has a quencher molecule towards 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.

[0182] Additionally, or alternatively, and in accordance with another aspect of the disclosed subject matter, specific probes, e.g., probes for specific target nucleic acids, different variants of a target nucleic acid and / or internal controls, are each labeled with a different fluorophore, thus allowing for simultaneous detection of a plurality of amplified products. For example, but not by way of limitation, a first probe can be labeled with a first fluorophore and the second probe can be labeled with a second fluorophore, wherein the first fluorophore and the second fluorophore are different. In certain embodiments, the first probe and the second probe can be labeled with the same fluorophore.

[0183] 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. ii. Digital Detection

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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- HC1, 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 mMNaCl; 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.

[0189] 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.

[0190] C. Samples

[0191] 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.

[0192] 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.

[0193] 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, nasopharyngeal fluid, urine, lymphatic fluid, cerebrospinal fluid, interstitial fluid, bronchoalveolar lavage 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).

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] In certain embodiments, the sample can be a pooled sample. For example, but not by way of limitation, samples from a plurality of individuals and / or a plurality of different samples can be pooled together to generate a pooled sample. In certain embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 or at least 24 samples from individuals, e.g., donors, can be pooled together to generate a pooled sample. In certain embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 or at least 24 different samples can be pooled together to generate a pooled sample.

[0200] Target nucleic acids can be isolated from a sample by any method known in the art. For example, but not by limitation, isolation of nucleic acids can incorporate the use of a variety of sample buffers, nucleic acid immobilization techniques e.g., immobilization on magnetic particles) and / or elution aspects. For purpose of illustration and not limitation, the methods of the disclosed subject matter can include an isolation process for isolating nucleic acids that comprises a lysis process, i.e., a sample lysis process. In certain embodiments, the sample lysis process includes combining one or more samples with a sample lysis buffer. In certain embodiments, the sample lysis buffer is a solution adapted to disrupt the membranes or walls of pathogens, infectious agents and / or cells present within a sample and release the contents of the pathogens, infectious agents and / or cells, e.g., nucleic acids present within pathogens, infectious agents, and / or cells. Additional 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.

[0201] III. Compositions

[0202] The present disclosure further provides compositions for performing the methods disclosed herein, e.g., the methods of Section II. For example, but not by way of limitation, the present disclosure provides compositions comprising one or more reagents, e.g., reagent compositions, for performing a method, e.g., a nucleic acid analysis, of the present disclosure.

[0203] In certain embodiments, a composition of the present disclosure includes a first probe and a second probe at the ratios and / or amounts described herein, e.g., at ratios and / or amounts described above in Section II. A and as described below. In certain embodiments, a composition includes a second probe at a concentration less than the concentration of the first probe. As disclosed herein, a composition of the present disclosure that includes a first probe and a second probe targeting variants of a gene sequence, where the second probe is present at a concentration significantly less than the concentration of the first probe, resulted in a synergistic improvement in the detection of a minority gene variant compared to the detection of the minority gene variant using the first probe or second probe alone.

[0204] In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 20 nM to about 300 nM. In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 50 nM to about 200 nM. In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 50 nM to about 170 nM. In certain embodiments a composition of the present disclosure includes a first probe at a concentration of about 50 nM to about 150 nM. In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 50 nM to about 120 nM. In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 60 nM to about 110 nM. In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 80 nM to about 175 nM. In certain embodiments, a composition of the present disclosure includes a first probe at a concentration of about 50 nM to about 150 nM. In certain embodiments, the composition of the present disclosure includes a first probe at a concentration of about 170 nM. In certain embodiments, the composition of the present disclosure includes a first probe at a concentration of about 120 nM. In certain embodiments, the composition of the present disclosure includes a first probe at a concentration of about 105 nM. In certain embodiments, the composition of the present disclosure includes a first probe at a concentration of about 85 nM. In certain embodiments, the composition of the present disclosure includes a first probe at a concentration of about 67.5 nM.

[0205] In certain embodiments, a composition of the present disclosure includes a second probe at a concentration of about 5 nM to about 200 nM. For example, but not by way of limitation, a composition of the present disclosure includes a second probe at a concentration of about 10 nM to about 100 nM. In certain embodiments, the composition of the present disclosure includes a second probe at a concentration of about 10 nM to about 70 nM. In certain embodiments, the composition of the present disclosure includes a second probe at a concentration of about 15 nM to about 80 nM. In certain embodiments, the composition of the present disclosure includes a second probe at a concentration of about 15 nM. In certain embodiments, the composition of the present disclosure includes a second probe at a concentration of about 25 nM. In certain embodiments, the composition of the present disclosure includes a second probe at a concentration of about 65 nM.

[0206] In certain embodiments, the present disclosure provides a composition comprising a first probe and a second probe, where the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.5 to about 20. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 10. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.5 to about 5, about 1.5 to about 4 or about 2 to about 5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.5 to about 10. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.5 to about 8. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.5 to about 5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.5. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 1.6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 2.7. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 3.4. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 4.7. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe can be about 8.0.

[0207] In certain embodiments, a composition of the present disclosure can include a first probe and a second probe, where the second probe is present in the composition at a concentration of about 10 nM to about 70 nM, the first probe is present in the composition at a concentration of about 50 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe in the composition is about 1.5 to about 10.

[0208] In certain embodiments, the present disclosure provides a composition that includes a first and a second probe that target nucleotide sequences that are variant. For example, but not by way of limitation, the first probe targets a nucleotide sequence of a genomic region (e.g., a gene), and the second probe targets a variant of the nucleotide sequence of the genomic region (e.g., the gene). In certain embodiments, the variant nucleotide sequence includes one or more (e.g. , two or more, three or more, four or more or five or more) nucleotide substitutions compared to the nucleotide sequence targeted by the first probe. In certain embodiments, the sequence of a nucleotide variant targeted by the second probe has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity the nucleotide sequence targeted by the first probe. As shown in Examples 1 and 2, the use of a first probe and a second probe targeting variants of a gene sequence resulted in a significant improvement in the detection of a minority gene variant compared to the detection of the minority gene variant using the first probe or second probe alone.

[0209] In certain embodiments, the second probe for use in the present disclosure, e.g., in a composition of the present disclosure, can have a nucleotide sequence that has an identity of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater to the first probe. In certain embodiments, the second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 93% or greater to the first probe. In certain embodiments, the second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 95% or greater to the first probe. In certain embodiments, the second probe for use in the present disclosure can have a nucleotide sequence that has an identity of about 98% or greater to the first probe. In certain embodiments, the second probe for use in the present disclosure can have a nucleotide sequence that has an identity to the first probe of about 90% to about 99%.

[0210] As shown in Example 1, the use of a second probe that has an identity to the first probe of about 95% to about 99% at a low concentration (e.g., at a concentration of about 10 nM to about 40 nM) resulted in a significant improvement in the detection of a minority gene variant compared to the detection of the minority gene variant using the first probe or second probe alone.

[0211] In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe, differs by no more than 9 nucleotides, differs by no more than 8 nucleotides, differs by no more than 7 nucleotides, differs by no more than 6 nucleotides, differs by no more than 5 nucleotides, differs by no more than 4 nucleotides, differs by no more than 3 nucleotides, differs by no more than 2 nucleotides or differs by no more than 1 nucleotide from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 3 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that is identical to or differs by no more than 2 nucleotides from the nucleotide sequence of the first probe. In certain embodiments, the second probe for use in the present disclosure can include a nucleotide sequence that differs by about 1 to about 10 nucleotides, e.g., about 1 to about 5 nucleotides, from the nucleotide sequence of the first probe.

[0212] In certain embodiments, the second probe for use in a composition of the present disclosure binds to a region (e.g., second region) of the amplicon that is located at a distance of about 500 nucleotides or less from the region of the amplicon (e.g., first region) bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 100 nucleotides or less from the region of the amplicon bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 50 nucleotides or less from the region of the amplicon bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 25 nucleotides or less from the region of the amplicon bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 20 nucleotides or less from the region of the amplicon bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 15 nucleotides or less from the region of the amplicon bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 10 nucleotides or less from the region of the amplicon bound by the first probe present in the composition. In certain embodiments, the second probe present in a composition of the present disclosure binds to a region of the amplicon that is located at a distance of about 5 nucleotides or less from the region of the amplicon bound by the first probe present in the composition.

[0213] In certain embodiments, a composition of the present disclosure includes one or more primers for amplifying a target nucleic acid. In certain embodiments, the composition includes at least two primers. In certain embodiments, a composition of the present disclosure includes a forward primer (e.g., at least two forward primers) and a reverse primer (e.g., at least two reverse primers) for amplifying a target nucleic acid. In certain embodiments, a composition of the present disclosure includes one forward and at least three reverse primers (e.g., at least four reverse primers) for amplifying a target nucleic acid.

[0214] In certain embodiments, the primer sequences and probe sequences disclosed herein can be modified to increase the sensitivity of a nucleic acid analysis of the present disclosure. For example, but not by way of limitation, one or more modifications can be introduced into the nucleotide sequences of the disclosed primer sequences and / or probe sequences to increase sensitivity, e.g, one or more, two or more, three or more, four or more, five or more modifications can be introduced into the nucleotide sequences of the disclosed primer sequences and / or probe sequences to increase sensitivity. In certain embodiments, the primer sequences and probe sequences disclosed herein can be modified to introduce one or more mismatches to the sequence of the complementary target nucleic acid, e.g., one or more, two or more, three or more, four or more or five or more mismatches to the sequence of the complementary target nucleic acid. In certain embodiments, a mismatch can be introduced towards the 3’ end of a primer or probe disclosed herein. In certain embodiments, a mismatch can be introduced towards the 3’ end of a probe disclosed herein. In certain embodiments, a mismatch can be introduced towards the 5’ end of a primer or probe disclosed herein. In certain embodiments, a mismatch can be introduced towards the 5’ end of a probe disclosed herein. In certain embodiments, the introduction of a mismatch in one or more primers, e.g., a forward primer and / or a reverse primer, of the present disclosure can reduce the interaction of the primer with a probe used in the same isothermal amplification reaction.

[0215] In certain embodiments, the primer sequences and / or probe sequences disclosed herein can be modified to introduce one or more non-canonical nucleotides, e.g., substituting one or more nucleotides of the presently disclosed primer sequences and / or probe sequences with a non-canonical nucleoside. Non-limiting examples of non-canonical nucleosides include inosine, xanthine hypoxanthine, isocytosine and isoguanine. In certain embodiments, the primer sequences and / or probe sequences disclosed herein can be modified to introduce one or more locked nucleic acids (LNAs), e.g., substitution of one or more nucleotides of the presently disclosed primer sequences and / or probe sequences with an LNA. In certain embodiments, a probe disclosed herein can be modified by modifying the linkage of the fluorophore and / or quencher to the probe to improve sensitivity.

[0216] In certain embodiments, a composition of the present disclosure, e.g., reagent composition, further 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 reducing agent, a creatine kinase, a nuclease, a crowding agent, ATP or an ATP analog, at least one recombinase loading protein, a first primer and, optionally, a second primer and a reverse transcriptase.

[0217] In certain embodiments, a composition of the present disclosure, e.g., reagent composition, further includes a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP and at least one primer (e.g., two primers). In certain embodiments, a composition of the present disclosure does not include a reverse transcriptase.

[0218] In certain embodiments, about 0.5% to about 15% weight to volume (w / v) of a crowding agent (e.g., PEG and / or trehalose) can be included in a composition of the present disclosure, e.g, reagent composition. In certain embodiments, the crowding agent can be present in a composition of the present disclosure, e.g., reagent composition, in an amount of about 1% to about 10% w / v.

[0219] In certain embodiments, about 5 mM to about 10 mM of a reducing agent e.g., DTT) can be included in a composition of the present disclosure, e.g., reagent composition.

[0220] In certain embodiments, about 50 mM to about 100 mM potassium acetate can be included in a composition of the present disclosure, e.g., reagent composition.

[0221] In certain embodiments, about 1 mM to about 5 mM dNTPs can be included in a composition of the present disclosure, e.g., reagent composition.

[0222] In certain embodiments, about 1 mM to about 10 mM of ATP, e.g., about 2 mM to about 5 mM ATP, can be included in a composition of the present disclosure, e.g., reagent composition.

[0223] In certain embodiments, about 20 mM to about 100 mM Phosphocreatine, e.g., about 40 mM to about 100 mM Phosphocreatine, can be included in a composition of the present disclosure, e.g., reagent composition.

[0224] In certain embodiments, about 5 mM to about 40 mM Mg Acetate, e.g., about 10 mM to about 40 mM Mg Acetate, can be included in a composition of the present disclosure, e.g., reagent composition.

[0225] In certain embodiments, about 0.01 mg / mL to about 10 mg / mL BSA can be included in a composition of the present disclosure, e.g., reagent composition.

[0226] In certain embodiments, about 5% to about 10% Glycerol can be included in a composition of the present disclosure, e.g., reagent composition.

[0227] 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 included in a composition of the present disclosure, e.g., reagent composition.

[0228] 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 included in a composition of the present disclosure, e.g., reagent composition.

[0229] 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 included in a composition of the present disclosure, e.g., reagent composition.

[0230] 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 included in a composition of the present disclosure, e.g., reagent composition. 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 included in a composition of the present disclosure, e.g., reagent composition.

[0231] In certain embodiments, about 0.01 mg / mL to about 0.5 mg / mL Exonuclease

[0232] III, e.g., about 0.1 mg / mL to about 0.5 mg / mL Exonuclease III, can be included in a composition of the present disclosure, e.g., reagent composition.

[0233] In certain embodiments, about 0.5 pg / mL to about 100.0 pg / mL of a reverse transcriptase (e.g., 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 included in a composition of the present disclosure, e.g., reagent composition.

[0234] In certain embodiments, a composition of the present disclosure can further include a buffer. Non-limiting examples of buffers include a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a HEPES buffer, a TRIS buffer, a MES buffer, a MOPS buffer and combinations thereof. In certain embodiments, a composition of the present disclosure includes about 5 mM to about 100 mM Tris-HCl at a pH of about 6.5-9.0, e.g., 8.3.

[0235] In certain embodiments, a composition of the present disclosure can be formulated in a dried form. In certain embodiments, a composition of the present disclosure can be freeze-dried (i.e., lyophilized). In certain embodiments, lyophilized reagents offer the advantage of not requiring refrigeration to maintain activity. For example, but not by way of limitation, a composition of reagents can be stored at room temperature. This advantage is especially useful in field conditions where access to refrigeration is limited. In certain embodiments, the composition of reagents can be freeze dried onto the bottom of a tube, container or on a bead (or another type of solid support). To perform a method of the present disclosure, the composition can be reconstituted in a buffer and, optionally, with a crowding reagent and subsequently used.

[0236] IV. Methods of Use

[0237] In certain embodiments, the methods of the present disclosure can be used to detect the presence or absence of a target nucleic acid, e.g., variants 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, e.g., variants of a target nucleic acid, in a sample.

[0238] In certain embodiments, the methods of the present disclosure can be used to amplify and detect target nucleic acids, e.g., variants of 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 target nucleic acids, e.g., variants of target nucleic acids, that are markers for a disease or disorder, e.g., to determine if a subject has the disease or disorder or if the subject is at risk of developing the disease or disorder, e.g., cancer.

[0239] In certain embodiments, the methods of the present disclosure can be used to amplify and detect target nucleic acids, e.g., variants of 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 methods of the present disclosure can be used to amplify and detect target nucleic acids, e.g., variants of target nucleic acids, that are derived from a pathogen, e.g., to determine the pathogen type, subgroup or subtype. In certain embodiments, methods of the present disclosure can be used to determine the specific virus subtype or subgroup that a subject is infected with. For example, but not by way of limitation, methods of the present disclosure can be used to determine the specific HIV-1 or HIV-2 subtype or subgroup infecting a subject. In certain embodiments, methods of the present disclosure can be used to detect an HEV variant present in a sample from a subject. In certain embodiments, methods of the present disclosure can be used to detect an HEV genotype variant present in a sample from a subject.

[0240] In certain embodiments, the methods of the present disclosure can be used to amplify and detect variants of target nucleic acids, where the target nucleic acid variants are genetic variants. In certain embodiments, the genetic variants can be variants of a pathogen, e.g., a virus. In certain embodiments, the genetic variants can be mutations observed in cancer or within cells of a cancer or tumor. In certain embodiments, the genetic variants can be mutations observed in cancer or within cells of a cancer or tumor that can inform cancer treatment (e.g., personalized treatment). In certain embodiments, the genetic variants could be associated with drug resistance, e.g., genetic variants in bacteria (e.g., S. aureus) or fungi (Candida and Aspergillus spp.) that can result in drug resistance.

[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 to determine if the sample includes target nucleic acids, e.g., variants of target nucleic acids, that are derived from a pathogen. 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 B19, 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 one or more new or emerging pathogens, viruses and / or agents.

[0245] 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.

[0246] In certain embodiments, the methods of the present disclosure can be used for the detection (e.g., presence or absence) or the quantification of two or more variants of a target nucleic acid in a sample. In certain embodiments, the methods of the present disclosure can be used to detect the presence of one or more variants of a target nucleic acid that is associated with, e.g., causes or correlates with, an infection, disease and / or disorder. For example, but not by way of limitation, the methods disclosed herein can be used to determine if a subject has a particular infection, disease and / or disorder based on the detection of one or more variants of a nucleic acid that are clinically relevant to the infection, disease and / or disorder.

[0247] In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid derived from a cancer and / or the genome of a subject. In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid that is associated with the development or progression of a cancer. In certain embodiments, the detection and / or quantification of one or more variants of a target nucleic acid associated with a cancer can assist in determining the best course of treatment for the subject with the cancer.

[0248] In certain embodiments, methods of the present disclosure can be used to detect and / or quantify low-frequency nucleic acid variations (e.g., minority variants). For example, but not by way of limitation, methods of the present disclosure can be used to detect and / or quantify low-frequency nucleic acid variations (e.g., minority variants) in samples that can contain low amounts of variant sequences.

[0249] In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid derived from a pathogen (e.g., a bacteria or virus) disclosed herein. In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a gene from a pathogen (e.g., a bacteria or virus). In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a gene from different groups or subgroups of a pathogen. In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a gene from different groups or subgroups of a bacteria. In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a gene from different groups or subgroups of a virus.

[0250] In certain embodiments, methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid derived from HIV-1 or HIV-2.

[0251] In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region, e.g. , the INT gene (e.g. , INT coding region of the POL gene), from different groups and / or clades of HIV-1. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from the groups M, N, O and / or P of HIV-1. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene group O of HIV-1. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from group N of HIV-1. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or a target nucleic acid, e.g., the POL gene, the gag gene, the env gene, the INT gene or the LTR (long terminal repeats) (e.g., 5’ LTR), from HIV-1 (e.g., group N), where the reagent composition for performing the method includes a first probe and a second probe, where the second probe is used in the nucleic acid analysis at a concentration of about 10 nM to about 80 nM, the first probe is used in the nucleic acid analysis at a concentration of about 60 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 5.

[0252] In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region, e.g, the POL gene, from different groups and / or clades of HIV-2. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from different HIV-2 subtypes HIV- 2A and / or HIV-2B. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from the HIV-2A subtype. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid from the HIV-2A subtype, e.g, the LTR region (long terminal repeats) (e.g., 5’ LTR) and / or the POL gene. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from the HIV-2B subtype, e.g., the LTR region (long terminal repeats) (e.g., 5’ LTR) and / or the POL gene. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene, e.g., the POL gene or the gag gene, from HIV-2 (e.g., subtypes HIV-2A and / or HIV-2B subtypes), where the reagent composition for performing the method includes a first probe and a second probe, where the second probe is used in the nucleic acid analysis at a concentration of about 10 nM to about 50 nM, the first probe is used in the nucleic acid analysis at a concentration of about 60 nM to about 90 nM and the ratio of the concentration of the first probe to the concentration of the second probe is about 2 to about 5.

[0253] In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region from HBV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene (e.g., a surface antigen-encoding gene (e.g., the S gene)) from HBV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from the genotypes (e.g., genotypes A-H) of HBV.

[0254] In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region from HCV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from HCV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid (e.g., gene) from the genotypes (e.g., genotypes 1-6) of HCV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a target nucleic acid (e.g., 5’ UTR) from the genotypes (e.g., genotypes 1-6) of HCV.

[0255] In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region from HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region from genotypes GT1-4, G7 and GT8 of HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from genotypes GT1-4 of HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from genotype GT1 of HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from genotype GT2 of HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene from genotype GT4 of HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene or genomic region from genotype GT3 of HEV. Non-limiting subtypes of genotype GT1 include GT la and GT lb and non-limiting subtype of genotype GT2 include GT2a and GT2b. In certain embodiments, subtypes of genotype GT3 include GT3a, GT3b, GT3c, GT3d, GT3e, GT3f and GT3i. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify a variant of a gene or genomic region from the GT3f subtype. In certain embodiments, the gene or genomic region is the 5’ UTR and / or the 0RF1 of HEV. In certain embodiments, the gene or genomic region is 0RF2 and / or ORF3 of HEV. In certain embodiments, the methods of the present disclosure can be used to detect and / or quantify variants of a gene (e.g., the ORF1) from HEV (e.g., an HEV subtype, e.g., subtype GT3f), where the reagent composition for performing the method includes a first probe and a second probe, and where the second probe is used in the nucleic acid analysis at a concentration of about 10 nM to about 50 nM, the first probe is used in the nucleic acid analysis at a concentration of about 60 nM to about 150 nM and the ratio of the concentration of the first probe to the concentration of the second probe is about 5 to about 10. In certain embodiments, the methods of the present disclosure can be used for the 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.

[0256] 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.

[0257] 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.

[0258] In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Babesia and Malaria.

[0259] In certain embodiments, the methods of the present disclosure can be used for multiplex analysis of Parvovirus B 19 and HAV. V. Kits

[0260] The present disclosure further provides kits for performing the methods disclosed herein. In certain embodiments, the present disclosure provides kits containing materials for performing a method of the present disclosure. In certain embodiments, a kit of the present disclosure can include a composition disclosed herein. Non-limiting examples of compositions for use in kits disclosed herein are provided in Section II. A and Section III.

[0261] 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 first probe and a second probe, e.g., at the amounts and ratios described herein. For example, but not by way of limitation, a kit of the present disclosure can include the first probe and the second probe in different containers. In certain embodiments, a kit of the present disclosure can include the first probe in a first container and a second probe in a second container.

[0262] In certain embodiments, a kit of the present disclosure includes a first probe and a second probe, e.g., in a reagent composition in a single container or the first probe in a first container and the second probe in a second container, at the ratios and / or amounts described herein. In certain embodiments, a kit of the present disclosure includes a second probe at a concentration less than the concentration of the first probe.

[0263] In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 10 nM to about 300 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 50 nM to about 200 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 50 nM to about 170 nM, e.g, in a reagent composition. In certain embodiments a kit of the present disclosure includes a first probe at a concentration of about 50 nM to about 150 nM, e.g, in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 50 nM to about 120 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 60 nM to about 110 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 80 nM to about 175 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 50 nM to about 150 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 170 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 120 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 105 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 85 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 67.5 nM, e.g., in a reagent composition.

[0264] In certain embodiments, a kit of the present disclosure includes a second probe at a concentration of about 5 nM to about 200 nM, e.g., in a reagent composition. For example, but not by way of limitation, a kit of the present disclosure includes a second probe at a concentration of about 10 nM to about 100 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a second probe at a concentration of about 10 nM to about 70 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a second probe at a concentration of about 15 nM to about 80 nM, e.g., in a reagent composition. In certain embodiments, a kit of the present disclosure includes a second probe at a concentration of about 15 nM, e.g., in a reagent composition. In certain embodiments, the kit of the present disclosure includes a second probe at a concentration of about 67.5 nM or about 105 nM, e.g., in a reagent composition. In certain embodiments, the kit of the present disclosure includes a second probe at a concentration of about 25 nM or about 65 nM, e.g., in a reagent composition.

[0265] In certain embodiments, a kit of the present disclosure includes a first probe at a concentration of about 20 nM to about 300 nM (e.g., about 50 nM to about 150 nM, e.g., about 67.5 nM, about 105 nM or about 120 nM) and a second probe at a concentration of about 5 nM to about 200 nM (e.g, about 10 nM to about 100 nM or about 10 nM to about 70 nM, e.g., about 25 nM or about 65 nM), e.g., in a reagent composition or in separate containers.

[0266] In certain embodiments, the present disclosure provides a kit comprising a reagent composition that includes a first probe and a second probe, where the ratio of the concentration of the first probe to the concentration of the second probe is about 3 to about 20. In certain embodiments, the present disclosure provides a kit comprising a reagent composition that includes a first probe and a second probe, where the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20, e.g., about 1.5 to about 6, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 5 or about 3 to about 6.

[0267] In certain embodiments, the present disclosure provides a kit comprising a reagent composition that includes a first probe and a second probe, where the second probe is present in the reagent composition at a concentration of about 10 nM to about 70 nM, the first probe is present in the reagent composition at a concentration of about 50 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe in the reagent composition is about 1.5 to about 10.

[0268] In certain embodiments, the second probe provided in a kit of the present disclosure, e.g, in a reagent composition, can have a nucleotide sequence that has an identity of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater to the first probe. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe from about 90% to about 99%. In certain embodiments, the second probe provided in a kit of the present disclosure, e.g, in a reagent composition, can include a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe, differs by no more than 9 nucleotides, differs by no more than 8 nucleotides, differs by no more than 7 nucleotides, differs by no more than 6 nucleotides, differs by no more than 5 nucleotides, differs by no more than 4 nucleotides, differs by no more than 3 nucleotides, differs by no more than 2 nucleotides or differs by no more than 1 nucleotide from the nucleotide sequence of the first probe. In certain embodiments, the second probe provided in a kit of the present disclosure, e.g., in a reagent composition, can have a nucleotide sequence that differs by about 1 to about 10 nucleotides, e.g., about 1 to about 5 nucleotides, from the nucleotide sequence of the first probe.

[0269] In certain embodiments, the second probe provided in a kit of the present disclosure, e.g., in a reagent composition, hybridizes to a region (e.g., second region) of the amplicon that is located at a distance of about 100 nucleotides or less from the region of the amplicon (e.g., first region) hybridized to by the first probe, e.g., present in the same a reagent composition or in a separate reagent composition. In certain embodiments, the second probe provided in a kit of the present disclosure, e.g., in a reagent composition, hybridizes to a region (e.g., second region) of the amplicon that is located at a distance of about 50 nucleotides or less from the region of the amplicon (e.g., first region) hybridized to by the first probe, e.g., present in the same a reagent composition or in a separate reagent composition.

[0270] In certain embodiments, the reagent composition of a kit of the present disclosure can further include one or more of the following (in any combination): at least one recombinase, at least one single-stranded DNA binding protein, at least one DNA polymerase, dNTPs, a buffer, a reducing agent, ATP or an ATP analog, at least one recombinase loading protein, a nuclease, creatine kinase, a first primer and, optionally, a second primer, a crowding agent and a reverse transcriptase. In certain embodiments, the reagent composition can further include a control nucleic acid, e.g., a reference nucleic acid. For example, but not by way of limitation, the reagent composition can further include one or more primers, e.g., a first primer and / or a second primer.

[0271] 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, creatine kinase, a nuclease, a reverse transcriptase and a template nucleic acid molecule, e.g., a single-stranded (e.g., RNA) or double stranded nucleic acid.

[0272] 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) (z.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).

[0273] In certain embodiments, a kit of the present disclosure further includes an additional reagent composition (e.g., a second reagent composition or a third 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).

[0274] In certain embodiments, a composition of the present disclosure further includes a recombinase, a single-stranded binding protein, a polymerase, dNTPs, ATP, a primer (e.g., two primers) and a template nucleic acid. In certain embodiments, a composition of the present disclosure does not include a reverse transcriptase. Alternatively, such reagents can be present in a different container than the first and / or second probe. In certain embodiments, these reagents can be lyophilized.

[0275] 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.

[0276] 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. In certain embodiments, the kit can include other materials desirable from a commercial and user standpoint, including other buffers and diluents.

[0277] VI. Systems

[0278] The present disclosure further provides systems for performing the 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 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 (e.g., area for performing the isothermal amplification process) and a nucleic acid detection area (e.g., area for detecting the amplified target nucleic acids). 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.

[0279] 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., reagent composition) described herein. Non-limiting examples of compositions for use in systems disclosed herein are provided in Section II. A and Section III.

[0280] 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) comprising a first probe and a second probe at the ratios and / or amounts described herein. 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) comprising a first probe and a second probe, where the second probe is present at a concentration less than the concentration of the first probe.

[0281] 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 10 nM to about 300 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 50 nM to about 200 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 50 nM to about 170 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 50 nM to about 150 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 50 nM to about 120 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 60 nM to about 110 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 80 nM to about 175 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 50 nM to about 150 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 170 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 120 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 105 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 85 nM. 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 67.5 nM.

[0282] 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) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 5 nM to about 200 nM. 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 composition (e.g., reagent composition) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 10 nM to about 100 nM. 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) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 10 nM to about 70 nM. 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) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 15 nM to about 80 nM. 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) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 15 nM. 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) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 67.5 nM or about 105 nM. 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) comprising a first probe and a second probe, where the composition includes the second probe at a concentration of about 25 nM or about 65 nM.

[0283] 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) comprising a first probe and a second probe, where the composition includes the first probe at a concentration of about 20 nM to about 300 nM (e.g., about 50 nM to about 150 nM, e.g., about 67.5 nM, about 105 nM or about 120 nM) and the second probe at a concentration of about 5 nM to about 200 nM (e.g, about 10 nM to about 100 nM or about 10 nM to about 70 nM, e.g., about 25 nM or about 65 nM), e.g., in a reagent composition or in separate containers or reservoirs.

[0284] 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) comprising a first probe and a second probe, where the ratio of the concentration of the first probe to the concentration of the second probe is about 3 to about 20. 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) comprising a first probe and a second probe, where ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20, e.g., about 1.5 to about 6, about 1.5 to about 5, about 1.5 to about 4, about 1.5 to about 3, about 2 to about 5, about 2 to about 4, about 2 to about 3, about 3 to about 5 or about 3 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 8. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 7. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 6. In certain embodiments, the ratio of the concentration of the first probe to the concentration of the second probe in the nucleic acid analysis can be about 1.5 to about 5.

[0285] In certain embodiments, a system of the present disclosure includes a reagent composition that includes a first probe and a second probe (e.g., in a container or reservoir in the system), where the second probe is present in the reagent composition at a concentration of about 10 nM to about 70 nM, the first probe is present in the reagent composition at a concentration of about 50 nM to about 170 nM and the ratio of the concentration of the first probe to the concentration of the second probe in the reagent composition is about 1.5 to about 10.

[0286] In certain embodiments, the second probe provided in a system of the present disclosure, e.g., in a reagent composition, can have a nucleotide sequence that has an identity of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater to the first probe. In certain embodiments, the second probe comprises a nucleotide sequence having an identity to the first probe from about 90% to about 99%. In certain embodiments, the second probe provided in a system of the present disclosure, e.g., in a reagent composition, can include a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe, differs by no more than 9 nucleotides, differs by no more than 8 nucleotides, differs by no more than 7 nucleotides, differs by no more than 6 nucleotides, differs by no more than 5 nucleotides, differs by no more than 4 nucleotides, differs by no more than 3 nucleotides, differs by no more than 2 nucleotides or differs by no more than 1 nucleotide from the nucleotide sequence of the first probe. In certain embodiments, the second probe provided in a system of the present disclosure, e.g, in a reagent composition, can have a nucleotide sequence that differs by about 1 to about 10 nucleotides, e.g, about 1 to about 5 nucleotides, from the nucleotide sequence of the first probe.

[0287] In certain embodiments, the second probe provided in composition present in a reservoir or container of a system of the present disclosure, e.g., in a reagent composition, binds to a region (e.g., second region) of the amplicon that is located at a distance of about 100 nucleotides or less from the region of the amplicon (e.g., first region) bound by the first probe, e.g., present in the same a reagent composition or in a separate reagent composition. In certain embodiments, the second probe provided in composition present in a reservoir or container of a system of the present disclosure, e.g., in a reagent composition, binds to a region (e.g., second region) of the amplicon that is located at a distance of about 50 nucleotides or less from the region of the amplicon (e.g., first region) bound by the first probe, e.g., present in the same a reagent composition or in a separate reagent composition.

[0288] In certain embodiments, the reagent composition present in a reservoir or container of a system of the present disclosure can further include one or more of the following (in any combination): at least one recombinase, at least one single-stranded DNA binding protein, at least one DNA polymerase, dNTPs, a buffer, a reducing agent, ATP or an ATP analog, at least one recombinase loading protein, a nuclease, creatine kinase, a first primer and, optionally, a second primer, a crowding agent and a reverse transcriptase. In certain embodiments, the reagent composition can further include a control nucleic acid, e.g., a reference nucleic acid. For example, but not by way of limitation, the reagent composition can further include one or more primers, e.g., a first primer and / or a second primer.

[0289] In certain embodiments, a system of the present disclosure can further include one or more additional reagent compositions (e.g., a second and / or third reagent composition, e.g., in one or more additional containers or reservoirs in the system) 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, creatine kinase, a nuclease, a reverse transcriptase and a template nucleic acid molecule, e.g., a single-stranded (e.g., RNA) or double stranded nucleic acid.

[0290] In certain embodiments, a system of the present disclosure further includes an additional reagent composition (e.g., a second reagent composition, e.g, in one or more additional containers or reservoirs in the system) that includes the protein components (PC) (i.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).

[0291] In certain embodiments, a system of the present disclosure further includes an additional reagent composition (e.g., a second reagent composition or a third reagent composition, e.g. , in one or more additional containers or reservoirs in the system) 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).

[0292] VII Exemplary Embodiments

[0293] A. The present disclosure provides a method for detecting one or more target nucleic acids in a sample, comprising: a. performing an isothermal amplification process; and b. detecting the one or more target nucleic acids with a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

[0294] B. The present disclosure provides a method for detecting one or more variants of a target nucleic acid in a sample, comprising: a. performing an isothermal amplification process; and b. detecting the one or more variants of the target nucleic acid with a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

[0295] C. The present disclosure provides a method for detecting one or more target nucleic acids in a sample, comprising: a. performing an isothermal amplification process comprising: i. contacting a sample with a reagent composition comprising a first probe and a second probe, wherein the concentration of the second probe in the reagent composition is less than the concentration of the first probe; and ii. amplifying the one or more target nucleic acids; and b. detecting the one or more target nucleic acids with the first probe and the second probe.

[0296] D. The present disclosure provides a method for detecting one or more variants of a target nucleic acid in a sample, comprising: a. performing an isothermal amplification process comprising: i. contacting a sample with a reagent composition comprising a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe; and ii. amplifying the one or more variants of the target nucleic acid; and b. detecting the one or more variants of the target nucleic acid with the first probe and the second probe.

[0297] DI. The method of any one of A-D, wherein the concentration of the second probe is about 5 nM to about 200 nM.

[0298] D2. The method of any one of A-Dl, wherein the concentration of the second probe is about 15 nM or about 80 nM.

[0299] D2-1. The method of any one of A-Dl, wherein the concentration of the second probe is about 15 nM or about 70 nM.

[0300] D3. The method of any one of A-D2-1, wherein the concentration of the first probe is about 20 nM to about 300 nM.

[0301] D3-1. The method of any one of A-D3, wherein the concentration of the first probe is about 60 nM to about 170 nM.

[0302] D4. The method of any one of A-D3-1, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20.

[0303] D4-1. The method of any one of A-D4-1, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10.

[0304] D4-2. The method of any one of A-D4-2, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 8.

[0305] D5. The method of any one of A-D4-5, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 5.

[0306] D6. The method of any one of A-D5, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater.

[0307] D7. The method of any one of A-D6, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 90% or greater. D7-1. The method of any one of A-D7, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 95% or greater.

[0308] D8. The method of any one of A-D7-1, wherein the second probe comprises a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe.

[0309] D8-1. The method of any one of A-D8, wherein the second probe comprises a nucleotide sequence that is identical to or differs by no more than 5 nucleotides from the nucleotide sequence of the first probe.

[0310] D8-2. The method of any one of A-D8-1, wherein the second probe binds to a region of an amplicon of the target nucleic acid that is located at a distance of about 100 nucleotides or less from the region of the amplicon bound by the first probe.

[0311] D8-3. The method of any one of A-D8-2, wherein the second probe binds to a region of an amplicon of the target nucleic acid that is located at a distance of about 50 nucleotides or less from the region of the amplicon bound by the first probe.

[0312] D9. The method of any one of A-D8-3, wherein the first probe is labeled with a first fhiorophore and the second probe is labeled with a second fluorophore.

[0313] DIO. The method of D9, wherein the first fluorophore and the second fluorophore are different.

[0314] Dl l. The method of D9, wherein the first fluorophore and the second fluorophore are the same.

[0315] D12. The method of any one of A-Dl l, 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).

[0316] D13. The method of D12, wherein the isothermal amplification process is RPA.

[0317] D14. The method of D12, wherein the isothermal amplification process is NEAR.

[0318] D15. The method of any one of A-D14, wherein the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid.

[0319] DI 6. The method of any one of A-D15, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Parvovirus B19, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Virus, Influenza, Babesia, Malaria, Usutu Virus or HEV.

[0320] DI 7. The method of A or C, wherein the target nucleic acid is a variant of the target nucleic acid.

[0321] DI 8. The method of any one of B and D-D 17, wherein the variant of the target nucleic acid is a minority variant of the target nucleic acid.

[0322] DI 8-1. The method of DI 8, wherein the variant of the target nucleic acid is a minority variant of a gene of HIV- 1.

[0323] DI 8-2. The method of DI 8, wherein the variant of the target nucleic acid is a minority variant of an INT gene of HIV- 1.

[0324] DI 8-3. The method of DI 8, wherein the variant of the target nucleic acid is a minority variant of a POL gene of HIV-2.

[0325] DI 8-4. The method of DI 8, wherein the variant of the target nucleic acid is a minority variant of a gene of HEV.

[0326] DI 8-5. The method of DI 8-4, wherein the variant of the target nucleic acid is a minority variant of the 0RF1 of HEV.

[0327] DI 9. The method of any one of A-D18-5, wherein the sample is a tissue sample.

[0328] D20. The method of D19, wherein the target nucleic acid is isolated from the tissue sample prior to amplification.

[0329] D21. The method of any one of A-D20, wherein the sample is a biological fluid.

[0330] D22. The method of D21 wherein the biological fluid is blood.

[0331] D23. The method of D21 or D22, wherein the target nucleic acid is isolated from the biological fluid prior to amplification.

[0332] D24. The method of any one of C-D23, wherein the reagent composition further comprises one or more of the following: a. a DNA polymerase; b. a recombinase; c. a recombinase loading protein; d. a single stranded binding protein; e. dNTPs or a mixture of dNTPs and ddNTPs; f. a reducing agent; g. creatine kinase; h. a nuclease, i. a crowding agent; j . at least one primer; and k. a reverse transcriptase.

[0333] D25. The method of any one of C-D24, wherein the reagent composition comprises about 5 mM to about 100 mM Tris-HCl at a pH of about 6.5-9.0.

[0334] D26. The method of any one of C-D25, wherein the reagent composition comprises 5 mM to about 10 mM of a reducing agent.

[0335] D27. The method of any one of C-D26, wherein the reagent composition comprises about 50 mM to about 100 mM potassium acetate.

[0336] D28. The method of any one of C-D27, wherein the reagent composition comprises about 1 mM to about 5 mM dNTPs.

[0337] D29. The method of any one of C-D28, wherein the reagent composition comprises about 1 mM to about 10 mM of ATP.

[0338] D30. The method of any one of C-D29, wherein the reagent composition comprises about 20 mM to about 100 mM Phosphocreatine.

[0339] D31. The method of any one of C-D30, wherein the reagent composition comprises about 5 mM to about 40 mM Mg Acetate.

[0340] D32. The method of any one of C-D31, wherein the reagent composition comprises about 0.01 mg / mL to about 10 mg / mL BSA.

[0341] D33. The method of any one of C-D32, wherein the reagent composition comprises about 5% to about 10% Glycerol.

[0342] D34. The method of any one of C-D33, wherein the reagent composition comprises about 0.01 mg / mL to about 0.5 mg / mL Creatine Kinase.

[0343] D35. The method of any one of C-D34, wherein the reagent composition comprises about 0.1 mg / mL to about 1.0 mg / mL UvsX.

[0344] D36. The method of any one of C-D35, wherein the reagent composition comprises about 0.01 mg / mL to about 0.25 mg / mL UvsY.

[0345] D37. The method of any one of C-D36, wherein the reagent composition comprises about 0.01 mg / mL to about 0.5 mg / mL DNA Polymerase.

[0346] D38. The method of any one of C-D37, wherein the reagent composition comprises about 0.1 mg / mL to about 2.0 mg / mL GP32.

[0347] D39. The method of any one of C-D38, wherein the reagent composition comprises about 0.01 mg / mL to about 0.5 mg / mL Exonuclease III.

[0348] D40. The method of any one of C-D39, wherein the reagent composition comprises about 0.5 pg / mL to about 100.0 pg / mL of a reverse transcriptase. D41. The method of any one of A-D40, wherein the first probe and the second probe detect variants of the same target nucleic acid.

[0349] E. The present disclosure provides a composition for detecting one or more target nucleic acids comprising a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

[0350] F. The present disclosure provides a composition for detecting one or more variants of a target nucleic acid comprising a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

[0351] Fl. The composition of E or F, wherein the concentration of the second probe is about 5 nM to about 200 nM.

[0352] Fl-1. The composition of Fl, wherein the concentration of the second probe is about 10 nM to about 80 nM.

[0353] F2. The composition of any one of E-Fl-1, wherein the concentration of the second probe is about 15 nM to about 80 nM or about 25 nM or about 65 nM.

[0354] F3. The composition of any one of E-F2, wherein the concentration of the first probe is about 20 nM to about 300 nM.

[0355] F3-1. The composition of any one of E-F3, wherein the concentration of the first probe is about 60 nM to about 180 nM.

[0356] F3-2. The composition of any one of E-F3-1, wherein the concentration of the first probe is about 80 nM to about 180 nM.

[0357] F4. The composition of any one of E-F3-2, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20.

[0358] F4-1. The composition of any one of E-F4, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10.

[0359] F4-2. The composition of any one of E-F4-1, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 8.

[0360] F5. The composition of any one of E-F4-2, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 5.

[0361] F6. The composition of any one of E-F5, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater.

[0362] F7. The composition of any one of E-F6, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 90% or greater.

[0363] F7-1. The composition of any one of E-F7, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 95% or greater. F8. The composition of any one of E-F7-1, wherein the second probe comprises a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe.

[0364] F9. The composition of any one of E-F8, wherein the second probe comprises a nucleotide sequence that is identical to or differs by no more than 5 nucleotides.

[0365] F9-1. The composition of any one of E-F9, wherein the second probe binds to a region of an amplicon of the target nucleic acid that is located at a distance of about 100 nucleotides or less from the region of the amplicon bound by the first probe.

[0366] F9-2. The composition of any one of E-F9-1, wherein the second probe binds to a region of an amplicon of the target nucleic acid that is located at a distance of about 50 nucleotides or less from the region of the amplicon bound by the first probe.

[0367] F10. The composition of any one of E-F9, wherein the first probe is labeled with a first fhiorophore and the second probe is labeled with a second fluorophore.

[0368] Fl 1. The composition of F10, wherein the first fluorophore and the second fluorophore are different.

[0369] F12. The composition of F10, wherein the first fluorophore and the second fluorophore are the same.

[0370] F13. The composition of any one of E-F12, wherein the composition further comprises one or more reagents for performing an isothermal amplification process.

[0371] F14. The composition of F13, 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).

[0372] F15. The composition of F14, wherein the isothermal amplification process is RPA.

[0373] F16. The composition of F14, wherein the isothermal amplification process is NEAR.

[0374] Fl 7. The composition of any one of E-F15, wherein the target nucleic acid is abacterial, eukaryotic or viral nucleic acid.

[0375] F18. The composition of any one of E-F15, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Par-vovirus Bl 9, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Vi-rus, Influenza, Babesia, Malaria, Usutu Virus or HEV. Fl 9. The composition of any one of E-F18, wherein the target nucleic acid is a variant of the target nucleic acid.

[0376] F20. The composition of any one of E-F19, wherein the variant of the target nucleic acid is a minority variant of the target nucleic acid.

[0377] F21. The composition of any one of E-F20, wherein the variant of the target nucleic acid is a minority variant of a gene of HIV-1.

[0378] F22. The composition of any one of E-F21, wherein the variant of the target nucleic acid is a minority variant of an INT gene of HIV- 1.

[0379] F23. The composition of any one of E-F20, wherein the variant of the target nucleic acid is a minority variant of a POL gene of HIV-2.

[0380] F24. The composition of any one of E-F20, wherein the variant of the target nucleic acid is a minority variant of a gene of HEV.

[0381] F24-1. The composition of any one of E-F24, wherein the variant of the target nucleic acid is a minority variant of the ORF1 of HEV.

[0382] F25. The composition of any one of E-F24-1, wherein the composition further comprises one or more of the following: a. a DNA polymerase; b. a recombinase; c. a recombinase loading protein; d. a single stranded binding protein; e. dNTPs or a mixture of dNTPs and ddNTPs; f. a reducing agent; g. creatine kinase; h. a nuclease, i. a crowding agent; j . at least one primer; and k. a reverse transcriptase.

[0383] F26. The method of any one of E-F25, wherein the reagent composition comprises 5 mM to about 10 mM of a reducing agent.

[0384] F27. The method of any one of E-F26, wherein the reagent composition comprises about 50 mM to about 100 mM potassium acetate.

[0385] F28. The method of any one of E-F27, wherein the reagent composition comprises about 1 mM to about 5 mM dNTPs. F29. The method of any one of E-F28, wherein the reagent composition comprises about 1 mM to about 10 mM of ATP.

[0386] F30. The method of any one of E-F29, wherein the reagent composition comprises about 20 mM to about 100 mM Phosphocreatine.

[0387] F31. The method of any one of E-F30, wherein the reagent composition comprises about 5 mM to about 40 mM Mg Acetate.

[0388] F32. The method of any one of E-F31, wherein the reagent composition comprises about 0.01 mg / mL to about 10 mg / mL BSA.

[0389] F33. The method of any one of E-F32, wherein the reagent composition comprises about 5% to about 10% Glycerol.

[0390] F34. The method of any one of E-F33, wherein the reagent composition comprises about 0.01 mg / mL to about 0.5 mg / mL Creatine Kinase.

[0391] F35. The method of any one of E-F34, wherein the reagent composition comprises about 0.1 mg / mL to about 1.0 mg / mL UvsX.

[0392] F36. The method of any one of E-F35, wherein the reagent composition comprises about 0.01 mg / mL to about 0.25 mg / mL UvsY.

[0393] F37. The method of any one of E-F36, wherein the reagent composition comprises about 0.01 mg / mL to about 0.5 mg / mL DNA Polymerase.

[0394] F38. The method of any one of E-F37, wherein the reagent composition comprises about 0.1 mg / mL to about 2.0 mg / mL GP32.

[0395] F39. The method of any one of E-F38, wherein the reagent composition comprises about 0.01 mg / mL to about 0.5 mg / mL Exonuclease III.

[0396] F40. The method of any one of E-F39, wherein the reagent composition comprises about 0.5 pg / mL to about 100.0 pg / mL of a reverse transcriptase.

[0397] F41. The method of any one of E-F40, wherein the first probe and the second probe detect variants of the same target nucleic acid.

[0398] F42. The method of any one of E-F41, wherein the reagent composition comprises about 5 mM to about 100 mM Tris-HCl at a pH of about 6.5-9.0.

[0399] G. The present disclosure provides a system for performing the method of any one of A-D23.

[0400] Gl. The system of G, wherein the system is automated.

[0401] H. The present disclosure provides a system comprising the composition of any one of E-F42.

[0402] HL The system of H, wherein the system is automated. I. The present disclosure provides a kit for performing the method of any one of A-D23.

[0403] J. The present disclosure provides a kit comprising the composition of any one of E- F42.

[0404] EXAMPLES

[0405] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0406] Example 1: Addition of a secondary probe improves RPA signal of HIV-1 and HIV-2 nucleic acid variants

[0407] This example discloses the addition of a secondary probe in an RPA reaction to improve the detection of different HIV-1 Groups. FIGS. 1A-1B illustrate exemplary results associated with the amplification and detection of HIV-1, M and N Groups within the INT amplicon. The integrase (INT) gene region and the Gag gene region of Human HIV-1 was amplified and detected by RPA using the primers, probes and concentrations shown in Table 1. “FP” represents forward primer, “RP” represents reverse primer and “P” represent probe in Table 1.

[0408] Table 1

[0409] The addition of the second INT probe (P2) improved HIV-1 Group N detection substantially as shown in FIG. IB. Group N targets are weakly detected by the nominal INT probe (P4) as shown in FIG. 1A. The addition of the second INT probe (P2) at a low concentration of 25 nM significantly improved Group N signal and variant detection earlier in the RPA process. Probe P2 and Probe P4 both have a length of about 48-49 nucleotides, both target the INT gene region and have a difference of two nucleotides between their nucleotide sequences. Probe P2 binds to the variant sequence of the INT gene region. As shown in FIG. 1A and FIG. IB, Group N is initially detected around cycle 17 of the RPA process in the presence of the P2 probe. In the absence of the P2 probe, Group N targets are not detected until around cycle 45. It was found that the addition of the second INT probe (P2) at a concentration of 65 nM with the nominal INT probe (P4) at a concentration of 105 nM also significantly improved Group N signal and variant detection earlier in the RPA process in an automated process.

[0410] Similar results were observed during an RPA process for the detection of HIV- 2 subtypes. FIGS. 2A-2C provide exemplary results demonstrating the addition of 25 nM of a second probe (P9) to an RPA process that includes a first probe (Pl). As shown in FIGS. 2A-2C, the addition of 25 nM of a second probe (P9) significantly improved the detection (sensitivity and MR) of the pol gene of both HIV-2A and HIV-2B subtypes at or below 2X limit of detection (LOD) of 20 cps / mL (FIG. 2C) compared to an RPA process in the absence of the second probe (P9) (FIG. 2A) or the presence of the second probe (P9) alone (FIG. 2B). Amplification of nucleic acid was performed using RPA with the primer oligonucleotides and probe oligonucleotides at the amounts specified in Table 2. The first probe (Pl) has a length of about 45-46 nucleotides and the second probe (P9) has a length of about 46-47 nucleotides. The first probe (Pl) and the second probe (P9) both target the pol gene and have a difference of 5 nucleotides between their nucleotide sequences. In particular, the first probe (Pl) includes 2 additional nucleotides at its 5’ end and the second probe include three additional nucleotides at its 3’ end. The second probe (P9) was designed to eliminate the 5’ nucleotide region of the pol gene that contains a mismatch between the nucleotide sequences of the HIV-2A and HIV- 2B subtypes. However, the use of the P9 probe alone did not improve the detection of the pol gene of the HIV-2B subtype (FIG. 2B) compared to an RPA process performed in the absence of the P9 (FIG. 2A). Surprisingly, it was the combined use of the first probe and the second probe that led to a significant improvement in the detection of the HIV-2B subtype (FIG. 2C).

[0411] Table 2

[0412] This example shows that the addition of a second probe at lower concentrations than the first probe surprising results in synergistic improvement in the detection of the amplicons in an RPA process.

[0413] Example 2: Addition of a secondary probe improves RPA signal of HEV variants

[0414] This example discloses the addition of a secondary probe in an RPA reaction to improve the detection of an HEV variant. FIG. 3A illustrates exemplary results associated with the amplification and detection of the HEV variant GT3f using a second probe. The 5’ UTR and the 0RF1 of HEV was amplified and detected by RPA using the primers, probes and concentrations shown in Table 3. As shown in FIG. 3B, the secondary probe is complementary to a sequence of the HEV amplicon that does not overlap with the HEV amplicon sequence that is complementary to the primary probe. In particular, the secondar probe hybridizes to the HEV GT3f variant (e.g., of the 0RF1 gene region).

[0415] Table 3

[0416] As shown in FIG. 3A, the presence of a secondary probe at a low concentration of 15 nM significantly improved detection of the HEV genetic variant GT3f compared to the use of the primary probe alone. In addition, the use of the secondary probe alone did not result in the detection of the HEV variant and a comparable increase in the concentration of the primary probe did not improve detection of the HEV variant. This example shows that the addition of a second probe at lower concentrations than the first probe surprising results in synergistic improvement in the detection of the amplicons in an RPA process. 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.

[0417] 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 method for detecting one or more target nucleic acids in a sample, comprising: a) performing an isothermal amplification process; and b) detecting the one or more target nucleic acids with a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

2. A method for detecting one or more variants of a target nucleic acid in a sample, comprising: a) performing an isothermal amplification process; and b) detecting the one or more variants of the target nucleic acid with a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

3. A method for detecting one or more target nucleic acids in a sample, comprising: a) performing an isothermal amplification process comprising: i. contacting a sample with a reagent composition comprising a first probe and a second probe, wherein the concentration of the second probe in the reagent composition is less than the concentration of the first probe; and ii. amplifying the one or more target nucleic acids; and b) detecting the one or more target nucleic acids with the first probe and the second probe.

4. A method for detecting one or more variants of a target nucleic acid in a sample, comprising: a) performing an isothermal amplification process comprising: i. contacting a sample with a reagent composition comprising a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe; and ii. amplifying the one or more variants of the target nucleic acid; and b) detecting the one or more variants of the target nucleic acid with the first probe and the second probe.

5. The method of any one of claims 1-4, wherein the concentration of the second probe is about 5 nM to about 200 nM.

6. The method of any one of claims 1-5, wherein the concentration of the second probe is about 15 nM or about 80 nM.

7. The method of any one of claims 1-6, wherein the concentration of the first probe is about 20 nM to about 300 nM.

8. The method of any one of claims 1-7, wherein the concentration of the first probe is about 60 nM to about 170 nM.

9. The method of any one of claims 1-8, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20.

10. The method of any one of claims 1-9, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 8.

11. The method of any one of claims 1-10, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 7.

12. The method of any one of claims 1-11, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 5.

13. The method of any one of claims 1-12, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater.

14. The method of any one of claims 1-13, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 90% or greater.

15. The method of any one of claims 1-14, wherein the second probe comprises a nucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe.

16. The method of any one of claims 1-15, wherein the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 100 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe.

17. The method of any one of claims 1-16, wherein the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 50 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe.

18. The method of any one of claims 1-17, wherein the first probe is labeled with a first fhiorophore and the second probe is labeled with a second fluorophore.

19. The method of claim 18, wherein the first fluorophore and the second fluorophore are different.

20. The method of claim 18, wherein the first fluorophore and the second fluorophore are the same.

21. The method of any one of claims 1-20, wherein 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).

22. The method of claim 21, wherein the isothermal amplification process is RPA.

23. The method of claim 21, wherein the isothermal amplification process is NEAR.

24. The method of any one of claims 1-23, wherein the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid.

25. The method of any one of claims 1-24, wherein the target nucleic acid is derived from SARS-CoV-2 (COVID-19), HIV-1, HIV-2, HBV, HCV, CMV, Parvovirus B19, HAV, Chlamydia, Gonorrhea, WNV, Zika Virus, Dengue Virus, Chikungunya Virus, Influenza, Babesia, Malaria, Usutu Virus or HEV.

26. The method of claim 1 or 3, wherein the target nucleic acid is a variant of the target nucleic acid.

27. The method of any one of claims 2 and 4-26, wherein the variant of the target nucleic acid is a minority variant of the target nucleic acid.

28. The method of claim 27, wherein the minority variant of the target nucleic acid is a minority variant of a target nucleic acid of HIV-1.

29. The method of claim 27, wherein the minority variant of the target nucleic acid is a minority variant of a target nucleic acid of HIV-2.

30. The method of claim 27, wherein the minority variant of the target nucleic acid is a minority variant of a target nucleic acid of HEV.

31. The method of any one of claims 1-30, wherein the sample is a tissue sample.

32. The method of claim 31, wherein the target nucleic acid is isolated from the tissue sample prior to amplification.

33. The method of any one of claims 1-30, wherein the sample is a biological fluid.

34. The method of claim 33, wherein the biological fluid is blood.

35. The method of claim 33 or 34, wherein the target nucleic acid is isolated from the biological fluid prior to amplification.

36. The method of any one of claims 3-35, wherein the reagent composition further comprises one or more of the following: a) a DNA polymerase; b) a recombinase; c) a recombinase loading protein; d) a single stranded binding protein;e) dNTPs or a mixture of dNTPs and ddNTPs; f) a reducing agent; g) creatine kinase; h) a nuclease, i) a crowding agent; j) at least one primer; and k) a reverse transcriptase.

37. A composition for detecting one or more target nucleic acids comprising a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

38. A composition for detecting one or more variants of a target nucleic acid comprising a first probe and a second probe, wherein the concentration of the second probe is less than the concentration of the first probe.

39. The composition of claim 37 or 38, wherein the concentration of the second probe is about 5 nM to about 200 nM.

40. The composition of any one of claims 37-39, wherein the concentration of the second probe is about 15 nM to about 80 nM or about 25 nM or about 65 nM.

41. The composition of any one of claims 37-40, wherein the concentration of the first probe is about 20 nM to about 300 nM.

42. The composition of any one of claims 37-41, wherein the concentration of the first probe is about 60 nM to about 170 nM.

43. The composition of any one of claims 37-42, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 20.

44. The composition of any one of claims 37-43, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 10.

45. The composition of any one of claims 37-44, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 8.

46. The composition of any one of claims 37-45, wherein the ratio of the concentration of the first probe to the concentration of the second probe is about 1.5 to about 5.

47. The composition of any one of claims 37-46, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 85% or greater.

48. The composition of any one of claims 37-47, wherein the second probe comprises a nucleotide sequence having an identity to the first probe of about 90% or greater.

49. The composition of any one of claims 37-48, wherein the second probe comprises anucleotide sequence that is identical to or differs by no more than 10 nucleotides from the nucleotide sequence of the first probe.

50. The composition of any one of claims 37-49, wherein the second probe comprises a nucleotide sequence that is identical to or differs by no more than 5 nucleotides.

51. The composition of any one of claims 37-50, wherein the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 100 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe.

52. The composition of any one of claims 37-51, wherein the second probe hybridizes to a region of the target nucleic acid that is located at a distance of about 50 nucleotides or less from the region of the target nucleic acid hybridized to by the first probe.

53. The composition of any one of claims 37-46, wherein the first probe is labeled with a first fluorophore and the second probe is labeled with a second fluorophore.

54. The composition of claim 53, wherein the first fluorophore and the second fluorophore are different.

55. The composition of claim 53, wherein the first fluorophore and the second fluorophore are the same.

56. The composition of any one of claims 37-55, wherein the composition further comprises one or more reagents for performing an isothermal amplification process.

57. The composition of claim 56, 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).

58. The composition of claim 57, wherein the isothermal amplification process is RPA.

59. The composition of claim 57, wherein the isothermal amplification process is NEAR.

60. The composition of any one of claims 37-59, wherein the composition further comprises one or more of the following: a) a DNA polymerase; b) a recombinase; c) a recombinase loading protein; d) a single stranded binding protein; e) dNTPs or a mixture of dNTPs and ddNTPs; f) a reducing agent;g) creatine kinase; h) a nuclease, i) a crowding agent; j) at least one primer; and k) a reverse transcriptase.

61. The composition of any one of claims 37-60, wherein the target nucleic acid is a bacterial, eukaryotic or viral nucleic acid.

62. The composition of any one of claims 37-61, 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.

63. The composition of any one of claims 37-62, wherein the target nucleic acid is a variant of the target nucleic acid.

64. The composition of claim 63, wherein the variant of the target nucleic acid is a minority variant of the target nucleic acid.

65. The composition of claim 64, wherein the minority variant of the target nucleic acid is a minority variant of a target nucleic acid of HIV- 1.

66. The composition of claim 64, wherein the minority variant of the target nucleic acid is a minority variant of a target nucleic acid of HIV-2.

67. The composition of claim 64, wherein the minority variant of the target nucleic acid is a minority variant of a target nucleic acid of HEV.

68. A system for performing the method of any one of claims 1-36.

69. The system of claim 68, wherein the system is automated.

70. A system comprising the composition of any one of claims 37-67.

71. The system of claim 70, wherein the system is automated.

72. A kit for performing the method of any one of claims 1-36.

73. A kit comprising the composition of any one of claims 37-67.

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