WO2025026366A1

WO2025026366A1 - Compositions and methods for nucleic acid detection

Info

Publication number: WO2025026366A1
Application number: PCT/CN2024/108944
Authority: WO
Inventors: Xiang Li; Kun Yang; Liping Guo; Huiying FENG
Original assignee: Coyote Bioscience Co Ltd
Current assignee: Coyote Bioscience Co Ltd
Priority date: 2023-07-31
Filing date: 2024-07-31
Publication date: 2025-02-06
Anticipated expiration: 2026-01-31

Abstract

Provided herein are systems, methods, compositions, and kits for analyte detection. The systems, methods, compositions, and kits can allow for multiplex analysis of multiple analytes. The compositions provided herein comprises various probes, enzymes, label moieties, quencher moieties, or primers for practicing the methods. Additionally, provided herein are analysis methods for analyte detections and the devices and/or systems for practicing the same.

Description

COMPOSITIONS AND METHODS FOR NUCLEIC ACID DETECTION

CROSS-REFERENCE

This application is a continuation of Patent Cooperation Treaty Patent Application No. PCT/CN2023/110321, filed July 31, 2023, which is entirely incorporated herein by reference.

BACKGROUND

Analyte detection has various applications in the fields of molecular biology and medicine. For example, nucleic acid detection provides information for diagnosing disease conditions to prevent or control disease transmission in a population. Additionally, nucleic acid detection can also facilitate diagnosing or prognosing a certain condition in a subject to facilitate therapeutic and treatment designs. Despite the advance of molecular biology technology, current analyte detection methods lack efficiency, modularity, and/or accuracy; requires laborious efforts; and/or are time-consuming.

SUMMARY

Provided herein are methods, compositions, and systems for analyte detections. The methods, compositions, and systems provided herein can allow multiplex detection of various nucleic acids. These methods, compositions, and systems are also modular, thus providing uniform platforms to analyze various types or subtypes of analytes. Additionally, the methods, compositions, and systems can be optimized for increased accuracy and speed, relative to the existing counterparts. Because the methods, compositions, and systems provided herein are simplified, they also require minimal efforts to implement.

Provided herein, are probes. In an aspect, a probe comprises, in a 5’ to 3’ direction, (1) a label moiety, a first template-binding nucleotide sequence, a second template-binding nucleotide sequence, and a quencher moiety or (2) the quencher moiety, the first template-binding nucleotide sequence, the second template-binding nucleotide sequence, and the label moiety; wherein the first and second template-binding nucleotide sequences binds two different template nucleotide sequences.

In some embodiments, each of the first and second template-binding nucleotide sequences is complementary to only one of the two different template nucleotide sequences. In some embodiments, the first and second template-binding nucleotide sequences are complementary to the two different template nucleotide sequences of two different template nucleic acid molecules. In some embodiments, the first template-binding nucleotide sequence is complementary to a non-naturally occurring nucleotide sequence. In some embodiments, the second template-binding nucleotide is complementary to a pathology-associated nucleotide sequence. In some embodiments, the pathology-associated nucleotide sequence comprises a nucleotide sequence of a pathogen. In some embodiments, the pathogen comprises a virus, bacterium, protozoan, fungus, or a combination thereof. In some embodiments, the pathology-associated nucleotide sequence comprises a sequence of a cell. In some embodiments, the cell comprises a eucaryotic cell. In some embodiments, the cell comprises a human cell. In some embodiments, the cell is associated with a disease condition. In some embodiments, the disease condition comprises a cancer, a genetic disorder, an infectious disease, or a combination thereof.

Provided herein, are probes. In an aspect, a probe comprises, in a 5’ to 3’ direction: (1) a label moiety, a nucleotide sequence comprising at least 25 nucleotides, and a quencher moiety; or (2) the quencher moiety, the nucleotide sequence comprising the at least 25 nucleotides, and the label moiety.

In some embodiments, the nucleotide sequence comprises at most 100 nucleotides. In some embodiments, the nucleotide sequence comprises at most 80 nucleotides. In some embodiments, the nucleotide sequence comprises at most 50 nucleotides. In some embodiments, the nucleotide sequence comprises at most 100 nucleotides, at most 80 nucleotides, or at most 50 nucleotides. In some embodiments, the nucleotide sequence comprises at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, or at least 32 nucleotides. In some embodiments, the nucleotide sequence comprises at least 32 nucleotides. In some embodiments, the label moiety comprises 2, 3, 4, 5, or more label moieties. In some embodiments, the label moiety comprises FAM, SYBR, JOE, VIC, NED, Cy3, TAMRA, ROX, Texas Red, Cy5, TET, HEX, Quasar 670, or Cy5.5. In some embodiments, the quencher moiety comprises 2, 3, 4, 5, or more quencher moieties. In some embodiments, the quencher moiety comprises Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, or BBQ 650. In some embodiments, the label moiety is configured to provide a label signal that is quenchable, absorbable, or alterable by the quencher moiety.

Provided herein, are methods for detecting a target nucleic acid. In an aspect, a method for detecting a target nucleic acid comprises: (a) providing a reaction mixture comprising (i) the probe described herein; and (ii) the target nucleic acid or an amplification product thereof, an upstream primer of the target nucleic acid, a downstream primer of the target nucleic acid, a second probe, and an enzyme; (b) detecting a label signal generated by the label moiety.

Provided herein, are compositions. In an aspect, a composition comprises the probe described herein and at least one of: a target nucleic acid or an amplification product thereof, an upstream primer, a downstream primer, a second probe, or an enzyme.

Provided herein, are compositions. In an aspect, a composition comprises: a first probe comprising (a) a nucleotide sequence and (b) a first label moiety and a first quencher moiety; and a second probe comprising a second label moiety or a second quencher moiety coupled thereto, wherein the first label moiety is coupled to a first terminal end of the first probe, and wherein the first quencher moiety is coupled to a second terminal end of the first probe different from the first terminal end.

In some embodiments, the first quencher moiety and the second quencher moiety are a same quencher moiety. In some embodiments, the first quencher moiety and the second quencher moiety are different quencher moieties. In some embodiments, the label moiety comprises 2, 3, 4, 5, or more label moieties. In some embodiments, the label moiety comprises FAM, SYBR, JOE, VIC, NED, Cy3, TAMRA, ROX, Texas Red, Cy5, TET, HEX, Quasar 670, or Cy5.5. In some embodiments, the second probe comprises the second quencher moiety coupled thereto. In some embodiments, the second quencher moiety comprises 2, 3, 4, 5, or more quencher moieties. In some embodiments, the second quencher moiety comprises Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, or BBQ 650. In some embodiments, the first label moiety is configured to provide a label signal that is quenchable, absorbable, or alterable by the first and second quencher moieties. In some embodiments, the second quencher moiety of the second probe is configured to quench, absorb, or alter a label signal generated by the first label moiety on the first probe. In some embodiments, the composition further comprises at least one of: a target nucleic acid or an amplification product thereof, an upstream primer, a downstream primer, or an enzyme.

Provided herein, are methods for detecting a target nucleic acid. In an aspect, a method for detecting a target nucleic acid comprises: (a) providing a reaction mixture comprising (i) the composition of described herein; and (ii) the target nucleic acid or an amplification product thereof, an upstream primer of the target nucleic acid, a downstream primer of target nucleic acid, and an enzyme; (b) detecting a label signal generated by the first label moiety.

Provided herein, are methods comprising: (a) hybridizing a first mediator probe to a target nucleic acid, wherein the first mediator probe comprises at least a nucleotide sequence complementary to a sequence of the target nucleic acid or an amplification product thereof, wherein the first mediator probe comprises, in a 5’ to 3’ direction, a first template-binding nucleotide sequence, a second template-binding nucleotide sequence, and a quencher moiety; (b) cleaving a cleaved fragment from the first mediator probe resulting in a first detectable signal or signal change, wherein the cleaved fragment comprises a second sequence is not complementary to a second sequence of the target nucleic acid; (c) hybridizing the cleaved fragment to a nucleotide sequence of the first reporter probe to generate a duplex molecule and result in a second detectable signal or signal change; (d) heating the duplex molecule to generate at least one third detectable signal or signal change; (e) identifying a presence, an absence, or an amount of the target nucleic acid by matching the at least one third detectable signal or signal change and a temperature or temperature range at which the at least one third detectable signal or signal change is generated.

In some embodiments, the method further comprises hybridizing an upstream primer or/and a downstream primer to the target nucleic acid to generate the amplification product of the target nucleic acid. In some embodiments, the method further comprises providing an enzyme.

In some embodiments, the enzyme comprises a comprises a nuclease activity or a nucleic acid polymerase activity. In some embodiments, the enzyme comprises a Flap nuclease. In some embodiments, the cleaved fragment is cleaved from the first mediator probe using the enzyme. In some embodiments, (c) comprises extending the cleaved fragment to generate the duplex molecule. In some embodiments, (c) comprises extending the cleaved fragment to generate the duplex molecule using the cleaved fragment as a primer in a nucleic acid polymerization reaction. In some embodiments, the (c) comprises extending the cleaved fragment to generate the duplex molecule using a sequence of the first reporter probe as a template in a nucleic acid polymerization reaction. In some embodiments, the nucleic acid polymerization reaction comprises a nucleic acid amplification reaction. In some embodiments, the nucleic acid amplification reaction comprises a polymerase chain reaction (PCR) . In some embodiments, the method further comprises detecting the first detectable signal or signal change. In some embodiments, the method further comprises detecting the second detectable signal or signal change. In some embodiments, the method further comprises detecting the third detectable signal or signal change. In some embodiments, the method further comprises repeating (a) - (e) , wherein a second mediator probe is used in place of the first mediator probe, wherein the second mediator probe comprises at least a nucleotide sequence complementary to a second sequence of the target nucleic acid or an amplification product thereof different from the nucleotide sequence of the target nucleic acid or the amplification product thereof, and wherein a cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to a second nucleotide sequence of the first reporter probe that is different from the nucleotide sequence of the first reporter probe. In some embodiments, the method further comprises repeating (a) - (e) , wherein a second mediator probe is used in place of the first mediator probe and a second reporter probe is used in place of a first reporter probe, wherein the second mediator probe comprises at least a nucleotide sequence complementary to a second sequence of the target nucleic acid or an amplification product thereof different from the nucleotide sequence of the target nucleic acid or the amplification product thereof, wherein the cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to sequence of the second reporter probe, wherein the first and second reporter probes are different probe molecules. In some embodiments, the cleaved fragment cleaved from the second mediator probe does not hybridize to the first reporter probe or the cleaved fragment cleaved from the first mediator probe does not hybridize to the second reporter probe. In some embodiments, the method further comprises repeating (a) - (e) , wherein a second mediator probe is used in place of the first mediator probe, wherein the second mediator probe comprises at least a nucleotide sequence complementary to a sequence of a second target nucleic acid or an amplification product thereof different from the target nucleic acid or the amplification product thereof, and wherein a cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to a second nucleotide sequence of the first reporter probe that is different from the nucleotide sequence of the first reporter probe. In some embodiments, a cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to a second nucleotide sequence of the first reporter probe that is different from the nucleotide sequence of the first reporter probe. In some embodiments, the first signal change generated when using the first mediator probe in (a) is the same as a first signal or signal change generated when using the second mediator probe in (a) . In some embodiments, the first signal change generated when using the first mediator probe in (a) is different from a first signal or signal change generated when using the second mediator probe in (a) . In some embodiments, the second signal change generated when using the first mediator probe in (c) is the same as a second signal or signal change generated when using the second mediator probe in (c) . In some embodiments, the second signal change generated when using the first mediator probe in (c) is different from a second signal or signal change generated when using the second mediator probe in (c) . In some embodiments, the third signal change generated when using the first mediator probe in (d) is the same as a third signal or signal change generated when using the second mediator probe in (d) . In some embodiments, the third signal change generated when using the first mediator probe in (d) is different from a third signal or signal change generated when using the second mediator probe in (d) . In some embodiments, the quencher moiety is coupled to a nucleotide that is at most about 10 nucleotides 3’ to a 3’ terminal nucleotide of the first template-binding nucleotide sequence. In some embodiments, the quencher moiety is coupled to a nucleotide that is at most about 7 nucleotides 3’ to the 3’ terminal nucleotide of the first template-binding nucleotide sequence. In some embodiments, the quencher moiety is coupled to a nucleotide that is at most about 5 nucleotides 3’ to the 3’ terminal nucleotide of the first template-binding nucleotide sequence.

Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein. Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative instances of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different instances, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 1 depicts an exemplary workflow of the method described herein.

FIG. 2 depicts another exemplary workflow of the method described herein.

FIG. 3 depicts a first exemplary method with the exemplary compositions as described herein.

FIG. 4 depicts a second exemplary method with the exemplary compositions as described herein.

FIG. 5 depicts a third exemplary method with the exemplary compositions as described herein.

FIG. 6 depicts a fourth exemplary method with the exemplary compositions for analyzing a target nucleic acid.

FIG. 7 depicts a fifth exemplary method with the exemplary compositions for analyzing a target nucleic acid.

FIG. 8 depicts sixth, seventh, or eighth exemplary methods with the exemplary compositions for analyzing a target nucleic acid.

FIG. 9A shows an exemplary quantification analysis of amplification signals using four fluorophores.

FIG. 9B shows an exemplary melting curve analysis using two mediator probes directed to two target nucleic acids.

FIG. 10 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.

FIGs. 11A-B show an exemplary quantification analysis of amplification signals using exemplary mediator/reporter probes described herein. FIGs. 11C-D show an exemplary melting curve analysis using exemplary mediator/reporter probes described herein.

FIG. 12 shows an exemplary quantification analyses and melting curve analyses using exemplary mediator probes described herein.

DETAILED DESCRIPTION

Introduction

Provided herein are methods, compositions, devices, systems, and kits for analyte detection. The methods may comprise the workflow 100 of FIG. 1. A first probe may bind a target analyte in step 101. In step 102, a fragment or segment of the first probe may be subsequently released. The released fragment or segment may subsequently bind a second probe in step 103 and generate a detectable signal in step 104. The compositions, devices, systems, and kits provided herein may be used to implement the methods described herein. The term “analyte” as used herein, generally refers to an object that is the subject of analysis, or an object, regardless of being the subject of analysis, that is directly or indirectly analyzed during a step of the methods described herein. An analyte may be naturally occurring. An analyte may be non-naturally occurring or synthetic. An analyte may be, originate from, and/or be derived from, a sample, such as a biological sample. In some examples, an analyte is or includes a molecule, macromolecule, nucleic acid, carbohydrate, lipid, antibody, antibody fragment, antigen, peptide, polypeptide, protein, macromolecular group, cell, tissue, biological particle, or an organism, or any engineered copy or variant thereof, or any combination thereof.

In an aspect, the methods may comprise the workflow 200 of FIG. 2. A mediator probe may be hybridized to a target nucleic acid in step 201. The mediator probe may comprise any of those described herein. In step 202, a fragment or segment of the mediator probe may be subsequently released. In step 202, the hybridization may also generate an intermediate product. The intermediate product may comprise any of those described herein. In some cases, step 202 may comprise using an enzyme to release the released fragment or segment from the mediator probe. The released fragment or segment may subsequently hybridize to a reporter probe in step 203 and subsequently generate a detectable signal in step 204. The reporter probe may comprise any of those described herein. In some cases, the detectable signal in step 204 may comprise an amplification signal, a hybridization signal, a denaturation signal, a combination thereof, or any of those described herein.

In some cases, step 202 may generate a detectable signal (e.g., an amplification signal, a hybridization signal, a denaturation signal, or a combination thereof) . In some cases, prior to step 201, an optional step 201.1 may comprise amplifying the target nucleic acid in an amplification reaction. The amplification may comprise at least a primer. For example, the amplification may comprise an upstream and/or a downstream primer. In some embodiments, optional step 201.1 may also comprise generation of a detectable signal described herein.

In some cases, prior to step 204, an optional step 203.1 may comprise amplifying the intermediate product in an amplification reaction. This amplification may generate a detectable signal described herein. In some cases, the detectable signal generated in any of the steps 201.1, 202, 203.1, and 204 may be the same as another step. In some cases, the detectable signal generated in any of the steps 201.1, 202, 203.1, and 204 may be different from another step.

In some cases, the method may allow multiplex detection of more than one target nucleic acids, as described herein. The compositions, devices, systems, and kits provided herein may be used to implement the methods described herein.

The terms “nucleic acid, ” “nucleic acid molecule, ” “nucleic acid sequence, ” “nucleic acid fragment, ” “oligonucleotide” and “polynucleotide, ” as used herein, generally refer to a polynucleotide that may have various lengths of bases, comprising, for example, deoxyribonucleotide, deoxyribonucleic acid (DNA) , ribonucleotide, or ribonucleic acid (RNA) , or analogs thereof. A nucleic acid may be single-stranded. A nucleic acid may be double-stranded. A nucleic acid may be partially double-stranded, such as to have at least one double-stranded region and at least one single-stranded region. A partially double-stranded nucleic acid may have one or more overhanging regions. A nucleic acid may comprise A nucleic acid can comprise a sequence of four natural nucleotide bases: adenine (A) ; cytosine (C) ; guanine (G) ; and thymine (T) (uracil (U) for thymine (T) when the nucleic acid is RNA) . A nucleic acid may include one or more nonstandard nucleotide (s) , nucleotide analog (s) and/or modified nucleotide (s) .

As used herein, the terms “hybridization” and “hybridizing” or “binding (between two nucleic acid molecules” refers to the process by which complementary single-stranded nucleic acid molecules form double-stranded nucleic acids. Two nucleic acid sequences that have substantially complementarity can hybridize (to each other) . The degree of complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used (e.g., temperature, pH, or ionic strength of the reaction mixture) .

The methods (and compositions, devices, systems, and kits for practicing the same) have various beneficial advantages over the existing methods. Because the methods may not require amplification or extension reaction-which can require an extended period of time to initiate and/or complete-for detectable signal generation, the methods may be implemented in a shorter period of time, relative to the existing methods. Because the methods can provide various signal detection opportunities with the various detectable signals that can be generated, and the various detectable signals can be designed to analyze various types or subtypes of target analytes, a substantial amount of time and reagents may not be used or wasted. For example, the methods can be designed such that detection of a type of target analytes in an earlier step of the methods will indicate whether a later step to detect various subtypes of the target analytes needs to be performed. Because various compositions for practicing the methods (such as the cleaved fragment or segment of the mediator probe, the reporter probe, the label/quencher moieties coupled thereto, or the enzymes or nucleotides) may be the same or substantially the same for detecting various analytes, the methods described (and systems/devices/kits for practicing the same) are modular for detecting various analytes with minimal modifications. Because of the same reasons, the experiment parameters (such as temperature, concentration of various reagents, buffer conditions, time, or any combination thereof) may also be the same for detecting various analytes, allowing the same or substantially the same set of reagents/devices/systems for practicing the methods. The methods described herein thus have efficient and wide application in diagnosing disease conditions to prevent or control disease transmission in a population or diagnosing or prognosing a certain condition in a subject to facilitate therapeutic and treatment designs.

Mediator probe

Provided herein are mediator probes. The mediator probes provided herein may facilitate generation of at least 1, 2, 3, 4, 5 or more detectable signals. The mediator probes provided herein may facilitate generation of at most 1, 2, 3, 4, or 5 detectable signals. The detectable signal may facilitate quantification and/or identification of a target analyte (such as a target nucleic acid) . The mediator probe may be cleaved. The mediator probe may hybridize to a target nucleic acid or an amplification product thereof. A cleaved fragment or segment of the mediator probe may be generated. The cleaved fragment or segment of the mediator probe may hybridize to another nucleic acid (such as at least one reporter probe) . The mediator probe or a cleaved fragment (or segment) thereof may be extended in an amplification reaction (e.g., when hybridized to a target nucleic acid or reporter probe, respectively) . In some cases, generation of a detectable signal using a mediator probe, or a cleaved fragment or segment thereof may comprise a hybridization reaction, amplification reaction, denaturation reaction, or any combination thereof.

The mediator probe may comprise a template-binding nucleotide sequence. The template-binding nucleotide sequence may comprise a nucleotide sequence that is capable of hybridizing to a template nucleic acid (e.g., a target nucleic acid, an amplification product thereof, a derivate product thereof, or any combination thereof) . Hence, a template-binding nucleotide sequence may also be a primer or a hybridization probe for an amplification reaction of the template nucleic acid. The template-binding nucleotide sequence may be complementary to a target nucleic acid. The template-binding nucleotide sequence may have at least about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence identity to a target nucleic acid. The template-binding nucleotide sequence may have at most about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence identity to a target nucleic acid. The template-binding nucleotide sequence may have at least about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a target nucleic acid. The template-binding nucleotide sequence may have at most about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a target nucleic acid. The sequence identity or complementarity of the template-binding nucleotide sequence and the target nucleic acid can be determined based on whether it is designed to hybridize a target nucleic acid in the method described herein.

As used herein, the terms “identical, ” “sequence identity, ” or “percent identity, ” when used with respect to two or more nucleic acid sequences, refer to two or more sequences that are the same or, alternatively, have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using any one or more of the following sequence comparison algorithms: Needleman-Wunsch (see, e.g., Needleman, Saul B. ; and Wunsch, Christian D. (1970) . “Ageneral method applicable to the search for similarities in the amino acid sequence of two proteins” Journal of Molecular Biology 48 (3) : 443-53) ; Smith-Waterman (see, e.g., Smith, Temple F. ; and Waterman, Michael S., “Identification of Common Molecular Subsequences” (1981) Journal of Molecular Biology 147: 195-197) ; or BLAST (Basic Local Alignment Search Tool; see, e.g., Altschul S F, Gish W, Miller W, Myers E W, Lipman D J, “Basic local alignment search tool” (1990) J Mol Biol 215 (3) : 403-410) . As used herein, the terms “substantially identical” or “substantial identity” when used with respect to two or more nucleic acid sequences, refer to two or more sequences or subsequences (such as biologically active fragments) that have at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Substantially identical sequences are typically considered to be homologous without reference to actual ancestry. In some embodiments, “substantial identity” exists over a region of the sequences being compared. In some embodiments, substantial identity exists over a region of at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, at least 150 residues in length, at least 200 residues in length, or greater than 200 residues in length. In some embodiments, the sequences being compared are substantially identical over the full length of the sequences being compared. Typically, substantially identical nucleic acid or protein sequences include less than 100%nucleotide or amino acid residue identity as such sequences would generally be considered “identical. ”

As used herein, the terms “sequence complementarity” or “complementary” when used with respect to two nucleic acid sequences, refer to two sequences that are complementary to each other, based on complementary canonical Watson-Crick base-pairing, in which one nucleic acid sequence, in a 5’ to 3’ direction, is aligned to the other nucleic acid sequence, in a 3’ to 5’ direction. In some cases, because nucleic acid molecules can be double-stranded, sequence identity and sequence complementarity can be used interchangeably. In some cases, because a complementary counter part of a nucleotide sequence can be immediately deduced based on the canonical Watson-Crick base-pairing, sequence identity and sequence complementarity can be used interchangeably.

The mediator probe may have more than one template-binding nucleotide sequence. For example, the mediator probe may have at least 2 template-binding nucleotide sequences. The two template-binding nucleotide sequences may be different. The two template-binding nucleotide sequences may have different nucleotide sequences. The two template-binding nucleotide sequences may be capable or bind two different nucleic acid sequences. The nucleotide sequences of two different template-binding nucleotide sequences may have at least about 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %or more sequence identity. The nucleotide sequences of two different template-binding nucleotide sequences may have at most about 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %or more sequence identity. The sequence identity of the two template-binding nucleotide sequences can be determined based on the different nucleic acid sequences they are designed to hybridize. For example, two template-binding nucleotide sequences may have a sequence identity that minimize the hybridization of one template-binding nucleotide sequence and the nucleic acid sequence designed to be bound by another template-binding nucleotide sequence; the hybridization between two template-binding nucleotide sequences; or any combination thereof. In other cases, the two template-binding nucleotide sequences may have the sequence or bind to the same sequence of a target nucleic acid.

The mediator probe may have at least 1, 2, 3, 4, 5 or more template-binding nucleotide sequences. The mediator probe may have at least 1, 2, 3, 4, or 5 template-binding nucleotide sequences. The mediator probe may have 1 template-binding nucleotide sequence. The mediator probe may have 2 template-binding nucleotide sequences. The mediator probe may have 3 template-binding nucleotide sequences. The mediator probe may have 4 template-binding nucleotide sequences. The mediator probe may have 5 template-binding nucleotide sequences. The number of template-binding nucleotide sequences can be determined based on the different nucleic acid sequences they are designed to hybridize. For example, if the method comprises two hybridizations or binding of template-binding nucleotide sequences to two different nucleic acid sequences, the mediator probe may comprise two template-binding nucleotide sequences.

In some cases, a template-binding nucleotide sequences may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. In some cases, a template-binding nucleotide sequences may have at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. In some cases, a template-binding nucleotide sequences may have 5 nucleotides. In some cases, a template-binding nucleotide sequences may have 6 nucleotides. In some cases, a template-binding nucleotide sequences may have 7 nucleotides. In some cases, a template-binding nucleotide sequences may have 8 nucleotides. In some cases, a template-binding nucleotide sequences may have 9 nucleotides. In some cases, a template-binding nucleotide sequences may have 10 nucleotides. In some cases, a template-binding nucleotide sequences may have 11 nucleotides. In some cases, a template-binding nucleotide sequences may have 12 nucleotides. In some cases, a template-binding nucleotide sequences may have 13 nucleotides. In some cases, a template-binding nucleotide sequences may have 14 nucleotides. In some cases, a template-binding nucleotide sequences may have 15 nucleotides. In some cases, a template-binding nucleotide sequences may have 16 nucleotides. In some cases, a template-binding nucleotide sequences may have 17 nucleotides. In some cases, a template-binding nucleotide sequences may have 18 nucleotides. In some cases, a template-binding nucleotide sequences may have 19 nucleotides. In some cases, a template-binding nucleotide sequences may have 20 nucleotides. In some cases, a template-binding nucleotide sequences may have 21 nucleotides. In some cases, a template-binding nucleotide sequences may have 22 nucleotides. In some cases, a template-binding nucleotide sequences may have 23 nucleotides. In some cases, a template-binding nucleotide sequences may have 24 nucleotides. In some cases, a template-binding nucleotide sequences may have 25 nucleotides. In some cases, a template-binding nucleotide sequences may have 26 nucleotides. In some cases, a template-binding nucleotide sequences may have 27 nucleotides. In some cases, a template-binding nucleotide sequences may have 28 nucleotides. In some cases, a template-binding nucleotide sequences may have 29 nucleotides. In some cases, a template-binding nucleotide sequences may have 30 nucleotides. In some cases, a template-binding nucleotide sequences may have 31 nucleotides. In some cases, a template-binding nucleotide sequences may have 32 nucleotides. In some cases, a template-binding nucleotide sequences may have 33 nucleotides. In some cases, a template-binding nucleotide sequences may have 34 nucleotides. In some cases, a template-binding nucleotide sequences may have 35 nucleotides. In some cases, a template-binding nucleotide sequences may have 36 nucleotides. In some cases, a template-binding nucleotide sequences may have 37 nucleotides. In some cases, a template-binding nucleotide sequences may have 38 nucleotides. In some cases, a template-binding nucleotide sequences may have 39 nucleotides. In some cases, a template-binding nucleotide sequences may have 40 nucleotides. In some cases, a template-binding nucleotide sequences may have 41 nucleotides. In some cases, a template-binding nucleotide sequences may have 42 nucleotides. In some cases, a template-binding nucleotide sequences may have 43 nucleotides. In some cases, a template-binding nucleotide sequences may have 44 nucleotides. In some cases, a template-binding nucleotide sequences may have 45 nucleotides. In some cases, a template-binding nucleotide sequences may have 46 nucleotides. In some cases, a template-binding nucleotide sequences may have 47 nucleotides. In some cases, a template-binding nucleotide sequences may have 48 nucleotides. In some cases, a template-binding nucleotide sequences may have 49 nucleotides. In some cases, a template-binding nucleotide sequences may have 50 nucleotides. The number of nucleotides of a template-binding nucleotide sequence may be dependent on the nucleic acid sequence it is designed to bind to, the nucleic acid sequences it is designed not to bind to, or a combination thereof. Additionally, the length of the template-binding nucleotide sequence may be dependent on the binding of the sequence and its binding target (the target nucleic acid or reporter probe) . For example, a long template-binding nucleotide sequence may have a beneficial advantage of increased binding specificity to its binding target (and thus the accuracy of the method for detecting the target) . A short template-binding nucleotide sequence may have a beneficial advantage of efficient release of the sequence with its binding target, thereby increasing the efficiency of the method for analyzing the target.

In some cases, a mediator probe may have two template-binding nucleotide sequences (e.g., a first template-binding nucleotide sequence and a second template-binding nucleotide sequence) . In some cases, the first template-binding nucleotide sequence may have 5-25 nucleotides, 6-24 nucleotides, 7-23 nucleotides, 8-22 nucleotides, 9-21 nucleotides, or 10-20 nucleotides. In some cases, the second template-binding nucleotide sequence may have 8-45 nucleotides, 9-44 nucleotides, 10-43 nucleotides, 11-42 nucleotides, 12-41 nucleotides, 13-40 nucleotides, 14-39 nucleotides, 15-38 nucleotides, 16-37 nucleotides, 17-36 nucleotides, or 18-35 nucleotides.

Two template-binding nucleotide sequences may be configured to be separated from each other. In some cases, the template-binding nucleotide sequences may be separated from each other using a cleavage reaction. In some cases, a mediator probe may hybridize to a target nucleic acid, and a cleaved fragment comprising one of the two template-binding nucleotide sequences may be generated by the cleavage reaction. In some cases, a mediator probe may hybridize to a target nucleic acid, and a cleaved fragment comprising only one of the two template-binding nucleotide sequences may be generated by the cleavage reaction and separated from the hybridized product of the other template-binding nucleotide sequence and the target nucleic acid. In some cases, a mediator probe may hybridize to a target nucleic acid with one of the two template-binding nucleotide sequences, and a cleaved fragment comprising the other template-binding nucleotide sequences may be generated by the cleavage reaction and separated from the hybridized product of the template-binding nucleotide sequence and the target nucleic acid. Hence, the other template-binding nucleotide sequence or the cleaved fragment may also be a primer or a hybridization probe for an amplification reaction of a nucleic acid sequence (such as those from a reporter probe described herein) . The cleavage reaction may comprise an enzyme described herein.

Two template-binding nucleotide sequences may be separated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more nucleotides. In some cases, two template-binding nucleotide sequences may be separated by a sequence with a cleavage site (such as one of the nucleases described herein) . In some cases, two template-binding nucleotide sequences may not be separated by a nucleotide (i.e., the two template-binding nucleotide sequences are contiguous) . In some cases, two template-binding nucleotide sequences may not be separated by a cleavage site or a sequence comprising thereof.

A mediator probe may comprise a label moiety, a quencher moiety, or a label moiety and a quencher moiety. A mediator probe may comprise a label moiety. A mediator probe may comprise a quencher moiety. A mediator probe may comprise a label moiety and a quencher moiety. The label moiety and quencher moiety may comprise any of those described herein.

A mediator probe may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more label moieties. A mediator probe may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 label moieties. A mediator probe may comprise 1 label moiety. A mediator probe may comprise 2 label moieties. A mediator probe may comprise 3 label moieties. A mediator probe may comprise 4 label moieties. A mediator probe may comprise 5 label moieties. The number of label moieties may be dependent on the intensity of a detectable signal generated.

A mediator probe may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more quencher moieties. A mediator probe may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quencher moieties. A mediator probe may comprise 1 quencher moiety. A mediator probe may comprise 2 quencher moieties. A mediator probe may comprise 3 quencher moieties. A mediator probe may comprise 4 quencher moieties. A mediator probe may comprise 5 quencher moieties. The number of quencher moieties may be dependent on the intensity of a detectable signal generated.

In some cases, the label moiety or quencher moiety may be coupled to the terminal ends of the mediator probe or the cleaved fragment thereof.

A mediator probe may comprise, in a 5’ to 3’ direction, a label moiety, a first template-binding nucleotide sequence, and a second template-binding nucleotide sequence. A mediator probe may comprise, in a 5’ to 3’ direction, a first template-binding nucleotide sequence, a label moiety, and a second template-binding nucleotide sequence. A mediator probe may comprise, in a 5’ to 3’ direction, a first template-binding nucleotide sequence, a second template-binding nucleotide sequence, and a label moiety. A mediator probe may comprise, in a 5’ to 3’ direction, a quencher moiety, a first template-binding nucleotide sequence, and a second template-binding nucleotide sequence. A mediator probe may comprise, in a 5’ to 3’ direction, a first template- binding nucleotide sequence, a quencher moiety, and a second template-binding nucleotide sequence. A mediator probe may comprise, in a 5’ to 3’ direction, a first template-binding nucleotide sequence, a second template-binding nucleotide sequence, and a quencher moiety.

The label moiety (ies) , quencher moiety (ies) , template-binding nucleotide sequence (s) of the mediator probe can be arranged in various configurations, as described herein. A mediator probe may comprise, in a 5’ to 3’ direction, a label moiety (LM) , a first template-binding nucleotide sequence (FT) , a second template-binding nucleotide sequence (ST) , and a quencher label moiety (QM) or 5’ -LM-FT-ST-QM-3’ , wherein “-” denotes a linkage between two moieties. A mediator probe may comprise 5’ -LM-FT-QM-ST-3’ . A mediator probe may comprise 5'-LM-QM-FT-ST-3’ . A mediator probe may comprise 5'-LM-FT-ST-QM-3’ . A mediator probe may comprise 5'-LM-ST-FT-QM-3’ . A mediator probe may comprise 5'-LM-QM-ST-FT-3’ . A mediator probe may comprise 5'-LM-ST-QM-FT-3’ . A mediator probe may comprise 5'-ST-FT-LM-QM-3’ . A mediator probe may comprise 5'-ST-LM-FT-QM-3’ . A mediator probe may comprise 5'-ST-FT-QM-LM-3’ . A mediator probe may comprise 5'-ST-QM-FT-LM-3’ . A mediator probe may comprise 5'-ST-LM-QM-FT-3’ . A mediator probe may comprise 5'-ST-QM-LM-FT-3’ . A mediator probe may comprise 5'-QM-FT-LM-ST-3’ . A mediator probe may comprise 5'-QM-LM-FT-ST-3’ . A mediator probe may comprise 5'-QM-FT-ST-LM-3’ . A mediator probe may comprise 5'-QM-ST-FT-LM-3’ . A mediator probe may comprise 5'-QM-LM-ST-FT-3’ . A mediator probe may comprise 5'-QM-ST-LM-FT-3’ . A mediator probe may comprise 5'-FT-LM-QM-ST-3’ . A mediator probe may comprise 5'-FT-QM-LM-ST-3’ . A mediator probe may comprise 5'-FT-LM-ST-QM-3’ . A mediator probe may comprise 5'-FT-ST-LM-QM-3’ . A mediator probe may comprise 5'-FT-QM-ST-LM-3’ . A mediator probe may comprise 5'-FT-ST-QM-LM-3’ .

A mediator probe may comprise, in a 5’ to 3’ direction, (1) a label moiety, a nucleotide sequence comprising at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, and a quencher moiety; or (2) the quencher moiety, the nucleotide sequence comprising the at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, and the label moiety. A mediator probe may have a nucleotide sequence of at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 nucleotides between a label moiety and a quencher moiety. In some cases, a mediator probe having more than one label moiety and/or more than one quencher moiety may have a nucleotide sequence of at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 nucleotides between a label moiety and an adjacent quencher moiety. In some cases, a mediator probe having more than one label moiety and/or more than one quencher moiety may have a nucleotide sequence of at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 nucleotides between a quencher moiety and an adjacent label moiety.

In some cases, the mediator probe may comprise a junction of the first template-binding nucleotide sequence and the second template-binding nucleotide sequence (e.g., the junction of a sequence that does not hybridize to the target nucleic acid and a sequence that hybridizes to the target nucleic acid, respectively) . In some cases, the junction of a mediator comprises the 3’ terminal nucleotide of the first template-binding nucleotide sequence. In some cases, the junction of a mediator comprises the 5’ terminal nucleotide of the second template-binding nucleotide sequence. In some cases, a label or quencher moiety is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides 3’ to the junction. In some cases, a label or quencher moiety is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides 3’ to the junction. In some cases, a quencher moiety is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides 3’ to the junction. In some cases, a quencher moiety is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides 3’ to the junction. In some cases, a label moiety is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides 3’ to the junction. In some cases, a label moiety is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides 3’ to the junction.

A cleaved fragment of the mediator probe may comprise a label moiety, a quencher moiety, or a label moiety and a quencher moiety. A cleaved fragment of the mediator probe may comprise a label moiety. A cleaved fragment of the mediator probe may comprise a quencher moiety. A cleaved fragment of the mediator probe may comprise a label moiety and a quencher moiety. A cleaved fragment of the mediator probe may comprise a label moiety and not a quencher moiety. A cleaved fragment of the mediator probe may comprise a quencher moiety and not a label moiety. A cleaved fragment of the mediator probe may comprise 5’ -LM-FT-3’ . A cleaved fragment of the mediator probe may comprise 5’ -FT-LM-3’ . A cleaved fragment of the mediator probe may comprise 5’ -QM-FT-3’ . A cleaved fragment of the mediator probe may comprise 5’ -FT-QM-3’ .

A cleaved fragment of the mediator probe may comprise 5’ -LM-FT-QM-3’ . A cleaved fragment of the mediator probe may comprise 5'-LM-QM-FT-3’ . A cleaved fragment of the mediator probe may comprise 5'-QM-LM-FT-3’ . A cleaved fragment of the mediator probe may comprise 5'-QM-FT-LM-3’ . A cleaved fragment of the mediator probe may comprise 5'-FT-LM-QM-3’ . A cleaved fragment of the mediator probe may comprise 5'-FT-QM-LM-3’ .

In other cases, a label moiety or quencher moiety may be coupled to the mediator probe or the cleaved fragment thereof at a nucleotide of any of the template-binding nucleotide sequence. For example, the label moiety or quencher moiety may be coupled to the nucleotide of the mediator probe or the cleaved fragment thereof using methods and moieties described herein. In some cases, the label moiety or quencher moiety may be coupled not at the terminal ends (5’ or 3’ ends of a nucleotide not engaging in a phosphodiester bond with another nucleotide) of the mediator probe or the cleaved fragment thereof. In some cases, the label moiety or quencher moiety may be coupled at a or at least one nucleotide of the template-binding nucleotide sequence of the mediator probe (or a fragment or derivative thereof) . In some cases, the label moiety or quencher moiety may be coupled both (1) at a or at least one nucleotide of the template-binding nucleotide sequence of the mediator probe (or a fragment or derivative thereof) and (2) any of the terminal ends of the mediator probe (or a fragment or derivative thereof) . Hence, the label/quencher moieties can be coupled to any nucleotide (terminal ends or specific sequences) of the configurations of the reporter probe described herein. In addition to the configurations of the mediator probe as described herein, the label and quencher moiety may also have the following configurations: (1) the quencher moiety may be coupled to a nucleotide of the first template-binding nucleotide sequence; (2) the quencher moiety may be coupled to a nucleotide of the second template-binding nucleotide sequence; (3) the label moiety may be coupled to a nucleotide of the first template-binding nucleotide sequence; (4) the label moiety may be coupled to a nucleotide of the second template-binding nucleotide sequence; or (5) any combinations of (1) - (4) .

In some instances, a first template-binding nucleotide sequence (e.g., the one at the 5’ end of the mediator probe, relative to a different template-binding nucleotide sequence (s) 3’ of the first template-binding nucleotide sequence) , may be separated from an upstream primer described herein by a distance. While the mediator probe (or a molecule comprising the template-binding nucleotide sequence such as the cleaved fragment) and the upstream primer may be two separate different molecules, the distance between them can be measured as the number of nucleotides that separates them when or if they hybridize to a same molecule (such as a template nucleic acid) . The distance may be measured as the number of nucleotides between the nucleotide at the 3’ terminus of the upstream primer and the nucleotide of the 5’ terminus of the mediator probe (or a molecule comprising the template-binding nucleotide sequence such as the cleaved fragment) . In some cases, the distance between the upstream primer and the mediator probe (or a molecule comprising the template-binding nucleotide sequence such as the cleaved fragment) is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000 or more nucleotides. In some cases, the distance between the upstream primer and the mediator probe (or a molecule comprising the template-binding nucleotide sequence such as the cleaved fragment) is at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1000 nucleotides.

Because the detectable signal generated may depend on the number, position, identity of label and quencher moieties as described herein contained in a mediator probe, a reporter probe, a fragment or derivative thereof generated using the methods described herein, or any combination thereof; the number, position, identity of label and quencher moieties of a mediator probe or a cleaved fragment/aderivative thereof can be adjusted based on the methods described herein.

In some instances, the mediator probe or the sequence thereof has a 3’ -OH terminus. The 3’-end of the mediator probe sequence may be configured to prevent it from being extended in a nucleic acid amplification reaction. For example, the 3’ -end of the mediator probe sequence can have a blocking group to inhibit its own extension in a nucleic acid amplification reaction. The 3’-end of a mediator probe or sequence thereof can be blocked by modifying the 3’ -OH of the terminal nucleotide of the mediator probe or sequence thereof. In some cases, the 3’ -end of the mediator probe or sequence thereof can be blocked by adding a chemical moiety to the 3’ -OH of the terminal nucleotide of the detection sequence. The chemical moiety can comprise biotin or an alkyl group or a combination thereof. In some cases, the 3’ -OH of the terminal nucleotide of the mediator probe can be blocked by removing or replacing the terminal nucleotide. Having a blocking group at the 3’ end of the mediator probe can have a beneficial advantage of not allowing 3’ extension of the mediator (e.g., in an amplification reaction) , thereby reducing any undesirable products being generated. In some instances, the 3’ -end of the mediator probe does not comprise a blocking group.

In some instances, one or more template-binding nucleotides of a mediator probe may comprise a sequence that is complementary to a naturally occurring nucleotide sequence. In some instances, one or more template-binding nucleotides of a mediator probe may comprise a sequence complementary to a non-naturally occurring nucleotide sequence. In some instances, a first template-binding nucleotide of a mediator probe comprises a sequence complementary to a non-naturally occurring nucleotide sequence, and a second template-binding nucleotide of the mediator probe comprises a sequence that is complementary to a naturally occurring nucleotide sequence. In some instances, the naturally occurring nucleotide sequence is a target nucleic acid disclosed herein or a fragment thereof. In some instances, the non-naturally occurring nucleotide sequence is part of a reporter probe disclosed herein.

In some cases, the mediator probe may have a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 6, 7, 12, or 13.

Any of the template-binding nucleotide sequences of the mediator probe may comprise a sequence that is complementary to any of those target nucleic acids described herein.

Reporter probe

Provided herein are reporter probes. The reporter probes (or derivatives thereof) provided herein may facilitate generation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more detectable signals. The reporter probes provided herein may facilitate generation of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 detectable signals. The detectable signal may facilitate quantification and/or identification of a target analyte (such as a target nucleic acid) . The reporter probe may hybridize to a cleaved fragment of a mediator probe. The reporter probe may be used as a template in a nucleic acid amplification reaction. In such a case, the reporter probe may be a template while the cleaved fragment of the mediator probe may be a primer. In other cases, a hybridized molecule of the reporter probe and the cleaved fragment of the mediator probe may be subjected to denaturation. Therefore, in some cases, generation of a detectable signal using the reporter probe may comprise a hybridization reaction, amplification reaction, denaturation reaction, or any combination thereof. The reporter probe may be a nucleic acid molecule.

The reporter probe may have a structure that is/has single-stranded; double stranded (i.e., comprising at least two nucleotides that are base-paired) ; both single-and double-stranded (i.e., comprising both single-and double-stranded region) ; linear; branched; circular; a hairpin-loop (or a stem-loop region) ; a pseudoknot; or any combination thereof. In some cases, the reporter probe may have more than one structure described herein. For example, the reporter probe may have a structure in one condition and a different structure in another condition. In some cases, two complementary nucleotide sequences can be located at the two terminal regions (5’ and 3’ end regions) of the reporter probe (or the sequence thereof) , so that the reporter probe can form a hairpin structure via complementary pairing of the two complementary nucleotide sequences. The arms of the hairpin structure may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. The arms of the hairpin structure may have at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. The arms of the hairpin structure may have 2-15, 3-7, 4-9, 5-10, or 6-12 nucleotides. For a reporter probe with the hairpin arms, when binding to the cleaved fragment of the mediator probe, the arms of the hairpin structure can denature (with or without using the cleaved fragment as a primer and the reporter probe as a template in a nucleic acid amplification reaction) , thereby separating the two arms (and the terminal ends, or the label/quencher moiety pair coupled to the terminal ends) . The separation of the two arms can facilitate the detectable signal as described herein.

In some cases, a reporter probe may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 or more nucleotides. In some cases, a reporter probe may have at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or 10000 nucleotides.

In some instances, the reporter probe comprises a sequence that is configured to hybridize or complementary to a sequence of a mediator probe. For example, the reporter probe may comprise a sequence (as used herein, such sequence may be referred as a “template sequence for hybridization of the reporter probe” ) that is configured to hybridize or complementary to a sequence of the template-binding nucleotide sequence or the cleaved fragment of the mediator probe. In some cases, the reporter probe may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different template sequences for hybridization of the reporter probe. In some cases, the reporter probe may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 different template sequences for hybridization of the reporter probe. When comprising more than one template sequences for hybridization of the reporter probe, the reporter probe may be used in the multiplex detection as described herein. Two template sequences for hybridization of the reporter probe may have an overlap of nucleotide sequences. For example, a first and second template sequences for hybridization of the reporter probe may have an overlap of 5 nucleotides if the two sequences share a same sequence of 5 nucleotides. In some cases, two template sequences for hybridization of the reporter probe may have an overlap of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. In some cases, two template sequences for hybridization of the reporter probe may have an overlap of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. Two template sequences for hybridization of the reporter probe may have an overlap of nucleotide sequences when they have at least an overlap. For example: Two template sequences for hybridization of the reporter probe may have a sequence of AAGGCCTT and AGGCCTTX, respectively; wherein A is adenosine, G is guanine, C is cytosine, T is thymine, and X is any of A, C, G, and T. In such case, the two template sequences for hybridization of the reporter probe have an offset of one nucleotide. In some cases, two template sequences for hybridization of the reporter probe may have an offset of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In some cases, two template sequences for hybridization of the reporter probe may have an offset of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

When used or configured to be used in a nucleic acid amplification reaction, a template sequence for hybridization of the reporter probe may have a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100 or more nucleotides. When used or configured to be used in a nucleic acid amplification reaction, a template sequence for hybridization of the reporter probe may have a length of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 100 nucleotides.

The template sequence for hybridization of the reporter probe may have at least about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a sequence of the template-binding nucleotide sequence or the cleaved fragment of the mediator probe. The template sequence for hybridization of the reporter probe may have at most about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence identity to a sequence of the template-binding nucleotide sequence or the cleaved fragment of the mediator probe. The template sequence for hybridization of the reporter probe may have at least about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a sequence of the template-binding nucleotide sequence or the cleaved fragment of the mediator probe. The template sequence for hybridization of the reporter probe may have at most about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a sequence of the template-binding nucleotide sequence or the cleaved fragment of the mediator probe. The sequence complementarity of the template sequence for hybridization of the reporter probe and the template-binding nucleotide sequence of a mediator probe (or a cleaved fragment or derivative thereof) may be determined based on a desirable hybridization behavior of thereof for using the methods described herein.

In some instances, the reporter probe comprises a sequence that is configured to be an extension template sequence when the reporter probe hybridizes with a sequence of the mediator probe. The reporter probe may comprise a sequence (as used herein, such sequence may be referred as a “template sequence for extension of the reporter probe” ) that is configured, in a nucleic acid amplification reaction, to serve as a template for nucleic acid polymerization when a sequence of the template-binding nucleotide sequence or the cleaved fragment of the mediator probe hybridizes to the template sequence for hybridization of the reporter probe. For example, if a polymerase chain reaction (PCR) using the cleaved fragment of the mediator probe as a primer molecule and the reporter probe as a template molecule, the cleaved fragment of the mediator probe may hybridize to the template sequence for hybridization of the reporter probe, add nucleotide into an extending polynucleotide chain based on the complementary base-pairing using the nucleotides of the template sequence for extension of the reporter probe.

In some cases, the reporter probe may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different template sequences for extension of the reporter probe. In some cases, the reporter probe may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 different template sequences for extension of the reporter probe. When comprising more than one template sequences for extension of the reporter probe, the reporter probe may be used in the multiplex detection as described herein. Two template sequences for extension of the reporter probe may have an overlap of nucleotide sequences. For example, a first and second template sequences for extension of the reporter probe may have an overlap of 5 nucleotides if the two sequences share a same sequence of 5 nucleotides. In some cases, two template sequences for extension of the reporter probe may have an overlap of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. In some cases, two template sequences for extension of the reporter probe may have an overlap of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. Two template sequences for extension of the reporter probe may have an overlap of nucleotide sequences when they have at least an overlap. For example: Two template sequences for extension of the reporter probe may have a sequence of AAGGCCTT and AGGCCTTX, respectively; wherein A is adenosine, G is guanine, C is cytosine, T is thymine, and X is any of A, C, G, and T. In such case, the two template sequences for extension of the reporter probe have an offset of one nucleotide. In some cases, two template sequences for extension of the reporter probe may have an offset of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more nucleotides. In some cases, two template sequences for extension of the reporter probe may have an offset of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 nucleotides.

When used or configured to be used in a nucleic acid amplification reaction, a template sequence for extension of the reporter probe may have a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000 or more nucleotides. When used or configured to be used in a nucleic acid amplification reaction, a template sequence for hybridization of the reporter probe may have a length of at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 5000 nucleotides.

A reporter probe may comprise a label moiety or a quencher moiety. A reporter probe may comprise a label moiety. A reporter probe may comprise a quencher moiety. A reporter probe may comprise a label moiety and a quencher moiety.

A reporter probe may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more label moieties. A reporter probe may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 label moieties. A reporter probe may comprise 1 label moiety. A reporter probe may comprise 2 label moieties. A reporter probe may comprise 3 label moieties. A reporter probe may comprise 4 label moieties. A reporter probe may comprise 5 label moieties. The number of label moieties may be dependent on the intensity of a detectable signal generated.

A reporter probe may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more quencher moieties. A reporter probe may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quencher moieties. A reporter probe may comprise 1 quencher moiety. A reporter probe may comprise 2 quencher moieties. A reporter probe may comprise 3 quencher moieties. A reporter probe may comprise 4 quencher moieties. A reporter probe may comprise 5 quencher moieties. The number of quencher moieties may be dependent on the intensity of a detectable signal generated.

In some cases, the label moiety or quencher moiety may be coupled to: (1) the terminal ends of the reporter probe or the derivative thereof; (2) at a nucleotide of a sequence of the reporter probe or the derivative thereof; or (1) and (2) . In some cases, the label moiety may be coupled to a 5’ end of the reporter probe or the derivative thereof. In some cases, the label moiety may be coupled to a 3’ end of the reporter probe or the derivative thereof. In some cases, the label moiety may be coupled at a nucleotide not at the 5’ or 3’ end of the reporter probe or the derivative thereof. In some cases, the label moiety may be coupled at a nucleotide at a sequence of the reporter probe or the derivative thereof. In some cases, the quencher moiety may be coupled to a 5’ end of the reporter probe or the derivative thereof. In some cases, the quencher moiety may be coupled to a 3’ end of the reporter probe or the derivative thereof. In some cases, the quencher moiety may be coupled at a nucleotide not at the 5’ or 3’ end of the reporter probe or the derivative thereof. In some cases, the quencher moiety may be coupled at a nucleotide at a sequence of the reporter probe or the derivative thereof. The label or quencher moiety can be coupled to any nucleotide or terminal end of the reporter probe, or the derivative thereof as described herein.

The label moiety (ies) , quencher moiety (ies) , template sequences for hybridization, or template sequences for extension of the reporter probe can be arranged in various configurations, as described herein. In some cases, the reporter probe may comprise, from 3’ -5’ , the template sequence for hybridization of the reporter probe (H) and the template sequence for extension of the reporter probe (E) . In some cases, the report probe may comprise 5’ -LM-E-H-QM-3’ . In some cases, the report probe may comprise 5’ -QM-E-H-LM-3’ . In some cases, the report probe may comprise 5’ -QM-LM-E-H-3’ . In some cases, the report probe may comprise 5’ -QM-E-LM-H-3’ . In some cases, the report probe may comprise 5’ -LM-E-H-QM-3’ . In some cases, the report probe may comprise 5’ -LM-QM-E-H-3’ . In some cases, the report probe may comprise 5’-LM-E-QM-H-3’ . The label/quencher moieties can be coupled to any nucleotide (terminal ends or specific sequences) of the configurations of the mediator probe described herein.

Because the detectable signal generated may depend on the number, position, identity of label and quencher moieties as described herein contained in a mediator probe, a reporter probe, a fragment or derivative therefor generated using the methods described herein, or any combination thereof; the number, position, identity of label and quencher moieties of a reporter probe or a derivative thereof can be adjusted based on the disclosure described herein.

In some instances, the reporter probe may comprise a sequence that is not complementary to a sequence of the mediator probe. Such a sequence may prevent the un-cleaved mediated probe from being used as a primer (and the reporter probe as a template) for a nucleic acid amplification reaction. Such an arrangement may have a beneficial advantage of increasing the hybridization specificity of the cleaved fragment of the mediator probe to the reporter probe. For example, the reporter probe may comprise the sequence that is not complementary to a sequence of the mediator probe 5’ to the template sequence for hybridization of the reporter probe. In some cases, the reporter probe may comprise the sequence that is not complementary to the second template-binding nucleotide sequence (that binds to the target nucleic acid or that is 3’ to the first template-binding nucleotide sequence of the reporter probe that can hybridize with the template sequence for hybridization of the reporter probe.

Although the un-cleaved mediator probe is capable of hybridizing to the reporter probe through the first template-binding nucleotide sequence, the second template-binding nucleotide sequence (that can bind the target nucleic acid) of the mediator probe that does not hybridize to the reporter probe may be located at the 3’ end of the first template-binding nucleotide sequence of the mediator probe such that the enzyme cannot extend the un-cleaved mediator probe that hybridizes to the reporter probe.

The reporter probe may also be modified. Such modification may allow the reporter probe (or the sequence thereof) to be resistant to nuclease activity (e.g., a nuclease activity, a 5’ nuclease activity, 5’ -3’ exonuclease activity, or any nuclease activity described herein) . For example, nuclease-resistant modifications can be introduced into the nucleotide backbone of the reporter probe. The nuclease-resistant modifications can comprise phosphorothioate bonds, alkyl phosphotriester bonds, aryl phosphotriester bonds, alkyl phosphonate bonds, aryl phosphine bonds, ester bond, hydrogenated phosphate bond, alkyl phosphoramidate bond, aryl phosphoramidate bond, 2’ -O-aminopropyl modification, 2’ -O-alkyl modification, 2’ -O-allyl modification, 2’ -O-butyl modification, 1- (4’ -thio-PD-ribofuranosyl) modification, or a combination thereof.

In some instances, the reporter probe or the sequence thereof has a 3’ -OH terminus. The 3’-end of the reporter probe sequence may be configured to prevent it from being extended in a nucleic acid amplification reaction. For example, the 3’ -end of the reporter probe sequence can have a blocking group to inhibit its own extension in a nucleic acid amplification reaction. The 3’-end of a reporter probe or sequence thereof can be blocked by modifying the 3’ -OH of the terminal nucleotide of the reporter probe or sequence thereof. In some cases, the 3’ -end of the reporter probe or sequence thereof can be blocked by adding a chemical moiety to the 3’ -OH of the terminal nucleotide of the detection sequence. The chemical moiety can comprise biotin or an alkyl group or a combination thereof. In some cases, the 3’ -OH of the terminal nucleotide of the reporter probe can be blocked by removing or replacing the terminal nucleotide.

In some cases, the reporter probe may have a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 8, 9, 14, or 15.

Upstream/downstream primers

Provided herein, are upstream and downstream primers. The upstream and downstream primers may be used to generate an amplification product or derivative of a template nucleic acid as described herein in an amplification described herein. Using the methods described herein, the upstream primer may facilitate the generation of the cleavage fragment of the mediator probe using the methods described herein. Additionally, generation of the amplification product or derivative of the template nucleic acid using the upstream/downstream primers may have a beneficial advantage of increasing the total amount of a sequence of the analyte (e.g., template nucleic acid) and increasing the sensitivity or accuracy of the detection of the analyte or template nucleic acid. In some other cases, the upstream (or downstream) primer may be used as a probe for generating the cleaved fragment of the mediator probe using the methods as described herein. The upstream (or downstream) primer may thus not be used in a nucleic acid amplification reaction.

In some cases, the upstream primer or downstream primer comprises a sequence that has at least 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence identity to a target nucleic acid. In some cases, the upstream primer or downstream primer comprises a sequence that has at most about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence identity to a target nucleic acid. In some cases, the upstream primer or downstream primer comprises a sequence that has at least about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a target nucleic acid. In some cases, the upstream primer or downstream primer comprises a sequence that has about 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, or 100 %sequence complementarity to a target nucleic acid.

In some cases, an upstream primer may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. In some cases, an upstream primer may have at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some cases, a downstream primer may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. In some cases, a downstream primer may have at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

In some cases, an upstream primer and a downstream primer may generate an amplification product of a target nucleic acid (also referred herein to as an amplicon) in an amplification reaction. The amplicon may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 1000 or more nucleotides. The amplicon may have at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, or 1000 nucleotides

The sequence identity/complementarity of the upstream or downstream primers, length of the upstream or downstream primers, and/or size of the amplicon can be designed based on the identity of the target nucleic acid as described herein.

In some cases, for each target nucleic acid, more than one upstream (or downstream) primer can be provided. For example, a first upstream/downstream primer may be used for amplification of the target nucleic acid, thereby increased the sensitivity of the detection method as described herein. Additionally, a second upstream may be used for generating a cleavage fragment of the mediator probe, as described herein, using the enzymes and methods described herein.

In some instances, the target nucleic acid is amplified by symmetrical amplification. The symmetrical amplification may comprise using equal amounts of the upstream and downstream primers for amplification for the target nucleic acid. In some cases, the target nucleic acid is amplified by asymmetric amplification. The asymmetric amplification may be performed using unequal amounts of upstream and downstream primers for a target nucleic acid. For example, in some cases, the upstream primer is in excess relative to the downstream primer, or the downstream primer is in excess relative to the upstream primer. The upstream primer may be at least about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the downstream primer. The upstream primer may be at most about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the downstream primer. The downstream primer may be at least about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the upstream primer. The downstream primer may be at most about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the upstream primer.

In some instances, a target nucleic acid may be amplified in a three-step amplification process. In the three-step amplification process, each round of nucleic acid amplification may comprise three steps: (1) nucleic acid denaturation at a first temperature, (2) nucleic acid annealing at a second temperature, and (3) nucleic acid extension at a third temperature. In some instances, a target nucleic acid is amplified in a two-step amplification process. The two-step amplification process, each round of nucleic acid amplification comprises two steps: (1) nucleic acid denaturation at a first temperature, and (2) nucleic acid annealing and extension at a second temperature. Suitable temperatures for nucleic acid denaturation, nucleic acid annealing, and nucleic acid extension can be readily determined by those skilled in the art by routine methods (see, e.g., Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) , which is herein incorporated by reference in its entirety.

In some cases, the upstream or downstream primers may be coupled to a label or quencher moiety as described herein. In such case, amplification or hybridization of the primers to a target nucleic acid (or amplification products thereof) can generate a detectable signal described herein, thereby allowing a detection event for quantification and/or identification of the target nucleic acid.

In some cases, the upstream primer may have a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 4, 10, 16, 20 or 24. In some cases, the downstream primer may have a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 5, 11, 17, 21, or 25.

Enzymes

Provided herein are enzymes. The enzymes may be used to induce the cleavage of the mediator probe to generate a cleavage fragment comprising the template-binding nucleotide sequences or parts thereof (e.g., one that is configured to hybridize to the reporter probe or has a sequence complementary to a sequence of the reporter probe) . The enzyme may have a nuclease activity (as described herein) that can induce cleavage of the mediator probe when: (1) the mediator probe hybridizes to the target nucleic acid (or a derivative thereof) and (2) the upstream primer hybridizes to the target nucleic acid (or a derivative thereof) . The enzyme may have a 5’ nuclease activity described herein. For example, in the methods described herein, when the mediator probe is in contact with the target nucleic acid using a second template-binding nucleotide sequence hybridizing with the target nucleic acid, a first template-binding nucleotide sequence does not hybridize with the target nucleic acid, maintaining a single-stranded structure. In some cases, an enzyme having the nuclease activity can be used to cleave the single-stranded first template-binding nucleotide sequence from the hybridized portion of the second template-binding nucleotide sequence of the mediator probe and the template nucleic acid, generating (and releasing) the cleaved fragment of the mediator probe comprising the first template-binding nucleotide sequence.

In some instances, cleavage of the mediator probe by the enzyme may be: (1) independent on an extension of the upstream primer (using the template nucleic acid as the template) or (2) dependent on in the extension of the upstream primer (using the template nucleic acid as the template) . In (1) , subsequent to the upstream primer and the mediator probe being hybridized to the target nucleic acid, if the upstream primer and the mediator probe are in close enough proximity, the enzyme with the nuclease activity (e.g., the 5’ nuclease activity) can induce cleavage of the mediator probe by binding to the upstream primer and cleaving the mediator probe without an extension (or an amplification reaction) . In (2) , subsequent to the hybridization with the target nucleic acid, if the upstream primer is not in close proximity with the mediator probe, a nucleic acid polymerase can be used to catalyze the extension or polymerization of the upstream primer using the target nucleic acid as a template in a nucleic acid amplification reaction. Subsequently, the enzyme with the nuclease activity (e.g., the 5’ nuclease activity) may be used to contact and bind to the upstream primer or the extension product thereof (hybridized to the target nucleic acid or amplification product thereof; and the induce cleavage of the mediator probe by binding to the upstream primer and cleaving the mediator probe without an extension (or an amplification reaction) . In (1) , the 3’ end of the upstream primer and 5’ end of the mediator probe may be contiguous (without a nucleotide in between the upstream primer and the mediator probe when/if both of them hybridize to the target nucleic acid) ; or have a distance of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In (2) , the 3’ end of the upstream primer and 5’ end of the mediator probe may have a distance of at least 31, 40, 50, 100, 200, 300, 400, 500, 1000 or more nucleotides. In (1) , the distance between the 3’ end of the upstream primer and 5’ end of the mediator probe may be 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotide (s) . In (2) , the 3’ end of the upstream primer and 5’ end of the mediator probe may have a distance of at most 31, 40, 50, 100, 200, 300, 400, 500, or 1000 nucleotides. Additionally, in (1) , the upstream primer may also be a probe without being used in an amplification reaction that is involved in the cleavage of the mediator probe. In some cases, in (1) , the upstream primer may also be a primer (being used in an amplification reaction of the target nucleic acid) and a probe (being used in an amplification reaction that is involved in the cleavage of the mediator probe) . For example, in (1) , the upstream primer may be: (1a) used to generate am amplification product of the target nucleic acid; and (1b) used to generate the cleavage fragment of the mediator probe in an extension-independent manner. (1a) may be carried out prior to (1b) . (1a) may be carried out simultaneously with (1b) .

Various methods and composition for using upstream oligonucleotides to induce cleavage of downstream oligonucleotides are described elsewhere in this disclosure. In some cases, the methods and/or compositions are described in U.S. Patent Nos: 5,210,015, 5,487,972, 5,691,142, 5,994,069 and 7,381,532; and U.S. Patent Application No: US 2008/0241838, each of which is herein incorporated by reference in its entirety.

In some cases, the cleavage site on the mediator probe is located at a junction of the first template-binding nucleotide sequence and the second template-binding nucleotide sequence. In such cases, the cleavage of the mediator probe by the enzyme will release the cleavage fragment comprising the entire first template-binding nucleotide sequence. In some cases, the cleavage site on the mediator probe is located within the 3’ -terminal region of the first template-binding nucleotide sequence.

In some instances, the enzyme may have endonuclease or exonuclease activity. The enzyme may have a 5’ exonuclease activity, 3’ exonuclease activity, or a combination thereof. In some cases, the enzyme may have a 5’ exonuclease activity. The enzyme may have a 3’ exonuclease activity. The enzyme may have a 5’ exonuclease activity and a 3’ exonuclease activity. The enzyme may have a nucleic acid polymerase that can be used in the nucleic acid amplification reaction as described herein. The enzyme may have a deoxyribonucleic acid (DNA) polymerase, a ribonucleic acid (RNA) polymerase, or a combination thereof. The enzyme may be a thermostable nuclease. The enzyme may have the nuclease activity and a polymerase activity. In some cases, the enzyme may comprise a thermostable DNA polymerase having a 5’ exonuclease. The uses of a nucleic acid polymerase with 5’ nuclease activity may have a beneficial advantage because the polymerase can both catalyze the extension of an upstream primer using the target nucleic acid as a template; and can induce the cleavage of the mediator probe (to release the nucleic acid comprising the template-binding nucleotide sequence that can hybridize or complementary to a sequence of the reporter probe) .

In some instances, the enzyme may comprise a thermostable DNA polymerase. The thermostable DNA polymerase may be a thermostable DNA polymerase from a bacteria. The thermostable DNA polymerase may be from a bacterial species comprising Aquifex aeolieus, Aquifex pyrophilus, Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus woesei, Pyrodictium occultum, Thermis flavus, Thermococcus barossi, Thermococcus gorgonarius, Thermococcus literalis, Thermococcus litoralis, Thermosipho africanus, Thermotoga maritima, Thermotoga maritima, Thermotoga neapolitana, Thermotoga neapolitana, Thermus antranildanii, Thermus aquaticus (Taq) , Thermus caldophllus, Thermus chliarophilus, Thermus filiformis, Thermus flavu, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus rubens, Thermus ruber, Thermus scotoductus, Thermus silvanus, Thermus thermophiles (Tth) , Thermus thermophllus, or a combination thereof.

Particularly preferably, the DNA polymerase with 5'nuclease activity is Taq polymerase. The thermostable DNA polymerase may be a Aquifex aeolieus polymerase. The thermostable DNA polymerase may be a Aquifex pyrophilus polymerase. The thermostable DNA polymerase may be a Pyrococcus abyssi polymerase. The thermostable DNA polymerase may be a Pyrococcus horikoshii polymerase. The thermostable DNA polymerase may be a Pyrococcus woesei polymerase. The thermostable DNA polymerase may be a Pyrodictium occultum polymerase. The thermostable DNA polymerase may be a Thermis flavuspolymerase. The thermostable DNA polymerase may be a Thermococcus barossi polymerase. The thermostable DNA polymerase may be a Thermococcus gorgonarius polymerase. The thermostable DNA polymerase may be a Thermococcus literalis polymerase. The thermostable DNA polymerase may be a Thermococcus litoralis polymerase. The thermostable DNA polymerase may be a Thermosipho africanus polymerase. The thermostable DNA polymerase may be a Thermotoga maritima polymerase. The thermostable DNA polymerase may be a Thermotoga maritima polymerase. The thermostable DNA polymerase may be a Thermotoga neapolitana polymerase. The thermostable DNA polymerase may be a Thermotoga neapolitana polymerase. The thermostable DNA polymerase may be a Thermus antranildanii polymerase. The thermostable DNA polymerase may be a Thermus aquaticus (Taq) polymerase. The thermostable DNA polymerase may be a Thermus caldophllus polymerase. The thermostable DNA polymerase may be a Thermus chliarophilus polymerase. The thermostable DNA polymerase may be a Thermus filiformis polymerase. The thermostable DNA polymerase may be a Thermus flavu polymerase. The thermostable DNA polymerase may be a Thermus igniterrae polymerase. The thermostable DNA polymerase may be a Thermus lacteus polymerase. The thermostable DNA polymerase may be a Thermus oshimai polymerase. The thermostable DNA polymerase may be a Thermus rubens polymerase. The thermostable DNA polymerase may be a Thermus ruber polymerase. The thermostable DNA polymerase may be a Thermus scotoductus polymerase. The thermostable DNA polymerase may be a Thermus silvanus polymerase. The thermostable DNA polymerase may be a Thermus thermophiles (Tth) polymerase. The thermostable DNA polymerase may be a Thermus thermophllus polymerase.

In some instances, the methods described herein may comprise a use of at least two different enzymes. For example, the two different enzymes may comprise a nuclease and a nucleic acid polymerase. In some cases, the two different enzymes may comprise a 5’ exonuclease and a DNA polymerase. In some cases, in the step of the cleavage of the mediator probe by the enzyme may be: (1) independent on an extension of the upstream primer (using the template nucleic acid as the template) or (2) dependent on in the extension of the upstream primer (using the template nucleic acid as the template) ; two different enzymes may be used in (1) , (2) , or (1) and (2) .

In some instances, the nuclease used as the enzyme described herein comprise Flap endonuclease (FEN) . FEN may comprise a nucleolytic enzyme that has both 5’ -3’ exonuclease activity and structure-specific endonuclease activity. For example, the endonuclease activity of FENs may target a DNA duplex (aDNA molecule that is at least partially double-stranded) comprising a single-stranded 5’ overhang on one of the strands (i.e., a “5 flap” ) . FEN can catalyze hydrolytic cleavage of the phosphodiester bond at the junction of single-and double- stranded DNA. In some cases, FENs can also have a 5’ -3’ exonucleases activity targeting the 5’ terminus of the flap strand and on “nicked” DNA substrates. In the methods described herein, FEN can cleave the junction between the first and second template-binding nucleotide sequences when the second template-binding nucleotide sequence hybridized with the target nucleic acid or the amplification product thereof. In some cases, the nuclease may not comprise Afu endonuclease.

In other cases, the enzyme may comprise a single-stranded restriction enzyme. When using such a nuclease in the method described herein, the mediator probe may comprise a cleavage site of the restriction enzyme 3’ of the first template-binding nucleotide sequences, wherein the restriction site (or a portion thereof) is located within the second template-binding nucleotide sequence. When a target nucleic acid is not present, the mediator probe may adopt a structure that masks the cleavage site from the restriction enzyme. For example, cleavage site may be part of a double-stranded structure via self-hybridization of the mediator probe (e.g., the mediator probe adopts a stem loop or hairpin structure that comprises the cleavage site or a portion thereof) . When a target nucleic acid is present, the second template-binding nucleotide sequence may hybridize with the target nucleic acid, remove the self-hybridized structure, and expose the cleavage site for the restriction enzyme, thereby allowing the generation of the cleavage fragment comprising the first template-binding nucleotide sequence.

Intermediate products

Provided herein are intermediate products generated using the methods and compositions described herein. The methods described herein can comprise hybridization, denaturation, nucleic acid amplification, or any combination thereof. Thus, various single-stranded, double stranded, partially single-stranded, or partially double-stranded nucleic acid intermediate products can be generated using the target nucleic acid, primers, mediator probes, reporter probes, or any combination thereof. For example, in some cases, the intermediate product may be generated using the mediator probe or cleaved fragment generated thereof; the reporter probe; or the hybridized product of the mediator probe or cleaved fragment generated thereof and the reporter probe, using the methods described herein.

In some cases, an intermediate product may comprise a duplex (anucleic acid molecule that is at least partially double-stranded) . In some cases, the intermediate product may comprise a duplex comprising two different probes. The two probes may comprise at least sequences that are complementary to each other. The two different probes may have a quencher/label moiety pair (a quencher moiety that can alter or absorb a detectable signal of the label moiety, as described herein) , wherein each member of the pairs is located on different strands of the duplex. For example: The first probe of the duplex may comprise a label moiety, and the second probe of the duplex may comprise a quencher moiety. The first probe of the duplex may comprise a label moiety but not a quencher moiety, and the second probe of the duplex may comprise the quencher moiety. The first probe of the duplex may comprise a label moiety, and the second probe of the duplex may comprise a quencher moiety but not the label moiety. The first probe of the duplex may comprise a label moiety and not a quencher moiety, and the second probe of the duplex may comprise the quencher moiety and not the label moiety. In some cases, any one strand of the duplex may comprise both a label moiety and a quencher moiety. The label and quencher moieties on the same strand may be a label/quencher pair. The label and quencher moieties on the same strand may not be a label/quencher pair. The first probe may be a portion of the mediator probe or the mediator probe. The second probe may be a portion of the reporter probe or the reporter probe. In other cases, the first probe may be a portion of the reporter probe or the reporter probe. The second probe may be a portion of the mediator probe or the mediator probe. In some cases, the duplex may comprise a cleaved fragment of the mediator probe and the reporter probe. The label or quencher moiety can be coupled to any nucleotide or portion thereof of the mediator probe or reporter probe, as described herein. For example: The label moiety may be coupled to the terminal end or sequence not at the terminal ends of the mediator probe.

The label moiety may be coupled to a 5’ end of the cleaved fragment of the mediator probe. The label moiety may be coupled to a 3’ end of the cleaved fragment of the mediator probe. The label moiety may be coupled to a nucleotide or sequence not at the 5’ end of the cleaved fragment of the mediator probe. The label moiety may be coupled to a nucleotide or sequence not at the 3’ end of the cleaved fragment of the mediator probe. The label moiety may not be coupled to a 5’ end of the cleaved fragment of the mediator probe. The label moiety may not be coupled to a 3’ end of the cleaved fragment of the mediator probe. The label moiety may not be coupled to a nucleotide or sequence not at the 5’ end of the cleaved fragment of the mediator probe. The label moiety may not be coupled to a nucleotide or sequence not at the 3’ end of the cleaved fragment of the mediator probe. The quencher moiety may be coupled to a 5’ end of the reporter probe. The quencher moiety may be coupled to a 3’ end of the reporter probe. The quencher moiety may be coupled to a nucleotide or sequence not at the 5’ end of the reporter probe. The quencher moiety may be coupled to a nucleotide or sequence not at the 3’ end of the reporter probe. The quencher moiety may not be coupled to a 5’ end of the reporter probe. The quencher moiety may not be coupled to a 3’ end of the reporter probe. The quencher moiety may not be coupled to a nucleotide or sequence not at the 5’ end of the reporter probe. The quencher moiety may not be coupled to a nucleotide or sequence not at the 3’ end of the reporter probe. The quencher moiety may be coupled to a 5’ end of the cleaved fragment of the mediator probe. The quencher moiety may be coupled to a 3’ end of the cleaved fragment of the mediator probe. The quencher moiety may be coupled to a nucleotide or sequence not at the 5’ end of the cleaved fragment of the mediator probe. The quencher moiety may be coupled to a nucleotide or sequence not at the 3’ end of the cleaved fragment of the mediator probe. The quencher moiety may not be coupled to a 5’ end of the cleaved fragment of the mediator probe. The quencher moiety may not be coupled to a 3’ end of the cleaved fragment of the mediator probe. The quencher moiety may not be coupled to a nucleotide or sequence not at the 5’ end of the cleaved fragment of the mediator probe. The quencher moiety may not be coupled to a nucleotide or sequence not at the 3’ end of the cleaved fragment of the mediator probe. The label moiety may be coupled to a 5’ end of the reporter probe. The label moiety may be coupled to a 3’end of the reporter probe. The label moiety may be coupled to a nucleotide or sequence not at the 5’ end of the reporter probe. The label moiety may be coupled to a nucleotide or sequence not at the 3’ end of the reporter probe. The label moiety may not be coupled to a 5’ end of the reporter probe. The label moiety may not be coupled to a 3’ end of the reporter probe. The label moiety may not be coupled to a nucleotide or sequence not at the 5’ end of the reporter probe. The label moiety may not be coupled to a nucleotide or sequence not at the 3’ end of the reporter probe.

The first strand of the duplex may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more label moieties. The first strand of the duplex comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 label moieties. The first strand of the duplex may comprise 1 label moiety. The first strand of the duplex may comprise 2 label moieties. The first strand of the duplex may comprise 3 label moieties. The first strand of the duplex may comprise 4 label moieties. The first strand of the duplex may comprise 5 label moieties. The second strand of the duplex may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more quencher moieties. The second strand of the duplex comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quencher moieties. The second strand of the duplex may comprise 1 quencher moiety. The second strand of the duplex may comprise 2 quencher moieties. The second strand of the duplex may comprise 3 quencher moieties. The second strand of the duplex may comprise 4 quencher moieties. The second strand of the duplex may comprise 5 quencher moieties. The first strand of the duplex may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more quencher moieties. The first strand of the duplex comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quencher moieties. The first strand of the duplex may comprise 1 quencher moiety. The first strand of the duplex may comprise 2 quencher moieties. The first strand of the duplex may comprise 3 quencher moieties. The first strand of the duplex may comprise 4 quencher moieties. The first strand of the duplex may comprise 5 quencher moieties. The second strand of the duplex may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more label moieties. The second strand of the duplex comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 label moieties. The second strand of the duplex may comprise 1 label moiety. The second strand of the duplex may comprise 2 label moieties. The second strand of the duplex may comprise 3 label moieties. The second strand of the duplex may comprise 4 label moieties. The second strand of the duplex may comprise 5 label moieties.

In some cases, the intermediate products may also comprise a hybridized product comprising a target nucleic acid hybridized with an upstream primer; a hybridized product comprising a target nucleic acid hybridized with a downstream primer; a hybridized product comprising a target nucleic acid hybridized with an upstream primer and a mediator probe; a hybridized product comprising a target nucleic acid hybridized with an upstream primer and a portion of mediator probe comprising only the second template-binding nucleotide sequence (that hybridizes or is configured to hybridize to the target nucleic acid with the first-template-binding nucleotide sequence cleaved off) ; a hybridized product comprising a target nucleic acid hybridized with a mediator probe; a hybridized product comprising the duplex comprising the reporter probe and the cleaved fragment of the mediator probe; an amplification product of the target nucleic acid; an amplification product of the upstream primer (e.g., one comprising the sequence of the upstream primer and at least a sequence of the target nucleic acid) ; an amplification product of the downstream primer (e.g., one comprising the sequence of the downstream primer and at least a sequence of the target nucleic acid) ; an amplification product of the cleaved fragment of the mediator probe (e.g., one comprising the sequence of the cleaved fragment of the mediator probe and at least a sequence of the reporter probe) ; an amplification product of the reporter probe; or any combination thereof. The intermediate product described herein may comprise any number, identity, or configuration of the label or quencher moiety as described herein based on the methods and compositions described herein. The intermediate product described herein may comprise any number, identity, or configuration of the sequence as described herein based on the methods and compositions described herein.

Label/quencher moieties

Provided herein are label and quencher moieties. The label or quencher moiety provided herein can be used in the methods described herein. The label moiety, the quencher moiety, or both the label and quencher moieties may be used to generate the detectable signal described herein, thereby allowing identification and/or quantification of the target nucleic acid. A label or quencher moiety may be coupled to a target nucleic acid, mediator probe, reporter probe, primers, or any derivative thereof generated thereof described in this disclosure. The label or quencher moiety may be coupled a nucleotide of the target nucleic acid, mediator probe, reporter probe, primers, or any derivative thereof generated thereof described in this disclosure. In some cases, the label or quencher moiety may be coupled a nucleotide at the terminal end of the target nucleic acid, mediator probe, reporter probe, primers, or any derivative thereof generated thereof described in this disclosure. In some cases, the label or quencher moiety may be coupled a nucleotide not at the terminal end of the target nucleic acid, mediator probe, reporter probe, primers, or any derivative thereof generated thereof described in this disclosure. In some cases, the label or quencher moiety may be coupled an atom of the phosphate group of a nucleotide. In some cases, the label or quencher moiety may be coupled an atom of the sugar group of a nucleotide. In some cases, the label or quencher moiety may be coupled an atom of the base group of a nucleotide. In some cases, the label or quencher moiety may be coupled an atom of a phosphodiester bond between two nucleotides. Specific alignment of coupled label or quencher moiety to a specific nucleotide position can be achieved using isoguanine (iso-dG) and 5′-methylisocytosine (iso-dC) as described herein.

A label moiety may comprise an optical moiety, an electrical moiety, a magnetic moiety, a thermal moiety, an acoustic moiety, or a combination thereof. A label moiety may comprise an optical moiety. A quencher moiety may comprise an optical moiety, an electrical moiety, a magnetic moiety, a thermal moiety, an acoustic moiety, or a combination thereof. A quencher moiety may comprise an optical moiety.

The term “coupled to, ” as used herein, generally refers to an association between two or more objects that may be temporary or substantially permanent. A first object may be reversibly or irreversibly coupled to a second object. For example, a nucleic acid molecule may be reversibly coupled to a label or quencher moiety. Coupling may encompass attachment, such as attachment of a first object to a second object. Coupling may comprise any interaction that affects an association between two objects, including, for example, a covalent bond, a non-covalent interaction, π-interaction, van der Waals force-based interactions, hydrophobic interaction, magnetic interaction, electromagnetic interaction, adsorption, or any other useful interaction. In some cases, the coupling may comprise attaching an adaptor comprising a label or quencher moiety to a nucleic acid molecule comprising the target nucleic acid, upstream/downstream primers, mediator probes the cleaved fragment of the mediator probe, the reporter probe, or combination thereof, by ligation or chemical means.

In some instances, a quenching moiety can absorb or quench a detectable signal generated by a label moiety. The quencher moiety may decrease or eliminate the signal intensity of the detectable signal (such that it is below the detection limit of the methods described herein) generated by the label moiety. In some cases, the quencher moiety may also alter the detection of the detectable signal generated by the label moiety. For example, a fluorescent label moiety may have a first emission spectrum of x (x can be a particular range of wavelength) for a first detectable signal. A quencher moiety may absorb the first detectable signal and generate a second detectable signal with a second emission spectrum that is different from x. In such case, the methods described herein may detect the first detectable signal, the second detectable signal, or a combination thereof. In some cases, a quencher moiety may minimize or eliminate a detectable signal generated by the label moiety. For example, a quencher moiety may decrease the signal intensity of a label moiety by at least about 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 %, 99.99 %or more. A quencher moiety may decrease the signal intensity of a label moiety by at most about 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.9 %, or 99.99 %. A quencher moiety may decrease the signal intensity of a label moiety by 100 %.

In some instances, a quencher moiety is configured to absorb, alter, quench, eliminate, or minimize a detectable signal generated by a label moiety. In some cases, a quencher moiety may be separated from the label moiety by a distance such that the quencher moiety can absorb, alter, quench, eliminate, or minimize a detectable signal generated by the label moiety. A label/quencher pair may thus form when they are placed within the distance.

A label/quencher moiety pair may have a beneficial advantage of a label moiety. For example, because a label/quencher moiety pair can absorb, alter, quench, eliminate, or minimize a detectable signal generated by the label moiety as described herein, the detectable signal can be detected as a decrease of the signal intensity of the detectable signal (or an increase of the signal intensity of a different detectable signal) , as opposed to detecting only the increase of the signal intensity of the detectable signal generated by the label moiety alone. In such cases, various reaction mixtures may have various levels of background signal as described herein, thereby preventing efficient or accurate measurement of only the increase of the signal intensity of the detectable signal generated by the label moiety alone. Thus, the detectable signal generated by the label/quencher moiety pair can thus provide efficient and accurate of signal detection. Additionally, the label/quencher moiety pair can allow generation of a detectable signal as described herein without a nucleic acid amplification or extension. For example, a reporter probe may comprise a molecular beacon in which the generation of a detectable signal requires the separation of a label and quencher moiety via a nucleic acid amplification or extension, such as those described in Faltin et al., Clin Chem. 2012 Nov; 58 (11) : 1546-56; Huang et al., Proc Natl Acad Sci U.S.A. 2022 Mar 1; 119 (9) : e2110672119; or U. S. Patent No.: 11,111,522, each of which is incorporated in its entirety.

In that case, using the label/quencher moiety pairing or the methods described herein; such a nucleic acid amplification or extension may be omitted, or one that requires a substantially decrease amount of time to separate the label and quencher moiety for generating the detectable signal.

The distance of the label and quencher moieties may be measured by the number of nucleotides separating them when they are coupled to a single-stranded nucleic acid molecule. The distance of the label and quencher moieties may be measured by the number of nucleotides separating them when they are coupled to two different strands of a double-stranded nucleic acid molecule, wherein distance refers to the number of nucleotides including the complementary base-paired nucleotide on the opposites strand. For example, if a label moiety is coupled to the 5’ end of one strand of double-stranded nucleic acid molecule, and a quencher moiety is coupled to the 3’ end of the other strand of double-stranded nucleic acid molecule, the distance between label and quencher moieties is “0. ” In some cases, the distance the label and quencher moieties is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more nucleotides. In some cases, the distance the label and quencher moieties is at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides

In some cases, the distance the label and quencher moieties is at least about 0.001 or more. In some cases, the distance the label and quencher moieties is at most about or

In some cases, a label moiety or quencher moiety may comprise Alexa ^TM (520) , Alexa^TM546 (570) , Alexa^TM594 (615) , B ODIPY558/568 (568) , BHQ1, BHQ2, BHQ3, Biosearch Blue (447) , BODIPY ^TMR (568) , BODIPY530/550 (550) , BODIPY564/570 (570) , BQ 650, C-Phycocyanin (648) , CAL Fluor Gold 540 (544) , CAL Fluor Orange 560 (559) , CAL Fluor Red 590 (591) , CAL Fluor Red 610 (610) , CAL Fluor Red 635 (637) , Calcein (517) , Calcium Crimson ^TM (615) , Calcium Green ^TM (533) , Calcium Orange^TM (576) , Cy2^TM (506) , Cy3, Cy3^TM (570) , Cy5, Cy5 . 5 (694) , Cy5 ^TM (670) , Cy5.5, Dabcyl, DiDilC (5) (665) , Dil (565) , Eclipse, FAM, FAM (520) , FITC (518) , Fluorescein (520) , Fluorescein-C3 (520) , FluorX ^TM (519) , HEX, HEX (556) , JOE, JOE (548) , Magnesium Green ^TM (531) , Magnesium Orange^TM (575) , MGB, NED, Nile Red (628) , Oregon Green ^TM500 (522) , Oregon Green^TM488 (524) , Phycoerythrin R&B (575) , Pulsar 650 (566) , PyroninY (580) , Quasar 570 (667) , Quasar 670, Quasar 670 (705) , Quasar 705 (610) , Rhodamine 110 (520) , Rhodamine 123 (529) , Rhodamine B (580) , Rhodamine Green ^TM (527) , Rhodamine Phalloidin (575) , Rhodamine Red^TM (590) , Ribo Green ^TM (525) , ROX, ROX (608) , R-phycocyanin (642) , SYBR, T0T03 (660) , TAMRA, TAMRA (582) , TET, TET (536) , Texas Red, Texas Red (615) , Thiadicarbocyanine (671) , TO-PRO^TM-3 (660) , TO-PRO^TM-l (533) , TOTOl (533) , TRITC (572) , VIC, YO-PRO^TM -l (509) , YO-PRO^TM-3 (631) , YOYO^TM-3 (631) , YOYO^TM-l (509) , or a combination thereof. The numbers in parentheses indicate the maximum emission wavelength in nanometer.

In some instances, depending on the pairing of the label and quencher moiety or the maximal excitation or emission wavelengths (or excitation/emission spectrum) , the chemical group described herein can be a label moiety or a quencher moiety. For example, FAM, SYBR, JOE, VIC, NED, Cy3, TAMRA, ROX, Texas Red, Cy5, TET, HEX, Quasar 670, Cy5.5, Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, or BBQ 650 may be a label moiety or a quencher moiety. A label moiety may comprise FAM, SYBR, JOE, VIC, NED, Cy3, TAMRA, ROX, Texas Red, Cy5, TET, HEX, Quasar 670, or Cy5.5. A label moiety may comprise FAM. A label moiety may comprise SYBR. A label moiety may comprise JOE. A label moiety may comprise VIC. A label moiety may comprise NED. A label moiety may comprise Cy3. A label moiety may comprise TAMRA. A label moiety may comprise ROX. A label moiety may comprise Texas Red. A label moiety may comprise Cy5. A label moiety may comprise TET. A label moiety may comprise HEX. A label moiety may comprise Quasar 670. A label moiety may comprise Cy5.5. A quencher moiety may comprise Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, or BBQ 650. A quencher moiety may comprise Dabcyl. A quencher moiety may comprise Eclipse. A quencher moiety may comprise MGB. A quencher moiety may comprise BHQ1. A quencher moiety may comprise BHQ2. A quencher moiety may comprise BHQ3. A quencher moiety may comprise BBQ 650.

In some instances, a label moiety or quencher may comprise an optical moiety. The optical moiety may comprise a fluorophore. In some instances, a label moiety may comprise a fluorophore. In such case, the detectable signal generated by the label moiety comprises fluorescence, and the quencher moiety comprises a molecule or group capable of absorbing/quenching the fluorescence. For example, a quencher moiety may comprise a second fluorophore capable of absorbing or quenching the fluorescence of the label moiety.

In some cases, the label moiety may have a maximum excitation wavelength of at least about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800 or more nm. In some cases, the label moiety may have a maximum excitation wavelength of at most about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 nm. In some cases, the label moiety may have a maximum emission wavelength of at least about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800 or more nm. In some cases, the label moiety may have a maximum emission wavelength of at most about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 nm.

In some cases, the label moiety may comprise ALEX-350, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 635, CAL Gold 540, CY3, CY5, CY5.5, FAM, HEX, JOE, Quasar 670, Quasar705, ROX, TAMRA, TET, TEXAS RED, VIC, or a combination thereof. In some cases, the label moiety may comprise ALEX-350. In some cases, the label moiety may comprise CAL Fluor Orange 560. In some cases, the label moiety may comprise CAL Fluor Red 590. In some cases, the label moiety may comprise CAL Fluor Red 610. In some cases, the label moiety may comprise CAL Fluor Red 635. In some cases, the label moiety may comprise CAL Gold 540. In some cases, the label moiety may comprise CY3. In some cases, the label moiety may comprise CY5. In some cases, the label moiety may comprise CY5.5. In some cases, the label moiety may comprise FAM. In some cases, the label moiety may comprise HEX. In some cases, the label moiety may comprise JOE. In some cases, the label moiety may comprise Quasar 670. In some cases, the label moiety may comprise Quasar705. In some cases, the label moiety may comprise ROX. In some cases, the label moiety may comprise TAMRA. In some cases, the label moiety may comprise TET. In some cases, the label moiety may comprise TEXAS RED. In some cases, the label moiety may comprise VIC.

In some cases, the quencher moiety may have a maximum excitation wavelength of at least about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800 or more nm. In some cases, the quencher moiety may have a maximum excitation wavelength of at most about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 nm. In some cases, the quencher moiety may have a maximum emission wavelength of at least about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800 or more nm. In some cases, the quencher moiety may have a maximum emission wavelength of at most about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 nm.

In some cases, the quencher moiety may absorb a detectable signal (light or fluorescence) with a wavelength of at least about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800 or more nm. In some cases, the quencher moiety may absorb a detectable signal (light or fluorescence) with a wavelength of at most about 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, or 800 nm.

In some cases, the quencher moiety may comprise DABCYL, BHQ, ECLIPSE, TAMRA, or a combination thereof. In some cases, the quencher moiety may comprise DABCYL. In some cases, the quencher moiety may comprise BHQ. In some cases, the quencher moiety may comprise ECLIPSE. In some cases, the quencher moiety may comprise TAMRA. BHQ may comprise BHQ-1, BHQ-2, or a combination thereof. BHQ may comprise BHQ-1. BHQ may comprise BHQ-2. BHQ may comprise BHQ-1, BHQ-2 and BHQ-3.

In some instances, a pair of label moiety and quencher moiety (or label/quencher moiety pair) may comprise any of those described herein, based on the excitation and emission spectrums (or maximum wavelength of the excitation or emission) of the label/quencher moieties. For example, a pair of label/quencher moieties may comprise a quencher moiety that has a maximum excitation wavelength that is substantially the same or the same as the maximum emission wavelength of a label moiety.

In some instances, pairings of label moiety and quencher moiety may comprise any of those described in Pesce et al., editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971) ; White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970) ; Berlman , Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971) ; Griffiths, Color AND Constitution of Oiganic Molecules (Academic Press , New York, 1976) ; Bishop, editor, Indicators (Peigamon Press, Oxford, 1972) ; Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992) ; Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949) ; Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, 6th Edition (Molecular Probes, Eugene, Oreg., 1996) ; or US Patents Nos: 3,996,345 and 4,351,760, each of which is herein incorporated by reference in its entirety.

In some cases, a label or quencher moiety may be coupled to any target nucleic acid, mediator probe, reporter probe, primers, or any derivative thereof described herein by chemical synthesis or coupling. In other cases, a label or quencher moiety may be coupled to any derivative of the target nucleic acid, mediator probe, reporter probe, or primers during a nucleic acid amplification reaction. For example, the label or quencher moiety may be coupled to a nucleotide. During a nucleic acid amplification reaction using the coupled nucleotide with the target nucleic acid, mediator probe, reporter probe, or primers, the coupled nucleotide can be incorporated into the amplification/extension products of the target nucleic acid, mediator probe, reporter probe, or primers.

Nucleic acid labeling dye can be used to detect any nucleic acids comprising target nucleic acid, mediator probe, reporter probe, primers, or any amplification products or derivatives thereof, in place of the label moiety coupled to the nucleic acids. For example, nucleic acid dyes (single-, double-, or single-and-double-strand specific) can be used to contact the nucleic acid, thereby detecting the nucleic acid. Such dyes can comprise SYBR Green^TM (I, II, GOLD) , Ethidium homodimer, PicoGreen^TM, OliGreen^TM and RiboGreen^TM quantitation reagents and their Quant-iT^TM, Applied Biosystems^TM SYBR^TM Safe DNA gel stain, CyQUANT^TM GR dye, cyanine dimer dyes, or any combination thereof.

Analyzing analytes

Provided herein methods and compositions for analyzing analytes. In some cases, the methods and compositions can be used to detect, identify, or quantify a target nucleic acid.

In some instances, the methods comprise detecting the detectable signal generated by the label or quencher moiety described herein. In the methods described herein, various detectable signals may be generated. For example, the label moiety may generate a first detectable signal. In some cases, the first detectable signal may be absorbed, minimized, eliminated, quenched, or altered by the quencher moiety. In some cases, a second detectable signal may thus be generated.

In some cases, when detecting a detectable signal, the detection method may detect: a presence of a signal (e.g., one generated by a label moiety or one generated by a label moiety and a quencher moiety) ; an increase of signal intensity of the signal; an absence of a signal (e.g., the signal being absorbed, minimized, eliminated, quenched, or altered by a quencher moiety) ; or a combination thereof.

In some cases, the detection method may comprise melting curve analysis (or melting analysis) . Melting curve analysis may comprise measuring the melting curve of a double-stranded nucleic acid molecule. Melting curve analysis may be used for detecting a presence of the double-stranded nucleic acid molecule. Melting curve analysis can analyze the dissociation or denaturation characteristics of double-stranded nucleic acid molecules during heating. For example, two nucleic acid molecules may form a double-stranded nucleic acid molecule at ambient temperature via complementary base-pairing, wherein one of the nucleic acid molecules has a label moiety and one has a quencher moiety (that pairs with the label moiety) , and wherein the label and quencher moiety may be in a distance that allow the quencher moiety to absorb, quench, eliminate, minimize, or alter a first detectable signal generated by the label moiety in the double-stranded molecule. Thus, the first detectable signal may not be detected when the two nucleic acid molecules are in the form of the double-stranded nucleic acid molecule. During heating of a population of the double-stranded nucleic acid molecules, the two nucleic acid molecules may begin to dissociate/denature in a subset of the population of the double-stranded nucleic acid molecules. Thus, in those cases, the quencher moiety on one strand is no longer able to absorb, quench, eliminate, minimize, or alter a first detectable signal generated by the label moiety on another strand, thereby allowing detection of the first detectable signal. As the temperature increases, the intensity of the first detected signal gradually increases, as the subset of the denatured double-stranded nucleic acid molecules increases. When the two strands of all the double-stranded nucleic acid molecule in a population of the double-stranded nucleic acid molecules are completely dissociated/denatured, the first detectable signal reaches the maximum intensity. Therefore, by detecting the first detectable signal generated during the heating or cooling process, the hybridization and resolution of the hybridization and resolution of two nucleic acid molecules/double-stranded nucleic molecules formed from thereof can be detected and analyzed. During the separation process, a curve of signal intensity changing with temperature is formed. Further, by performing derivative analysis on the obtained curve, a curve with the signal intensity change rate as the ordinate and temperature as the abscissa can be obtained (that is, the melting curve of the double-stranded nucleic acid molecules) . The peak in the melting curve is the melting peak, and the corresponding temperature is the melting point (Tm value; or melting peak) of the double-stranded nucleic acid molecules. Therefore, by detecting the Tm value of the double-stranded nucleic acid molecules, the presence (or absence) of any of the two nucleic acid molecules (or the double-stranded nucleic acid molecule that forms by thereof as described herein) can be determined. In the methods described herein, for example, in a double-stranded nucleic acid molecule comprising a cleaved fragment and a reporter probe, since the cleaved fragment is generated only in the presence of a target nucleic acid, the melting curve analysis can be used to identify the presence (or absence of a target nucleic acid) . Methods and procedures for performing melting curve analysis are described in Lyon et al., The Journal of Molecular Diagnostics 2009, 11 (2) : 93-101) , which is herein incorporated by reference in its entirety.

In other cases, melting curve analysis may be carried out wherein a decrease of signal intensity is used to determine the melting temperature and melting peak. For example, a nucleic acid molecule may comprise both label and quencher moieties of a label/quencher moiety pair. In such cases, when hybridized to another nucleic acid molecule as a double-stranded nucleic acid molecule, the label and quencher moieties may be separated with a distance such that the quencher no longer be able to absorb, minimize, eliminate, quench, or alter the detectable signal of the label moiety. However, when dissociated, the label and quencher moieties may now be in a distance such that that the quencher can absorb, minimize, eliminate, quench, or alter the detectable signal of the label moiety, thereby altering or minimizing or eliminating the detectable signal generated by the label moiety. Hence the melting curve analysis may identify a negative melting peak (i.e., the temperature with the lowest signal intensity) .

Melting curve analysis may comprise measuring the melting curve of an intermediate product described herein. Melting curve analysis may comprise measuring the melting curve of a duplex comprising the cleaved fragment of the mediator probe and the reporter probe. Melting curve analysis may comprise measuring the melting curve of a duplex comprising a mediator probe and a target nucleic acid (or an amplification product thereof) . Melting curve analysis may comprise measuring the melting curve of a duplex comprising upstream primer and a target nucleic acid (or an amplification product thereof) . Melting curve analysis may comprise measuring the melting curve of a duplex comprising downstream primer and a target nucleic acid (or an amplification product thereof) .

The melting curve analysis in the methods described herein may be used for multiplexing analysis. In some cases, a melting peak of a melting curve analysis may correspond to a particular target nucleic acid. In some cases, the melting curve analysis may generate at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more melting peaks. In some cases, the melting curve analysis may generate at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 more melting peaks. For multiplexing analysis, two melting peaks may have a temperature difference. In some cases, the temperature difference of two melting peaks may be at least about 0.1 ℃, 0.2 ℃, 0.3 ℃, 0.4 ℃, 0.5 ℃, 0.6 ℃, 0.7 ℃, 0.8 ℃, 0.9 ℃, 1 ℃, 1.1 ℃, 1.2 ℃, 1.3 ℃, 1.4 ℃, 1.5 ℃, 1.6 ℃, 1.7 ℃, 1.8 ℃, 1.9 ℃, 2 ℃, 2.1 ℃, 2.2 ℃, 2.3 ℃, 2.4 ℃, 2.5 ℃, 2.6 ℃, 2.7 ℃, 2.8 ℃, 2.9 ℃, 3 ℃, 3.1 ℃, 3.2 ℃, 3.3 ℃, 3.4 ℃, 3.5 ℃, 3.6 ℃, 3.7 ℃, 3.8 ℃, 3.9 ℃, 4 ℃, 4.1 ℃, 4.2 ℃, 4.3 ℃, 4.4 ℃, 4.5 ℃, 4.6 ℃, 4.7 ℃, 4.8 ℃, 4.9 ℃, 5 ℃, 5.5 ℃, 6 ℃, 6.5 ℃, 7 ℃, 7.5 ℃, 8 ℃, 8.5 ℃, 9 ℃, 9.5 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃ or more. In some cases, the temperature difference of two melting peaks may be at most about 0.1 ℃, 0.2 ℃, 0.3 ℃, 0.4 ℃, 0.5 ℃, 0.6 ℃, 0.7 ℃, 0.8 ℃, 0.9 ℃, 1 ℃, 1.1 ℃, 1.2 ℃, 1.3 ℃, 1.4 ℃, 1.5 ℃, 1.6 ℃, 1.7 ℃, 1.8 ℃, 1.9 ℃, 2 ℃, 2.1 ℃, 2.2 ℃, 2.3 ℃, 2.4 ℃, 2.5 ℃, 2.6 ℃, 2.7 ℃, 2.8 ℃, 2.9 ℃, 3 ℃, 3.1 ℃, 3.2 ℃, 3.3 ℃, 3.4 ℃, 3.5 ℃, 3.6 ℃, 3.7 ℃, 3.8 ℃, 3.9 ℃, 4 ℃, 4.1 ℃, 4.2 ℃, 4.3 ℃, 4.4 ℃, 4.5 ℃, 4.6 ℃, 4.7 ℃, 4.8 ℃, 4.9 ℃, 5 ℃, 5.5 ℃, 6 ℃, 6.5 ℃, 7 ℃, 7.5 ℃, 8 ℃, 8.5 ℃, 9 ℃, 9.5 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, or 15 ℃.

Provided herein, are methods for increasing the detection sensitivity in the identification of target nucleic acids using the compositions described herein. In the melting curve analysis, identification or determination of a melting peak as described herein can identify a presence of a target nucleic acid. The generation of a melting peak can depend on the number of the complementary nucleotides between two hybridized nucleic acid molecules. By decreasing the number of complementary nucleotides between the two hybridized nucleic acid molecules, a melting peak with a higher signal intensity can be generated, therefore increasing the sensitivity to identify the melting peak.

In an illustrative example, two populations of equal numbers of double-stranded nucleic acid molecules, one with 5 pairs of complementary of nucleotides (nucleic acid A) and one with 100 pairs of complementary of nucleotides (nucleic acid B) , are subjected to the melting curve analysis as described herein. In nucleic acids A and B, one nucleic acid strand has a label moiety coupled to the 5’ end and another nucleic acid strand has a paired quencher moiety coupled to the 3’ end, forming a label/quencher moiety pair. The label moiety and quencher moiety are configured to be separated by the denaturation of the double-stranded form into the single-stranded form. When the temperature is increased toward the Tm of nucleic acid A, most molecules in the populations of nucleic acid As will be segregated toward either the completely hybridized double-stranded form or completely dissociated single-stranded form, with only a minor population of partially hybridized or dissociated forms, because only a relatively small number of possible partially hybridized or dissociated forms of nucleic acid As (the possible partially hybridized or dissociated forms of nucleic acid As can comprise partially hybridized double-stranded molecules with 1, 2, 3, or 4 complementary-paired nucleotides) . The signal intensities generated in the melting curve analysis using the population of nucleic acids A will then develop from a complete lack of signal intensity (completely hybridized doble-stranded form with the label moiety quencher by the paired quencher moiety) to a maximum signal intensity (completely dissociated single-stranded form with the label moiety not quenched by the paired quencher moiety; also the melting peak) within a relative small temperature range (the temperature range at which the completely hybridized doble-stranded form transitioning into the partially hybridized or dissociated form and then into the completely dissociated form) . In contrast, as compared to the nucleic acids A, when the temperature is increased toward the Tm of nucleic acids B, only a minor portion of the molecules in the populations of nucleic acids Bs will be segregated toward either the completely hybridized double-stranded form or completely dissociated single-stranded form, with a majority of the population exists as the partially hybridized or dissociated nucleic acids Bs, because a relatively large number of nucleic acid Bs exists as partially hybridized or dissociated forms with the relatively large number of complementary nucleotides (the possible partially hybridized or dissociated forms of nucleic acid As can comprise partially hybridized double-stranded molecules with 1-99 complementary-paired nucleotides) . The signal intensities generated in the melting curve analysis using the nucleic acid B will then develop from a complete lack of signal intensity (completely hybridized doble-stranded form with the label moiety quencher by the paired quencher moiety) to a maximum signal intensity (completely dissociated single-stranded form with the label moiety not quenched by the paired quencher moiety; also the melting peak) within a relative large temperature range because of the relatively large number of the partially hybridized or dissociated form of the nucleic acid B. Because the populations of nucleic acids A and B have the same number of label and quencher moieties, the total amount of signal intensity generated by both populations should be the same (or substantially the same) . With a lower temperature range of the melting curve directed to the hybridized or dissociated forms, the melting curve of nucleic acid A may appear with a higher signal intensity at the melting peak with a steeper slope, relative to that of nucleic acid B, such as those depicted in Table 1 or Example 2. Thus, decreasing the number of complementary nucleotides between each strand of a double-stranded nucleic acid molecule can increase the detection sensitivity of the melting curve analysis.

In some cases, the methods may comprise quantification of a nucleic acid molecule (e.g., a target nucleic acid described herein) . For example, a first nucleic acid molecule may be used as a primer to amplify a second nucleic acid molecule as a template in a nucleic acid amplification reaction. The first nucleic acid molecule may comprise the mediator probe or the cleaved fragment thereof, upstream primer, downstream primer, or a combination thereof. The second nucleic acid molecule or template may comprise the target nucleic acid molecule. The second nucleic acid molecule or template may comprise the reporter probe.

The nucleic acid amplification reaction may generate a first detectable signal. For example: (1) A label moiety may be incorporated into the amplification product; (2) a label/quencher moiety pair may form (for example, a label moiety may be coupled to the primer and the quencher moiety may be coupled to the template; or a quencher moiety may be coupled to the primer and the label moiety may be coupled to the template) ; or (3) a strand-specific dye may be incorporated into the amplification product (whether or not a pairing quencher moiety is used to absorb, minimize, eliminate, quench, or alter a detectable signal of the dye) . By measuring the signal intensity of the first detectable signal during the cycles of the nucleic acid amplification reaction, the nucleic acid molecule cane be quantified. Such methods can be used to quantify the target nucleic acid as described herein.

In other cases, the first nucleic acid molecule may be used as a probe. During the nucleic amplification reaction of the second nucleic acid (e.g., with the upstream or downstream primer) , the probe will hybridize with the second nucleic acid, or the amplification product generated thereof, thereby generating a detectable signal as described herein. In some cases, the method may not use a nucleic acid amplification of the target nucleic acid during the hybridization of the probe to a target nucleic acid. For example, the probe may hybridize to the target nucleic acid for quantification. When quantifying using the probe hybridization to the target nucleic acid, the probe may comprise a detectable moiety that is configured to generate the detectable signal only when hybridized to the target nucleic acid (e.g., the probe may have a label moiety that is specific to the hybridized double-stranded product or form a label/quencher pair with a target nucleic acid that is coupled to the quencher moiety described herein) .

The detectable signal generated by the quantification methods described herein may be used to identify or qualitatively determine a presence or absence of a target nucleic acid molecule. A presence or absence of the detectable signal may determine a presence or absence of the target nucleic acid, respectively.

Using the first nucleic acid molecule as a probe, the method may also generate the detectable signal if the probe hybridizes to a reporter probe. For example, the mediator probe may be used as the probe. The mediator probe may hybridize to the target nucleic acid molecule (or an amplification product thereof if a nucleic acid amplification reaction is used) . Such a hybridization may or may not generate a detectable signal. The hybridized mediator probe may be cleaved by the enzyme and methods described herein. The cleaved fragment of the mediator probe may then hybridize to a reporter probe, thereby generating a detectable signal. The detectable signal may then be used to qualitatively or quantitatively analyze the target nucleic acid.

The amplification product may generate a first detectable signal. For example: (1) A label moiety may be incorporated into the amplification product; (2) a label/quencher moiety pair may form (for example, a label moiety may be coupled to the primer and the quencher moiety may be coupled to the template; or a quencher moiety may be coupled to the primer and the label moiety may be coupled to the template) ; or (3) a strand-specific dye may be incorporated into the amplification product (whether or not a pairing quencher moiety is used to absorb, minimize, eliminate, quench, or alter a detectable signal of the dye) . By measuring the signal intensity of the first detectable signal during the cycles of the nucleic acid amplification reaction, the nucleic acid molecule cane be quantified. Such methods can be used to quantify the target nucleic acid as described herein.

In some cases, the methods may comprise sequencing of a nucleic acid molecule (e.g., a target nucleic acid described herein) . For example, a first nucleic acid molecule may be used as a primer or probe to hybridize a second nucleic acid molecule or using the second nucleic acid molecule as a first template in a nucleic acid amplification reaction. The first nucleic acid molecule may comprise the mediator probe, upstream primer, downstream primer, or a combination thereof. The primer or probe may be configured to hybridize to a sequence specific to a sequence or region of a first template among other templates but do not bind to the corresponding sequences or regions of the other templates. For example, the first template may comprise a polymorphism, mutation, or change in a particular sequence or region, relative to those of the other templates. The sequence of the first template may be associated with a particular pathogen or disease described herein. In the sequencing method as described herein, the primer or probe (such as the mediator probe) may comprise a sequence (such as the second template-binding nucleotide sequence of the mediator probe that is configured to hybridize with the target nucleic acid) that is identical or complementary to the sequence or region of the first template (such as the target nucleic acid) . The sequence of the primer or probe may be configured to bind only the sequence or region of the first template but not the corresponding sequences or regions of the other templates. In some cases, the first nucleic acid molecule is used as the mediator probe to contact the first template, the cleaved fragment is generated that can hybridize with the reporter probe, thereby generating a detectable signal as described herein. In other cases, the first nucleic acid may be used as a probe. During a target nucleic acid amplification reaction (for example, using the upstream/downstream primer) , the probe will hybridize to the target nucleic acid and the amplification generated thereof, thereby generating a detectable signal as described herein. In either case, the first nucleic acid molecule can only generate a detectable signal when it contacts the first template or a sequence of the first template.

In some cases, the detectable signal may be generated using the reporter probe. For example, a mediator probe may comprise a second template-binding nucleotide sequence specific to a particular template nucleic acid (such as one associated with a pathogen or disease) . Hybridization of the mediator probe to the particular template nucleic acid then will facilitate the generation of the cleaved fragment for hybridizing to the reporter probe and subsequent detectable signal generation. Thus, in some cases, melting curve analysis can be used in the sequencing methods as described herein.

When referring to nucleic acid or sequence thereof, a “corresponding” counterpart may comprise a nucleic acid or sequence with a substantial sequence identity but with at least a change, mutation, or polymorphism. When the method is used to identify or quantify a particular nucleic acid (such as one associated with a particular pathogen or disease) , the corresponding counterpart may not be associated with that particular pathogen or disease.

In some cases, the method may comprise contacting the first nucleic acid molecule with the second nucleic acid molecule individually. For example, a sample may comprise only the sequence of the second nucleic acid molecule. In some cases, the method may comprise contacting the first nucleic acid molecule with the second nucleic acid molecule and a pool of corresponding nucleic acid molecules of the second nucleic acid molecule.

Melting curve analysis, quantification, or sequencing may be carried out on a same reaction mixture comprising any of the composition or intermediate products described herein. Melting curve analysis may be carried out prior to the quantification. Melting curve analysis may be carried out simultaneously with the quantification. Melting curve analysis may be carried out subsequent to the quantification. Melting curve analysis may be carried out without the quantification. Quantification may be carried out without the melting curve analysis. Melting curve analysis may be carried out prior to the sequencing. Melting curve analysis may be carried out simultaneously with the sequencing. Melting curve analysis may be carried out subsequent to the sequencing. Melting curve analysis may be carried out without the sequencing. Sequencing may be carried out without the melting curve analysis. Quantification may be carried out prior to the sequencing. Quantification may be carried out simultaneously with the sequencing. Quantification may be carried out subsequent to the sequencing. Quantification may be carried out without the sequencing. Sequencing may be carried out without the quantification.

Because the methods described herein can generate multiple detectable signals, carrying out multiple analysis steps as described herein (e.g., melting curve analysis, quantification (or qualitative identification) , or sequencing) can have beneficial advantages. For example, a group of target nucleic acids associated with a particular group/genus of pathogens or diseases (or risks thereof) may first be identified using a first detectable signal, and a subsequent step to identify or quantify particular subgroup of species of pathogens or diseases may then carried out. In such case, the methods may use less reagents by not carrying out the subsequent step if a group/genus of pathogens or diseases is not identified to be present or present in a significant amount in a sample (s) . Additionally, such methods can also increase the speed of the analysis (since some of the steps may not needed to be carried out) .

The quantification, qualitative analysis, and/or sequencing analyses may detect the amplification signal or hybridization signal, as described herein. The melting curve analysis, qualitative analysis, and/or sequencing analyses may detect the hybridization signal or denaturation signal, as described herein.

In the methods described herein, the detectable signals may be normalized. For example, the detectable signal may be subtracted, divided, summated, or multiplied by another detectable. The another detectable signal may comprise a background signal. In some cases, the detectable signal may be subtracted or divided by the background signal. Background signals may comprise the signal detected in a reaction mixture lacking at least one of the mediator probe (or cleaved fragment thereof) , reporter probe, enzyme, nucleotide, label moiety, quencher moiety, upstream primer, downstream primer, a component that allows for the nucleic acid amplification reaction, hybridization of any two nucleic acids, a reaction condition (such as buffer, conditions of the buffer, temperature, time, co-factors, or a combination thereof) .

Multiplexing

The methods, compositions, devices, systems, or reagents described herein can allow multiplexing analysis of analytes. “Multiplexing” or “multiplex” analysis as used herein referring to analyzing more than one analyte, simultaneously or subsequently, in a single or same reaction mixture or reaction container (i.e., a container in which a reaction mixture is contained within) . For example, multiple target nucleic acids may be analyzed within a single or same reaction mixture or within a single or same reaction container.

In some instances, the multiplexing methods may comprise analyzing at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more target nucleic acids. In some instances, the multiplexing methods may comprise analyzing at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 target nucleic acids. In some instances, the multiplexing methods may comprise generating at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more detectable signals. In some instances, the multiplexing methods may comprise generating at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 detectable signals.

For example, the multiplexing method may comprise using at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more reporter probes. The multiplexing method may comprise using at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 reporter probes.

One target nucleic acid may be analyzed using at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more reporter probes, mediator probes (or cleaved fragments thereof) , detectable signaled thereof, or any combinations thereof. One target nucleic acid may be analyzed using at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500 reporter probes, mediator probes (or cleaved fragments thereof) , detectable signaled thereof, or any combinations thereof.

In some cases, the multiplexing method may comprise using at least 2 mediator probes and at least 2 reporter probes. A first mediator probe may hybridize a first template nucleic acid, and a second mediator probe may hybridize a second template nucleic acid. A first and second cleaved fragments may be generated from the hybridized products of the first/second mediator probe/target nucleic acids, respectively, using the methods described herein. The first and second cleaved fragments may then hybridize to a first and second reporter probes, respectively. A first and second detectable signal may then be generated, using the methods described herein, for analyzing the first and second target nucleic acids, respectively.

In some cases, the multiplexing method may comprise using at least N mediator probes at least M reporter probes, wherein N and M are integers, and wherein M < N. For example, in a method for analyzing at least two target nucleic acids, a first mediator probe may hybridize a first template nucleic acid, and a second mediator probe may hybridize a second template nucleic acid. A first and second cleaved fragments may be generated from the hybridized products of the first/second mediator probe/target nucleic acids, respectively, using the methods described herein. The first and second cleaved fragments may then hybridize to one reporter probe at different nucleotide positions (such as with an offset of the template sequences for hybridization of the reporter probe described elsewhere in this disclosure) . A first and second detectable signal may then be generated, using the methods described herein, for analyzing the first and second target nucleic acids, respectively. In some cases, in the multiplexing methods, N may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more, wherein N and M are integers, and wherein M < N. In some cases, in the multiplexing methods, N may be at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500, wherein N and M are integers, and wherein M < N. In some cases, in the multiplexing methods, M may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more, wherein N and M are integers, and wherein M < N. In some cases, in the multiplexing methods, M may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, or 500, wherein N and M are integers, and wherein M < N.

In some cases, due to the offset of the template sequences for hybridization of the reporter probe, at least 2 cleaved fragments of the mediator probe may hybridize to two different nucleotide positions on a same reporter probe. Subsequent nucleic acid amplifications using the cleaved fragments as primer and the reporter probe as template, at least 2 amplification products with different lengths can be generated. The at least 2 amplification products may be analyzed using the melting curved analysis as described herein.

In some cases, the difference in lengths of the amplification products generated may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000 or more nucleotides. In some cases, the difference in lengths of the amplification products generated may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000 nucleotides.

In some cases, the amplification products with different lengths may also be generated using the multiplexing method comprising at least 2 mediator probes and at least 2 reporter probes. For example, the template sequences for extension of the reporter probe of the two reporter probes may have different lengths, thereby generating the amplification products with different lengths when using two different mediator probes (or the cleaved fragment thereof) as primers and the two reporter probes.

In some cases, at least two mediator probes, or the cleaved fragments thereof, or a combination thereof; may comprise or be coupled to at least two different label moieties, quencher moieties, or label/quencher moiety pairs, as described herein. The hybridized products (or the denatured product) or the amplification products generated from the at least two mediator probes or the cleaved fragments thereof and the reporter probe (or a least two different reporter probes) may each generate a different or differentiable detectable signal, thereby allowing analysis of the at least two target nucleic acids.

In some cases, at least two different hybridized products of at least two cleaved fragments of at least two mediator probes and at least two different template sequences for hybridization (or at least two different amplification products generated using thereof in nucleic acid amplification reactions) may at least two different label/quencher moiety pairs, thereby allowing generation of at least two different or differentiable detectable signals for analysis using the methods described herein. In some cases, the two different cleaved fragments of at least two mediator probes may each have a different label or quencher moiety, and the two template sequences for hybridization may have two different quencher or label moiety, respectively, thereby allowing generation of the two different label/quencher moiety pairs. In some cases, the two different cleaved fragments of at least two mediator probes may each have a different label or quencher moiety, and the two template sequences for hybridization may have a same quencher or label moiety that would allow generation of the two different label/quencher moiety pairs, using the methods disclosed herein (e.g., the two different label/quencher moiety pair may have different maximum emission wavelength or differentiable emission spectrums) .

In some cases, the two template sequences for hybridization of the reporter probe may be present on two different reporter probes.

In some cases, the two template sequences for hybridization of the reporter probe may be present on a same reporter probe. For example, the reporter probe may have at least one different label or quencher moiety at each template sequence for hybridization of the reporter probe. Hybridization of the cleaved fragments of the mediator probe may thus bring the label or quencher moieties (one from the cleaved fragment of the mediator probe and one from the reporter probe) within distances that allow generations of two different or differentiable label/quencher moiety pairs.

In some cases, two different label or quencher moieties may be incorporated into two different amplification products generated using two different cleaved fragments of the mediator probe and two template sequences for hybridization of the reporter probe (that can be present on the same reporter probe or two different reporter probes) . For example, each of the two template sequences for hybridization of the reporter probe may be placed 3’ to two template sequences for extension of the reporter probe, each with a different modified nucleotide that can only base pair with a modified complementary nucleotide. During nucleic acid amplification reactions when the two different cleaved fragments of the mediator probe hybridized to the two template sequences for hybridization of the reporter probe, only the base-pairable modified nucleotide can be incorporated into the nascent strand of the two different cleaved fragments of the mediator probe, because two different modified nucleotides are present within the two template sequences for extension of the reporter probe. When the two base-pairable modified nucleotides comprise or are coupled to two different label/quencher moieties, the amplification products generated in the nucleic acid amplification reaction will thus comprise two different or differentiable label/quencher moiety pairs.

In other cases, the upstream/downstream primers may be used in the multiplexing methods described herein. For example, at least two upstream or downstream primers, the amplification products generated using the at least two upstream or downstream primers, or a combination thereof; may comprise or be coupled to at least two different label moieties, quencher moieties, or label/quencher moiety pairs, as described herein. The hybridized products (or the denatured product) or the amplification products generated from the at least two upstream or downstream primers (and at least two target nucleic acids) may each generate a different or differentiable detectable signal, thereby allowing analysis of the at least two target nucleic acids.

In the multiplexing methods described herein, a reporter probe and at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mediator probes may be used in a single composition or reaction mixture. In some cases, the reporter probe may be in molar excess relative to the mediator probe. For example, the reporter probe may be at least about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the reporter probe. For example, the reporter probe may be at most about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the reporter probe. The molar excess of the reporter probe relative to the mediator probe can have a beneficial advantage because the overall reaction mixture will contain sufficient amounts of reporter probe to hybridize to the cleaved fragments of the mediator probes for practicing the methods described herein.

Target nucleic acids

Provided herein target nucleic acids that can be detected using the methods, compositions, devices, systems, or reagents described elsewhere in this disclosure. “Target nucleic acid” as used herein can refer to as a nucleic acid molecule (s) or a particular nucleotide sequence (s) .

The target nucleic acid may comprise a nucleic acid (or sequence thereof) . The target nucleic acid may comprise DNA, RNA, or any combination thereof. The target nucleic acid may be derived form a pathogen. A pathogen may be a prokaryotic or eukaryotic cell. The target nucleic acid may be derived from a cell associated with a disease (or obtained from a subject having or suspected of having the disease) . A pathogen may cause or be associated with a disease condition.

When the sample to be detected or the target nucleic acid is RNA, prior to carrying out the method described herein, a nucleic acid amplification reaction can be carried out to obtain complementary DNA (cDNA) using the RNA as a template. The nucleic acid amplification reaction can comprise a reverse transcription reaction. The reverse transcription reaction is described in, for example, Joseph Sam-brook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) , which is herein incorporated by reference in its entirety.

The pathogen may be archaea, bacteria, viruses (or viroids) , fungi, yeasts, plants, or protozoa. In some cases, a presence (or absence) of a particular nucleic acid sequence may indicate that the cell is associated with a pathogen or the subject in which the cell is obtained from has the pathogen. Virus (or viroid) pathogens may comprise hepatitis B, adenovirus, papillomavirus, poxvirus, herpesvirus (e.g., herpes simplex virus) , varicella zoster virus, Epstein-Barr virus, cytomegalovirus, new coronavirus, acute respiratory syndrome coronavirus 2, respiratory syncytial virus, Epstein-Barr virus, hepatitis virus, human immunodeficiency virus (HIV) , Human T-cell lymphotropic virus type 1 (HTLV-1) , influenza virus (influenza virus A, influenza virus B, and/or influenza virus C) , Dengue virus, hepatitis C virus, hepatitis E virus, ebolavirus, lyssavirus, West Nile virus, respiratory syncytial virus (RSV) , parainfluenza virus (PIV) , human metapneumovirus (hMPV) , human rhinovirus (HRV) , severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) , middle east respiratory syndrome coronavirus (MERS-CoV) , measles virus, or polio virus. Bacteria pathogens may comprise Streptococcus pyogenes, coliform, Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Clostridium difficile, Mycobacterium tuberculosis, Bordetella pertussis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Legionella pneumophila, Neisseria meningitidis, Listeria monocytogenes, Borrelia burgdorferi, Vibrio cholerae, Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Campylobacter, Vibrio parahaemolyticus, Bacillus cereus, or Bacillus anthracis. A pathogen may comprise a parasite. Nonlimiting examples of a parasite comprises a protozoan, a helminth, or an ectoparasite. Protozoa are microscopic, one-celled organisms that can be free-living or parasitic in nature. Protozoa pathogens may comprise four groups based on their mode of movement. Protozoa pathogens may comprise Sarcodina (ameba, e.g., Entamoeba) , Mastigophora (flagellates, e.g., Giardia, Leishmania) , Ciliophora (ciliates, e.g., Balantidium) , and Sporozoa (e.g., Plasmodium, Cryptosporidium) . In some embodiments, Plasmodia comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale. Helminths are large, multicellular organisms that can be either free-living or parasitic in nature. Nonlimiting examples of helminths can include, but are not limited to, flatworms (also called as platyhelminths, e.g., trematodes and cestodes) , thorny-headed worms (e.g., acanthocephalins) , and roundworms (also called as nematodes) . Nonlimiting examples of ectoparasites can include blood-sucking arthropods such as mosquitoes, ticks, fleas, lice, and mites. Pathogenic yeasts may comprise Trichophyton, Microsporum, Epidermophyton, Trichophyton rubrum, Epidermophyton floccosum, Aspergillus, Histoplasma capsulatum, Coccidioides, Blastomyces, Cryptococcus neoformans, Cryptococcus gattii, Candida (C. ) albicans, C. glabrata, C. tropicalis, C. stellatoidea, C. glabrata, C. krusei, C. parapsilosis, C. guilliermondii, C. viswanathii, C. lusitaniae, or Rhodotorula mucilaginosa.

A pathogen may cause or be associated with an infectious disease. The infectious disease may comprise AIDS/HIV (Acquired Immune Deficiency Syndrome) , Amebiasis, Anthrax, Asbestosis, Asthma, Avian Influenza (Bird flu) , Babesiosis, Bird flu (Avian influenza) , Botulism, Bronchiectasis, Bronchitis, Brucellosis, Campylobacter infection, Chancroid, Chickenpox (Varicella) , Chlamydia infections, Cholera, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD) , Ciguatera Fish Poisoning, Coccidioidomycosis, Colorado Tick Fever, Common Cold, COVID-19 -Coronavirus, Croup, Cryptosporidiosis, Cystic Fibrosis, Cysticercosis, Dengue Fever, Diphtheria, Domoic Acid Poisoning (Amnesic Shellfish Poisoning) , E. coli Infections, Ebola Virus (see also Viral Hemorrhagic Fever) , Ehrlichiosis, Gastroenteritis, Viral, German Measles (Rubella) , Giardia Infection, Glanders, Gonococcal Infection (Gonorrhea) , Gonorrhea, Haemophilus Influenzae Serotype B Disease (Hib) , Hand-Foot-and-Mouth Disease, Hantavirus, Hantavirus Infections, Hepatitis A, Hepatitis B, Hepatitis C, Human Immunodeficiency Virus (HIV/AIDS) , Idiopathic Pulmonary Fibrosis (IPF) , Influenza, Influenza (Flu) , Lassa Fever (see also Viral Hemorrhagic Fever) , Legionellosis (Legionnaire’s disease) , Leprosy (Hansen’s Disease) , Leptospirosis, Listeriosis, Long COVID, Lung Cancer, Lymphogranuloma Venereum (LGV) , Malaria, Marburg Virus Hemorrhagic Fever (see also Viral Hemorrhagic Fever) , Measles, Melioidosis, Meningitis, Meningococcal disease, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) , Monkeypox (guidance for providers) , Mumps, Non-Gonococcal Urethritis, Norovirus infection (Norwalk and Norwalk-like virus infection) , Novel Coronavirus (COVID-19) , Pandemic Flu, Paralytic Shellfish Poisoning, Pertussis, Pertussis (Whooping Cough) , Plague, Pleurisy, Pneumococcal Disease, Pneumonia, Polio, Psittacosis, Pulmonary Embolism, Pulmonary Hypertension, Rabies, Relapsing Fever, Respiratory Syncytial Virus (RSV) , Rocky Mountain Spotted Fever, Rubella (German Measles) , Salmonellosis, Sarcoidosis, Scombroid fish poisoning, Shigellosis, Sleep Apnea, Smallpox, Spirometry, Sudden Infant Death Syndrome (SIDS) , Syphilis, Tetanus, Toxoplasmosis, Trichinosis (Trichinellosis) , Tuberculosis, Tuberculosis (TB) , Tularemia, Typhoid fever, Typhus, Varicella (Chickenpox) , Viral Gastroenteritis and Norovirus, West Nile Virus, Whooping cough (Pertussis) , Yellow Fever, Yersiniosis (Yersinia enterocolitica) , Zika Virus, human respiratory syncytial virus, mycoplasma pneumoniae, or a combination thereof.

The infectious disease may comprise a respiratory disease. The respiratory disease may comprise Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD) , Common Cold, COVID-19 -Coronavirus, Croup, Cystic Fibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis (IPF) , Influenza, Long COVID, Lung Cancer, Pandemic Flu, Pertussis, Pleurisy, Pneumonia, Pulmonary Embolism, Pulmonary Hypertension, Respiratory Syncytial Virus (RSV) , Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS) , Tuberculosis, human respiratory syncytial virus, mycoplasma pneumoniae, or a combination thereof. In some cases, the target nucleic acid (or a sample) may comprise a sequence of influenza A virus, influenza B virus, human respiratory syncytial virus, mycoplasma pneumoniae, coronavirus, or a combination thereof.

The target nucleic acid may be derived from a cell associated with a disease (or obtained from a subject having or suspected of having the disease) . In some cases, a presence (or absence) of a particular nucleic acid sequence may indicate that the cell is associated with a disease or risk thereof or the subject in which the cell is obtained from has the disease or risk thereof.

The disease may be a cancer, a genetic disorder, an infectious disease (as described herein) , or a combination thereof. For example, a cancer, in some instances, can comprise malignant cell type, such as those found in a solid tumor or a hematological tumor. In some cases, a cancer can comprise a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, gallbladder, skin, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. In some cases, a cancer can comprise hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. In some cases, a cancer can also comprise carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung) , cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer) , pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, gallbladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, renal cell carcinoma, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, head and neck squamous cell carcinoma, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanomas, nodular melanomas, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL) ; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s macroglobulinemia) , chronic lymphocytic leukemia (CLL) , acute lymphoblastic leukemia (ALL) , Hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML) and chronic myeloblastic leukemia.

In some cases, a cancer can comprise neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's ; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some cases, the target nucleic acid does not comprise exons 18-21 of human EGFR. In some cases, the target nucleic acid does not comprise a sequence of human EGFR. In some cases, the target nucleic acid does not comprise a sequence of EGFR. In some cases, the target nucleic acid does not comprise a sequence associated with EGFR-associated disease condition (such as a cancer) .

The target nucleic acid may comprise cell-free nucleic acid molecules, such as cell-free DNA or cell-free RNA. Cell-free nucleic acid molecules may be fetal in origin (via fluid taken from a pregnant subject) or may be derived from tissue of the subject itself.

Various methods

Provided herein are various methods and compositions.

A method may comprise 300 of FIG. 3 for analyzing target nucleic acid (s) and the composition (s) for practicing the method. In step 301, upstream primer 3011 and downstream primer 3012 are used to hybridize and/or amplify target nucleic acid 3014. Target nucleic acid 3014 may comprise its amplification product generated with upstream primer 3011 and downstream primer 3012. Mediator probe 3013 comprises a second template-binding nucleotide sequence 30133 complementary to a sequence of target nucleic acid 3014. Mediator probe 3013 comprises first template-binding nucleotide sequence 30132 that is configured to not hybridize to target nucleic acid 3014. Mediator probe 3013 additionally comprises label moiety 30131 and quencher moiety 30134. Between first template-binding nucleotide sequence 30132 and second template-binding nucleotide sequence 30133, mediator probe 3013 can additionally comprise another quencher moiety (the same or different from quencher moiety 30134; not shown) . The binding of mediator probe 3013 and upstream primer 3011 triggers the cleavage of mediator probe 3013, generating cleavage fragment 3021 (having or not having the quencher moiety that is not shown) , with or without an enzyme described herein (not shown) . The cleavage reaction may additionally comprise the extension of a nascent chain (not shown) of upstream primer 3011 in a nucleic acid amplification described herein using nucleotides described herein (not shown) . The reactions of step 301 can generate a detectable signal 3015. In step 302, cleavage fragment 3021 hybridize to template sequence for hybridization of the reporter probe 30221 of reporter probe 3022, generating hybridized product 3023. Reporter probe 3022 comprises quencher moiety 30223, forming a label/quencher moiety pair with label moiety 30131. The hybridization reaction may further generate detectable signal 3024. In step 303, a nucleic acid amplification using cleavage fragment 3021 as a primer and reporter probe 3022 as a template extends a polynucleotide 30311 based on base-pairing to template sequence for extension of the reporter probe 30222, forming a nascent chain 30312 and a duplex comprising reporter probe 3022 and nascent chain 30312. The reactions in step 303 can generate detectable signal 3032.

In method 300, quencher moieties 30314 and 30223 are different quencher moieties. In a variation of method 300, quencher moieties 30314 and 30223 are a same quencher moiety. In a variation of method 300, quencher moiety 30134 and label moiety 30131 may form a label/quencher moiety pair. In a variation of method 300, quencher moiety 30134 and label moiety 30131 may not form a label/quencher moiety pair. In a variation of method 300, quencher moiety 30223 and label moiety 30131 may form a label/quencher moiety pair. In a variation of method 300, quencher moiety 30223 and label moiety 30131 may not form a label/quencher moiety pair.

In a variation of method 300, label moiety 30131 is replaced with a quencher moiety, and quencher moieties 30314 and 30223 are replaced with a same or different label moieties. The label and quencher moieties in this variation of method 300 can form a label/quencher pair. In another variation of this variation of method 300, label and quencher moieties may not form a label/quencher pair. The label moieties on mediator probe 3013 and reporter probe 3022 in these variations of method 300 are the same label moiety or different label moieties.

In a variation of method 300, step 301 comprises detecting detectable signal 3015. In a variation of method 300, step 302 comprises detecting detectable signal 3024. In a variation of method 300, step 303 comprises detecting detectable signal 3032. In a variation of method 300, step 301 comprises not detecting detectable signal 3015. In a variation of method 300, step 302 comprises not detecting detectable signal 3024. In a variation of method 300, step 303 comprises not detecting detectable signal 3032. In a variation of method 300, step 301 comprises detecting detectable signal 3015, and step 303 comprises detecting detectable signal 3032. In a variation of method 300, step 301 comprises detecting detectable signal 3015, step 302 comprises not detecting detectable signal 3024, and step 303 comprises detecting detectable signal 3032. In a variation of method 300, step 301 comprises detecting detectable signal 3015, step 302 comprises detecting detectable signal 3024, and step 303 comprises detecting detectable signal 3032. In a variation of method 300, step 301 comprises not detecting detectable signal 3015, step 302 comprises not detecting detectable signal 3024, and step 303 comprises detecting detectable signal 3032. Step 301 or 302 can comprise quantification or qualitative identification or sequencing as described herein. Step 303 can comprise melting curve analysis as described herein. Step 303 can comprise sequencing as described herein.

A method may comprise 400 of FIG. 4 for analyzing target nucleic acid (s) and the composition (s) for practicing the method. In step 401, upstream primers 4011/4011a and downstream primers 4012/4012a are used to hybridize and/or amplify target nucleic acids 4014/4014a, respectively. Target nucleic acids 4014/4014a may comprise their amplification products generated with upstream primers 4011/4012a and downstream primers 4012/4012a, respectively. Mediator probes 4013/4013a comprise second template-binding nucleotide sequences 40133/40133a that can hybridize to complementary sequences of target nucleic acids 4014/4014a, respectively. Mediator probes 4013/4013a comprise first template-binding nucleotide sequences 40132/40132a that are configured to not hybridize to target nucleic acids 4014/4014a, respectively. Mediator probes 4013/4013a additionally comprise label moieties 40131/40131a and quencher moieties 40134/40134a, respectively. Between first template-binding nucleotide sequences 40132/40132a and second template-binding nucleotide sequences 40133/40133a, mediator probes 4013/4013a can additionally comprise another quencher moiety (the same or different from quencher moieties 40134/40134a; not shown) , respectively. The binding of mediator probes 4013/4013a and upstream primers 4011/4011a triggers the cleavage of mediator probes 4013/4013a, generating cleavage fragments 4021/4021a (having or not having the quencher moiety that is not shown) , with or without an enzyme described herein (not shown) , respectively. The cleavage reaction may additionally comprise the extension of a nascent chain (not shown) of the upstream primers 4011/4011a in a nucleic acid amplification described herein using nucleotides described herein (not shown) . The reactions of step 401 can generate detectable signals 4015/4015a. In step 402, cleavage fragments 4021/4021a hybridize to template sequences for hybridization of the reporter probe 40221/40221a of reporter probe 4022, generating hybridized products 4023/4023a, respectively. Reporter probe 4022 comprises a quencher moiety 40223, forming a label/quencher moiety pair with label moieties 40131/40131a, respectively. The hybridization reaction may further generate detectable signals 4024/4024a. In step 403, a nucleic acid amplification using cleavage fragments 4021/4021a as a primer and reporter probe 4022 as a template extends polynucleotides 40311/40311a based on base-pairing to template sequence for extension of the reporter probe 40222 including sequence oftemplate sequences for hybridization of the reporter probe 40221 and/or 40221a, forming nascent chains 40312/40312a and duplexes comprising reporter probe 4022 and nascent chains 40312/40312a, respectively. The reactions in step 403 can generate detectable signals 4032/4032a.

In method 400, quencher moieties 40134/40134a and 40223 are different quencher moieties. In a variation of method 400, quencher moieties 40134/40134a and 40223 are a same quencher moiety. In a variation of method 400, quencher moieties 40134/40134a and label moieties 40131/40131a may form a label/quencher moiety pair. In a variation of method 400, quencher moieties 40134/40134a and label moieties 40131/40131a may not form a label/quencher moiety pair, respectively. In a variation of method 400, quencher moiety 40223 and label moieties 40131/40131a may form a label/quencher moiety pair, respectively. In a variation of method 400, quencher moiety 40223 and label moieties 40131/40131a may not form a label/quencher moiety pair, respectively.

In a variation of method 400, label moieties 40131/40131a are replaced with a quencher moiety, and quencher moieties 40314/40134a and 40223 are replaced with a same or different label moieties. The label and quencher moieties in this variation of method 400 can form a label/quencher pair. In another variation of this variation of method 400, label and quencher moieties may not form a label/quencher pair. The label moieties on mediator probe 40131/40131a and reporter probe 4022 in these variations of method 400 are the same label moiety or different label moieties.

In a variation of method 400, step 401 comprises detecting detectable signals 4015/4015a. In a variation of method 400, step 402 comprises detecting detectable signals 4024/4024a. In a variation of method 400, step 403 comprises detecting detectable signals 4032/4032a. In a variation of method 400, step 401 comprises not detecting detectable signals 4015/4015a. In a variation of method 400, step 402 comprises not detecting detectable signals 4024/4024a. In a variation of method 400, step 403 comprises not detecting detectable signals 4032/4032a. In a variation of method 400, step 401 comprises detecting detectable signals 4015/4015a, and step 403 comprises detecting detectable signals 4032/4032a. In a variation of method 400, step 401 comprises detecting detectable signals 4015/4015a, step 402 comprises not detecting detectable signals 4024/4024a, and step 403 comprises detecting detectable signals 4032/4032a. In a variation of method 400, step 401 comprises detecting detectable signals 4015/4015a, step 402 comprises detecting detectable signals 4024/4024a, and step 403 comprises detecting detectable signals 4032/4032a. In a variation of method 400, step 401 comprises not detecting detectable signals 4015/4015a, step 402 comprises not detecting detectable signals 4024/4024a, and step 403 comprises detecting detectable signals 4032/4032a.

Step 401 or 402 can comprise quantification or qualitative identification or sequencing as described herein. Step 403 can comprise melting curve analysis as described herein. Step 403 can comprise sequencing as described herein.

A method may comprise 500 of FIG. 5 for analyzing target nucleic acid (s) and the composition (s) for practicing the method. In step 501, upstream primers 5011/5011a and downstream primers 5012/5012a are used to hybridize and/or amplify target nucleic acids 5014/5014a, respectively. Target nucleic acids 5014/5014a may comprise their amplification products generated with upstream primers 5011/5012a and downstream primers 5012/5012a, respectively. Mediator probes 5013/5013a comprise second template-binding nucleotide sequences 50133/50133a that can hybridize to a complementary sequence of target nucleic acids 5014/5014a, respectively. Mediator probes 5013/5013a comprise first template-binding nucleotide sequence 50132/50132a that are configured not to hybridize to target nucleic acids 5014/5014a, respectively. Mediator probes 5013/5013a additionally comprise label moieties 50131/50131a (with different maximal excitation or emission wavelengths) and quencher moieties 50134/50134a, respectively. Between first template-binding nucleotide sequences 50132/50132a and second template-binding nucleotide sequences 50133/50133a, mediator probes 5013/5013a can additionally comprise another quencher moiety (the same or different from quencher moieties 50134/50134a; not shown) . The binding of mediator probes 5013/5013a and upstream primers 5011/5011a triggers the cleavage of mediator probes 5013/5013a, generating cleavage fragments 5021/5021a (having or not having the quencher moiety that is not shown) , with or without an enzyme described herein (not shown) . The cleavage reaction may additionally comprise the extension of a nascent chain (not shown) of upstream primers 5011/5011a in a nucleic acid amplification described herein using nucleotides described herein (not shown) . The reactions of step 501 can generate detectable signals 5015/5015a. In step 502, cleavage fragments 5021/5021a hybridize to template sequences for hybridization of the reporter probe 50221/50221a of reporter probes 5022/5022a, generating hybridized products 5023/5023a. Reporter probes 5022/5022a comprise a quencher moieties 50223/50223a, forming a label/quencher moiety pair with label moieties 50131/50131a, respectively. The hybridization reaction may further generate detectable signals 5024/5024a. In step 503, a nucleic acid amplification using cleavage fragments 5021/5021a as a primer and reporter probes 5022/5022a as a template extends polynucleotides 50311/50311a based on base-pairing to template sequences for extension of reporter probes 50222/50222a including sequences oftemplate sequence for hybridization of the reporter probe 50221a/50221a, forming nascent chains 50312/50312a and duplexes comprising reporter probe 5022/5022a and nascent chain 50312/50312a, respectively. The reactions in step 503 can generate detectable signals 5032/5032a.

In method 500, label moieties 50131 and 50131a have different maximal emission wavelengths. In a variation of method 500, quencher moieties 50134, 50134a, 50223, and 50223a have different maximal emission wavelengths or different maximal excitation wavelengths. Thus, in these variations of method 500, different label/quencher moiety pairs with different maximal emission wavelengths or different maximal excitation wavelengths can be generated, thereby generating different or differentiable detectable signals for each target nucleic acids. In method 500, label moieties 50131 and 50131a have the same maximal emission wavelengths. In a variation of method 500, any of quencher moieties 50134, 50134a, 50223, and/or 50223a have the same maximal emission wavelengths maximal excitation wavelengths.

In a variation of method 500, label moieties 50131/50131a are replaced with a quencher moiety, and quencher moieties 50314/50134a and 50223/50223a are replaced with a same or different label moieties. The label and quencher moieties in this variation of method 500 can form a label/quencher pair. In another variation of this variation of method 500, label and quencher moieties may not form a label/quencher pair. The label moieties on mediator probe 50131/50131a and reporter probe 5022/5022a in these variations of method 500 are the same label moiety or different label moieties.

In a variation of method 500, step 501 comprises detecting detectable signals 5015/5015a. In a variation of method 500, step 502 comprises detecting detectable signals 5024/5024a. In a variation of method 500, step 503 comprises detecting detectable signals 5032/5032a. In a variation of method 500, step 501 comprises not detecting detectable signals 5015/5015a. In a variation of method 500, step 502 comprises not detecting detectable signals 5024/5024a. In a variation of method 500, step 503 comprises not detecting detectable signals 5032/5032a. In a variation of method 500, step 501 comprises detecting detectable signals 5015/5015a, and step 503 comprises detecting detectable signals 5032/5032a. In a variation of method 500, step 501 comprises detecting detectable signals 5015/5015a, step 502 comprises not detecting detectable signals 5024/5024a, and step 503 comprises detecting detectable signals 5032/5032a. In a variation of method 500, step 501 comprises detecting detectable signals 5015/5015a, step 502 comprises detecting detectable signals 5024/5024a, and step 503 comprises detecting detectable signals 5032/5032a. In a variation of method 500, step 501 comprises not detecting detectable signals 5015/5015a, step 502 comprises not detecting detectable signals 5024/5024a, and step 503 comprises detecting detectable signals 5032/5032a. Step 501 or 502 can comprise quantification or qualitative identification or sequencing as described herein. Step 503 can comprise melting curve analysis as described herein. Step 503 can comprise sequencing as described herein.

A method may comprise method 600 of FIG. 6 for analyzing target nucleic acid (s) and the composition (s) for practicing the method. In step 601, upstream primer 6011 and downstream primer 6012 are used to hybridize and/or amplify target nucleic acid 6014. Target nucleic acid 6014 may comprise its amplification product generated with upstream primer 6011 and downstream primer 6012. Invasion probe 6013 comprises a template-binding nucleotide sequence 60133 complementary to a sequence of target nucleic acid 6014. Invasion probe 6013 additionally comprises label moiety 60131 and quencher moiety 60132. Invasion probe 6013 can additionally comprise another quencher moiety coupled to a nucleotide of template-binding nucleotide sequence 60133, or that quencher moiety 60132 is coupled to a nucleotide of template-binding nucleotide sequence 60133 but not at the terminal end (such as the 3’ end of invasion probe 6013) . The binding of invasion probe 6013 and upstream primer 6011 triggers the cleavage of invasion probe 6013, with or without an enzyme described herein (not shown) , separating the label-quencher moiety pair formed with label moiety 60131 and quencher moiety 60132. The reactions in step 601 can generate detectable signal 6015.

In some cases, the invasion probe may have a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 18, 19, 22, or 23.

Methods may comprise 701, 702, and/or 703 of FIG. 7 for analyzing at least 2 or more than 2 target nucleic acids. Method 701 comprises combining methods 300 as disclosed herein (such as those depicted in FIG. 3; referred to as sub-method 300 in method 701) and 600 disclosed herein (such as those depicted in 600; referred to as sub-method 600 in method 701) . In method 701, target nucleic acids 3014 and 6014 (as described using FIGs. 3 and 6) have different nucleic acid sequences or are two different template nucleic acids or the amplification products thereof. Label moieties 30131 and 60131 are different label moieties or can generate different differentiable detectable signals (e.g., detectable signals 3015, 3024, and/or 3032 are differentiable from detectable signal 6015) . In method 701, step 601 of sub-method 600 is carried out simultaneously with step 301 of sub-method 300. In this step, detectable signal 6015 is first detected. Subsequently, steps 302 and 303 of sub-method 300 are carried out according to the methods described herein (such as those described in FIG. 3) , and detectable signal 3032 (and/or 3024) is detected.

Method 702 comprises combining methods 400 as disclosed herein (such as those depicted in FIG. 4; referred to as sub-method 400 in method 702) and 600 disclosed herein (such as those depicted in 600; referred to as sub-method 600 in method 702) . In method 702, target nucleic acids 4014, 4014a, and 6014 (as described using FIGs. 4 and 6) have different nucleic acid sequences or are three different template nucleic acids or the amplification products thereof. Label moieties 40131, 40131a, and 60131 are different label moieties or can generate different differentiable detectable signals (e.g., detectable signals 4015, 4015a, 4024, 4024a, 4032, and/or 4032a are differentiable from detectable signal 6015) . In method 702, step 601 of sub-method 600 is carried out simultaneously with step 401 of sub-method 400. In this step, detectable signal 6015 is first detected. Subsequently, steps 402 and 403 of sub-method 400 are carried out accordingly the methods described herein (such as those described in FIG. 4) , and detectable signals 4032 and 4032a (and/or 4024 and 4024a) are detected.

Method 703 comprises combining methods 500 as disclosed herein (such as those depicted in FIG. 5; referred to as sub-method 500 in method 703) and 600 disclosed herein (such as those depicted in 600; referred to as sub-method 600 in method 702) . In method 702, target nucleic acids 5014, 5014a, and 6014 (as described using FIGs. 5 and 6) have different nucleic acid sequences or are three different template nucleic acids or the amplification products thereof. Label moieties 50131, 50131a, and 60131 are different label moieties or can generate different differentiable detectable signals (e.g., detectable signals 5015, 5015a, 5024, 5024a, 5032, and/or 5032a are differentiable from detectable signal 6015) . In method 703, step 601 of sub-method 600 is carried out simultaneously with step 501 of sub-method 500. In this step, detectable signal 6015 is first detected. Subsequently, steps 502 and 503 of sub-method 500 are carried out accordingly the methods described herein (such as those described in FIG. 5) , and detectable signals 5032 and 5032a (and/or 5024 and 5024a) are detected.

The methods described herein for detecting multiple target nucleic acids using different methods for generating different detectable signal in different steps, such as those described in methods 701-703, can have various beneficial advantages. The methods can be used to determine if a sample is associated with a specific condition (s) by determining if the sample comprises a specific target nucleic acid (s) . In some cases, the sample may be suspected of being associated with a disease condition among various disease conditions. For example, the sample may be obtained from a subject showing common symptoms of various disease conditions. In one example (for illustrating the beneficial advantage of the method) , the subject may have common symptoms associated with respiratory diseases as described herein. To determine if the subject is associated with one specific form of the respiratory infection (such as Covid infection by a specific Covid virus strain) , methods 701-703 may be adopted for using sub-method 601 to determine whether the sample comprises Covid, flu, or cold pathogen. Once determined if the sample comprises Covid virus, sub-methods 300, 400, and 500 will be adopted to determine which specific Covid virus strain the sample comprises. By using such methods, wastes of reagents can be minimized. For example, subsequent steps 302-303, 402-403, or 502-503 (or including steps 301, 401, and/or 501) that specifically designed to analyze fly and cold pathogen strains need not be carried out. In another example, when determining whether the sample is associated with a disease condition among various disease conditions, only one specific disease condition requires analysis steps 302-303, 402-403, or 502-503 (or steps 301, 401, and/or 501) , while for other disease conditions, sub-method 601 is sufficient for determining if the associated pathogen is present. In another example, sub-method 601 may be adopted for quality control purposes. For example, sub-method 601 may be adopted determining if a control target nucleic acid is present. If the control target nucleic acid is not determined to be present, the quality of the nucleic acids in the sample may not be suitable for subsequent analyses (such as the nucleic acid may have been degraded) . In this cases, once determined if the sample does not pass the quality control analysis, steps 302-303, 402-403, or 502-503 do not need to be carried out.

In the methods described herein, when contacting two nucleic acid molecules for hybridization, the contacting may comprise subjecting the two nucleic acid molecules in conditions sufficient for hybridization. For example, two single-stranded nucleic acid molecules having substantial complementary sequences can hybridize under appropriate hybridization conditions. Such hybridization conditions may comprise temperature, pH value, composition and ionic strength of the hybridization buffer, or any combinations thereof; and may be determined according to the length and GC content of the two complementary nucleic acid molecules. For example, low stringency hybridization conditions may be employed when the two complementary nucleic acid molecules are relatively short in length and /or have relatively low GC content. When the length of the two complementary nucleic acid molecules is relatively long and /or the GC content is relatively high, high stringency hybridization conditions can be used. Such hybridization conditions are described in, for example, Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) ; and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y. (1999) , which is herein incorporated by reference in its entirety.

In the methods described herein, when subject a nucleic acid molecule for nucleic acid amplification reaction, the subjecting may comprise subjecting the nucleic acid molecule in a condition sufficient for the nucleic acid amplification reaction, as described herein. Such conditions can be determined by conventional methods, such as those described in Sambrook et al. Such conditions can permit the amplification of the nucleic acid molecule, using the methods described herein.

In the methods described herein, when cleaving a nucleic acid molecule, the cleaving can comprise subjecting the nucleic acid molecule in a condition sufficient for the cleavage of the nucleic acid. Such conditions can permit the nucleic acid molecule being cleaved, by the methods described herein.

Any of the methods, compositions, devices, systems, or reagents can be combined for various methods to achieve any of the beneficial advantages described herein. In some cases, any of the methods, compositions, devices, systems, or reagents can be combined with Faltin et al., Huang et al., Jianping et al., (Heliyon . 2022 Nov 26; 8 (11) : e11856) , U.S. Patent No.: 11,111,522, or U.S. Patent No.: 10,519,489, each of which is incorporated in its entirety, for achieving any of the beneficial advantages described herein. In some cases, when using the mediator probe and/or upstream/downstream primers described herein to hybridize to the target nucleic acid or generating the cleaved fragment of the mediator probe as described herein, the method does not use an additional probe to hybridize to the target nucleic acids or to facilitate the generation of the cleaved fragment of the mediator probe. For example, the method described herein may not use an invasion, such as those described in Jianping et al. Without using such additional probes, the methods described herein have the beneficial advantages of reducing the amounts of reagents used.

Biological sample

A biological sample refers to any sample derived from a subject or specimen from the subject. A biological sample may comprise the target nucleic acid. In some instances, the method may comprise contacting the mediator probe, reporter probe, enzymes, nucleotides, upstream/downstream primers, or a combination thereof with biological sample comprising the target nucleic acid, without having to extracting, isolating, purifying, fractionating the target nucleic acid from the biological sample.

The subject may comprise a pathogen or an animal suspected having or suspected of having a disease. The animal may be a human. The biological sample can be a fluid, tissue, collection of cells, hair, or feces obtained from the animal. The fluid can be blood, saliva, urine, or sweat. The tissue can be from an organ, a tissue, a mass of cellular material, or a tumor. The biological sample can be a cellular sample or cell-free sample. A biological sample may comprise the target nucleic acid. Further, samples may be extracted from variety of animal fluids containing cell free sequences, including but not limited to blood, serum, plasma, vitreous, sputum, urine, tears, perspiration, saliva, semen, mucosal excretions, mucus, spinal fluid, amniotic fluid, or lymph fluid.

Nucleotide

Provided herein are nucleotides. The nucleotide may be used in the methods described herein. A target nucleic acid, a mediator probe (or a cleaved fragment thereof) , a reporter probe, or a combination thereof may comprise the nucleotide described herein. In some cases, the nucleotide may be used in the nucleic acid amplification reaction to be incorporated into an amplification or derivative product of the target nucleic acid, a mediator probe (or a cleaved fragment thereof) , a reporter probe, or a combination thereof. In other cases, the nucleotide may be incorporated into the target nucleic acid, a mediator probe (or a cleaved fragment thereof) , a reporter probe, or a combination thereof by chemical methods.

A nucleotide may comprise a nucleotide or nucleotide analog. The nucleotide may be naturally occurring or non-naturally occurring. The nucleotide may include a canonical base or a non-canonical base. The nucleotide may comprise an alternative base. The nucleotide may comprise or be coupled to a label moiety or quencher moiety. The nucleotide may include a modified polyphosphate chain (such as a triphosphate coupled to a fluorophore) . The nucleotide may be terminated (e.g., reversibly terminated) . Nonstandard nucleotides, nucleotide analogs, and/or modified analogs may comprise diaminopurine, a 5-methylcytosine, a 5-hydroxymethyl cytosine, a deoxyhypoxanthine, inosine, 1- (2'-deoxy-β-D-ribofuranosyl ) -3-nitrate pyrrole, 5-nitroindole, locked nucleic acid (LNA) , 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5’ -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v) , wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v) , 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2, 6-diaminopurine, ethynyl nucleotide bases, 1-propynyl nucleotide bases, azido nucleotide bases, phosphoroselenoate nucleic acids and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety.

Nucleic acids may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide) , sugar moiety or phosphate backbone. Nucleic acids may also contain amine -modified groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS) .

In some cases, nucleotides may be used to incorporate a quencher moiety into a nucleic acid described herein. For example, interaction between two modified nucleotides, isoguanine (iso-dG) and 5′-methylisocytosine (iso-dC) may be used to incorporate a quencher moiety into an extension or amplification product of a mediator probe, reporter probe, target nucleic acid, or any combination thereof. One primer is synthesized with an iso-dC residue as the 5’ -terminal nucleotide and a label moiety (such as a fluorophore) at the 5’ -end; the second primer is unlabeled. During nucleic acid amplification (such as PCR) , the labeled primer is annealed and extended, becoming part of the template used during the next round of amplification. During subsequent rounds of amplification, iso-dGTP, which is available in the nucleotide mix as quencher-labeled iso-dGTP, pairs specifically with iso-dC and is incorporated. The close proximity of the quencher and the label moiety on the opposite strand quenches the fluorescent signal.

Compositions

Provided herein compositions comprising any of the sample or biological sample, target nucleic acid, upstream primer, downstream primer, mediator probe, cleaved fragment of the mediator probe, reporter probe, enzyme, nucleotide, label/quencher moiety, or a combination thereof; for practicing the methods as described herein.

In some cases, the composition may comprise the sample or biological sample upstream primer, downstream primer, mediator probe

In some cases, the composition may comprise a probe set. The probe set may comprise at least one mediator probe. The probe set may comprise at least one reporter probe. The probe set may comprise at least one upstream primer. The probe set may comprise at least one downstream probe. The probe set may comprise at least one mediator probe and at least one reporter probe. The probe set may comprise at least one mediator probe and at least one upstream primer. The probe set may comprise at least one mediator probe and at least one downstream primer. The probe set may comprise at least one mediator probe, at least one reporter probe, at least one upstream primer. The probe set may comprise at least one mediator probe, at least one reporter probe, at least one downstream primer. The probe set may comprise at least one mediator probe, at least one reporter probe, at least one downstream primer. The probe set may comprise at least one mediator probe, at least one reporter probe, at least one upstream primer, and at least one downstream primer. The probe set may comprise at least one reporter probe and at least one upstream primer. The probe set may comprise at least one reporter probe and at least one downstream primer. The probe set may comprise at least one reporter probe, at least one reporter probe, at least one downstream primer. The probe set may comprise at least one upstream primer and at least one downstream primer.

The composition may comprise the probe set described herein; and at least one sample or biological sample, at least one target nucleic acid, at least one enzyme, at least one nucleotide, or any combination thereof. The composition may comprise the probe set and at least one sample or biological sample. The composition may comprise the probe set and at least one target nucleic acid. The composition may comprise the probe set and at least one enzyme. The composition may comprise the probe set and at least one nucleotide. The composition may comprise the probe set, at least one sample or biological sample, and at least one enzyme. The composition may comprise the probe set and at least one sample or biological sample, and at least one nucleotide. The composition may comprise the probe set, at least one sample or biological sample, at least one enzyme, and at least one nucleotide. The composition may comprise the probe set, at least one target nucleic acid, at least one enzyme, and at least one nucleotide. The composition may comprise the sample or biological sample and the enzyme. The composition may comprise the sample or biological sample and the nucleotide. The composition may comprise the sample or biological sample, the nucleotide, and the enzyme. The composition may comprise the target nucleic acid and the enzyme. The composition may comprise the target nucleic acid and the nucleotide. The composition may comprise the target nucleic acid, the nucleotide, and the enzyme.

The composition may further comprise control primers or probes. Control primers or probes may be used as a positive or negative control for the methods described herein. Control primers or probes may be used as identification or quantification purposes. Control primers or probes may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more primers or probes. Control primers or probes may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 primers or probes. Control primers or probes may have sequences that are 50 %, 60 %, 70 %, 80 %, 90 %, or 100 %identical or complementary to the target nucleic acids. Control primers or probes may have sequences that are not identical or complementary to the target nucleic acids. Sequence identity or complementary of the control primers or probes can depend on the purposes (whether it is a positive or negative control or is configured to hybridize or not hybridize to a target nucleic acid) . Control primers or probes may have a length of about 5, 10, 20, 30, 40, 50 100, 200, 500 nucleotides.

The composition may comprise a probe comprising a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 1-25. The composition may comprise a probe comprising a sequence that is at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 %, or 100 %sequence identity to any one of SEQ ID NOs: 4-25.

The composition may further comprise buffer or co-factors (ions or detergents) for practicing the methods described herein, such as those for hybridization or nucleic acid amplification. In some cases, the composition may further comprise buffer or co-factors (ions or detergents) for storage of any composition, probe, primer, nucleic acid, enzyme, nucleotide, or any combination thereof, as described herein. When generating any of the compositions described herein, any components of the compositions can be added simultaneously or sequentially.

The composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more mediator probes. The composition may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 mediator probes.

The composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more upstream primers. The composition may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 upstream primers.

The composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more downstream primers. The composition may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 downstream primers.

The composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more upstream/downstream primer pairs (for hybridizing/amplifying a particular target nucleic acid or sequence thereof) . The composition may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 upstream/downstream primer pairs.

The composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more reporter probes. The composition may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 reporter probes.

The composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 or more target nucleic acids. The composition may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 target nucleic acids.

The composition may have a volume of at least about 1 picoliter (pL) , 10 pL, 100 pL, 1 nanoliter (nL) , 10 nL, 100 nL, 1 microliter (μL) , 10 μL, 100 μL, 1 milliliter (mL) , 10 mL, 100 mL or more. The composition may have a volume of at most about 1 picoliter (pL) , 10 pL, 100 pL, 1 nanoliter (nL) , 10 nL, 100 nL, 1 microliter (μL) , 10 μL, 100 μL, 1 milliliter (mL) , 10 mL, or 100 mL.

The reaction mixture of the composition may have a volume of at least about 1 picoliter (pL) , 10 pL, 100 pL, 1 nanoliter (nL) , 10 nL, 100 nL, 1 microliter (μL) , 10 μL, 100 μL, 1 milliliter (mL) , 10 mL, 100 mL or more. The reaction mixture of the composition may have a volume of at most about 1 picoliter (pL) , 10 pL, 100 pL, 1 nanoliter (nL) , 10 nL, 100 nL, 1 microliter (μL) , 10 μL, 100 μL, 1 milliliter (mL) , 10 mL, or 100 mL.

The mediator probe within the composition may have a concentration of at least about 1 picomolar (pM) , 10 pM, 100 pM, 1 nanomolar (nM) , 10 nM, 100 nM, 1 micromolar (μM) , 10 μM, 100 μM, 1 millimolar (mM) , 10 mM, 100 mM, 1 molar (M) , 10 M, 100 M or more. The mediator probe within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The reporter probe within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The reporter probe within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The upstream primer within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The upstream primer within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The downstream primer within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The downstream primer within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The enzyme within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The enzyme within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The nucleotide within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The nucleotide within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The target nucleic acid within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The target nucleic acid within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The sample or biological sample within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The sample or biological sample within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

The sample or biological sample within a composition may have a weight of at least about 1 picogram (pg) , 10 pg, 100 pg, 1 nanogram (ng) , 10 ng, 100 ng, 1 microgram (μg) , 10 μg, 100 μg, 1 milligram (mg) , 10 mg, 100 mg or more. The sample or biological sample within a composition may have a weight of at most about 1 picogram (pg) , 10 pg, 100 pg, 1 nanogram (ng) , 10 ng, 100 ng, 1 microgram (μg) , 10 μg, 100 μg, 1 milligram (mg) , 10 mg, or 100 mg.

The buffer of the composition may have a volume of at least about 1 picoliter (pL) , 10 pL, 100 pL, 1 nanoliter (nL) , 10 nL, 100 nL, 1 microliter (μL) , 10 μL, 100 μL, 1 milliliter (mL) , 10 mL, 100 mL or more. The buffer of composition may have a volume of at most about 1 picoliter (pL) , 10 pL, 100 pL, 1 nanoliter (nL) , 10 nL, 100 nL, 1 microliter (μL) , 10 μL, 100 μL, 1 milliliter (mL) , 10 mL, or 100 mL.

The co-factors within the composition may have a concentration of at least about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, 100 M or more. The co-factors within the composition may have a concentration of at most about 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, 1 M, 10 M, or 100 M.

In some instances, a kit may comprise the mediator probes, reporter probes, upstream primers, downstream primers, control primers, devices, enzymes, label moieties, quencher moieties, nucleotides, buffers, co-factors, or any combinations thereof, as disclosed in this disclosure.

Nucleic acid amplification reaction

The terms “amplifying, ” “amplification, ” and “nucleic acid amplification” are used interchangeably and generally refer to generating one or more copies of a nucleic acid or a template, or an extension product of a nucleic acid or a template. Amplification of DNA can comprise generating one or more copies of a DNA molecule, or an extension product of a DNA or a DNA template. Amplification of a nucleic acid may be linear, exponential, or a combination thereof. Amplification of RNA can comprise generating one or more copies DNA copy of the RNA or an extension product of the RNA. Nucleic acid amplification reaction can comprise reverse transcription, primer extension, polymerase chain reaction (PCR) , ligase chain reaction (LCR) , helicase-dependent amplification, asymmetric amplification, rolling circle amplification (RCA) , recombinase polymerase reaction (RPA) , loop mediated isothermal amplification (LAMP) , nucleic acid sequence-based amplification (NASBA) , self-sustained sequence replication (3SR) , and multiple displacement amplification (MDA) . Where PCR is used, any form of PCR may be used, with non-limiting examples that include real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR (ePCR or emPCR) , dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR, and touchdown PCR. Amplification can be conducted in a reaction mixture comprising various components (e.g., mediator probes, reporter probes, primers, target nucleic acids, samples, nucleotides, enzymes, or co-factors) that facilitate the nucleic acid amplification.

Co-factors can comprise magnesium-ion, manganese-ion and isocitrate buffers. Additional examples of such buffers are described in Tabor, S. et al. C.C. PNAS, 1989, 86, 4076-4080 and U.S. Patent Nos. 5,409,811 and 5,674,716, each of which is herein incorporated by reference in its entirety. Useful methods for clonal amplification from single molecules include rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19: 225-232 (1998) , which is incorporated herein by reference) , bridge PCR (Adams and Kron, Method for Performing Amplification of Nucleic Acid with Two Primers Bound to a Single Solid Support, Mosaic Technologies, Inc. (Winter Hill, Mass. ) ; Whitehead Institute for Biomedical Research, Cambridge, Mass., (1997) ; Adessi et al., Nucl. Acids Res. 28: E87 (2000) ; Pemov et al., Nucl. Acids Res. 33: e11 (2005) ; or U.S. Pat. No. 5,641,658, each of which is incorporated herein by reference) , or ligation to bead-based adapter libraries (Brenner et al., Nat. Biotechnol. 18: 630-634 (2000) ; Brenner et al., Proc. Natl. Acad. Sci. USA 97: 1665-1670 (2000) ) ; Reinartz, et al., Brief Funct. Genomic Proteomic 1: 95-104 (2002) , each of which is incorporated herein by reference) . Amplification products from a nucleic acid may be identical or substantially identical.

The methods described herein may comprise using 5’ tail sequences (e.g., sequences that are 5’ to the upstream/downstream primers or mediator probe) for minimizing or eliminating primer dimer formation. For example, the upstream/downstream primers may or may each comprise the 5’ tail sequence (s) . In the initial cycles of the nucleic acid amplification, sequences 3’to the 5’ tail sequence in the upstream or downstream primers may be used for priming/hybridizing the target nucleic acid. Subsequently, primers having only the 5’ tail sequences of the upstream or downstream primers may be used for subsequent cycles of the nucleic acid amplification reaction. In some cases, the primers with only the 5’ tail sequences of the upstream or downstream primers may be in molar excess of the upstream/downstream primers.

For example, the primers with only the 5’ tail sequences of the upstream or downstream primers may be at least about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the upstream or downstream primers. In some cases, the primers with only the 5’ tail sequences of the upstream or downstream primers may be at most about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the upstream or downstream primers. The molar excess of the reporter probe relative to the mediator probe can have a beneficial advantage because of high local concentration of complementary sequences derived from the 5’ tail sequences. This can increase the annealing of the 5’ tail sequence and the complementary counterparts of the templates and gives rise to secondary structures that outcompetes the annealing of further primers with only the 5’ tail sequences, thereby preventing the formulation or accumulation of non-specific primer dimers. In some cases, the methods using the 5’ tail sequences may be those described in Brownie et al., Nucleic Acids Research, Volume 25, Issue 16, 1 August 1997, Pages 3235–3241, which is herein incorporated by reference in its entirety. Additionally, the primers with only the 5’ tail sequences may also be used in the nucleic acid amplification reaction of the reporter probe (as a template) and the cleaved fragment of the mediator probe (as a primer) . For example, an additional mediator probe may comprise the 5’ tail sequence 5’ of the first template-binding nucleotide sequence of the mediator probe (or the cleaved fragment thereof) . In some cases, the primer with only the 5’ tail sequence of the first template-binding nucleotide sequence of the mediator probe (or the cleaved fragment thereof) may be at least about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the mediator probe (or the cleaved fragment thereof) . In some cases, the primer with only the 5’ tail sequence of the first template-binding nucleotide sequence of the mediator probe (or the cleaved fragment thereof) may be at most about 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 200 %, 300 %, 400 %, 500 %, 85 %, 90 %, 95 %, 100 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold more than the mediator probe (or the cleaved fragment thereof) .

Devices and systems

Provided herein, are devices and systems for practicing the methods described herein. The device may comprise a thermocycler. In some cases, the device may be configured to control the temperature and time for carrying out the steps of the methods described herein. In some cases, the device may further comprise a detector that can detect the detectable signal generated by the methods described herein. For example, the device can comprise a fluorometer. The fluorometer may be configured to detect the detectable signal generated by the methods described herein generated in real time.

The systems described herein may comprise a computer control systems that are programmed to implement methods of the disclosure. FIG. 10 shows a computer system 1001 that is programmed or otherwise configured to implement methods of the disclosure, such as to control the systems described herein (e.g., reagent dispensing, detecting, etc. ) and collect, receive, and/or analyze the detectable signal. The computer system 1001 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

The computer system 1001 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1005, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1001 also includes memory or memory location 1010 (e.g., random-access memory, read-only memory, flash memory) , electronic storage unit 1015 (e.g., hard disk) , communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage and/or electronic display adapters. The memory 1010, storage unit 1015, interface 1020 and peripheral devices 1025 are in communication with the CPU 1005 through a communication bus (solid lines) , such as a motherboard. The storage unit 1015 can be a data storage unit (or data repository) for storing data. The computer system 1001 can be operatively coupled to a computer network ( “network” ) 1030 with the aid of the communication interface 1020. The network 1030 can be the Internet, an isolated or substantially isolated internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1030 in some cases is a telecommunication and/or data network. The network 1030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1030, in some cases with the aid of the computer system 1001, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1001 to behave as a client or a server.

The CPU 1005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. The instructions can be directed to the CPU 1005, which can subsequently program or otherwise configure the CPU 1005 to implement methods of the present disclosure. Examples of operations performed by the CPU 1005 can include fetch, decode, execute, and writeback.

The CPU 1005 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1001 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC) .

The storage unit 1015 can store files, such as drivers, libraries and saved programs. The storage unit 1015 can store user data, e.g., user preferences and user programs. The computer system 1001 in some cases can include one or more additional data storage units that are external to the computer system 1001, such as located on a remote server that is in communication with the computer system 1001 through an intranet or the Internet.

The computer system 1001 can communicate with one or more remote computer systems through the network 1030. For instance, the computer system 1001 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC) , slate or tablet PC’s (e.g., iPad, Galaxy Tab) , telephones, Smart phones (e.g., iPhone, Android-enabled device, ) , or personal digital assistants. The user can access the computer system 1001 via the network 1030.

Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1001, such as, for example, on the memory 1010 or electronic storage unit 1015. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1005. In some cases, the code can be retrieved from the storage unit 1015 and stored on the memory 1010 for ready access by the processor 1005. In some situations, the electronic storage unit 1015 can be precluded, and machine-executable instructions are stored on memory 1010.

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

Aspects of the systems and methods provided herein, such as the computer system 1001, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

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

The computer system 1001 can include or be in communication with an electronic display 835 that comprises a user interface (UI) 1040 for providing, for example, data generated by the fluorescence measurements. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1005. The algorithm can, for example, analyzing the analytes using the methods described herein.

As used herein, the singular forms “a, ” “an, ” and “the” include the plural reference unless the context clearly dictates otherwise.

When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range is encompassed within the scope of the present disclosure. Where the stated range includes upper or lower limits, ranges excluding either of those included limits are also included in the present disclosure.

Also disclosed herein are control mediator probes, reporter probes, target nucleic acids, nucleotides, or constituents of the reaction mixtures for generating positive or negative controls depending on the application of the methods to be carried out.

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

EXAMPLES

Example 1: Detecting target nucleic acids

FIG. 6 shows a cartoon schematic using the methods and compositions described herein.

A mediator probe comprising a from 5’ to 3’ : a label moiety, a first template-binding nucleotide sequence not complementary to the target nucleic acid, a junction, a second template-binding nucleotide sequence complementary to the target nucleic acid, a second quencher moiety (at the junction or within the second template-binding nucleotide sequence, and a first quencher moiety) . The label and first/second quencher moieties form label-quencher moiety pairs. When hybridizing to the target nucleic acid, alongside with the upstream primer; a cleavage reaction is triggered to cleave a cleaved fragment of the mediator probe comprising the first template-binding nucleotide sequence from the mediator probe with about 10-15 nucleotides in length. The cleavage also generates a first detectable signal, because the label moiety is no longer quenched, altered, or absorbed by any of the quencher moiety. FIG. 9A shows the first detectable signals generated using the method described in this example having FAM, HEX, ROX, and Cy5 as label moieties each coupled to one of four mediator probes. Four distinguishable detectable signals were observed using the label moieties described herein.

The cleaved fragment of the mediator probe is then used as a primer and hybridize to the reporter probe that contains a quencher moiety that can form a label/quencher pair with the label moiety coupled to the cleaved fragment of the mediator probe. A nucleic acid amplification reaction is carried out, thus generating a nascent strand using the reporter probe as a template. The duplex then forms comprising the nascent strand and the reporter probe.

When the cleaved fragment of the mediator probe hybridizes to the reporter probe (when the nascent strand hybridizes to the reporter probe) , the quencher moiety of the reporter probe quenches, alters, or eliminates any detectable signal generated by the label moiety (such as the first detectable signal) and can generate a second detectable signal (if the quencher moiety can emit a signal such as a fluorescence when contacting a label moiety in a label/quencher moiety pair) .

The duplex is then subjected to heating and cooling in a melting curve analysis. During denaturation of the duplex, the quencher moiety can no longer form the label/quencher moiety pair with the label moiety. Because the second detectable signal depends on the pairing of the label moiety of the cleaved fragment of the mediator probe and the quencher moiety of the reporter probe, the denaturation of the duplex then decreases the signal intensity of the second detectable signal. Because the label moiety of the cleaved fragment of the reporter probe is now not quenched, a third detectable signal is then generated and gradually increases as the temperature increases.

FIGs. 9B shows the melting curve analysis using the signal intensity of the third detectable signal generated by the label moiety of the cleaved fragment of the mediator probe and the reporter probe, using the method described herein. Two Tm peaks were observed, suggesting identification of at least two different target nucleic acids.

Example 2: Increasing detection sensitivity of target nucleic acid detection

Provided herein, are methods to increase the detection sensitivity of the methods as described herein.

A mediator probe comprising the sequence of SEQ ID NO: 1 was subjected to the melting curve analysis with either a reporter probe comprising the sequence of SEQ ID NOs: 2 or 3, respectively. Using the method described herein, the mediator probe was hybridized to a target nucleic acid complementary to the second template-binding sequence of SEQ ID NO: 1 (shown in italic in Table 1 below) and cleaved to release the cleaved fragment comprising the first template-binding sequence of SEQ ID NO: 1 (shown in bold in Table 1) . The cleaved fragment then hybridized with the reporter probe. A melting curve analysis was carried out to the resultant double-stranded nucleic acid molecule. At the 5’ end of the template sequence for hybridization of the reporter probe, a complementary G and a non-complementary C was present in SEQ ID NOs: 2 and 3, respectively. FIGs. 11A-B show the real-time PCR reactions for quantification analysis of amplification signals using SEQ ID NOs: 2 and 3 had similar relative fluorescence units (RFU) increase along with the amplification cycle. However, FIGs. 11C-D show that the melting curve of the reaction using SEQ ID NO: 3 had a maximal derivative reporter (ΔRn) about 2 times higher than that of the reaction using SEQ ID NO: 2. Additionally, the melting curve of the reaction using SEQ ID NO: 3 had a steeper slope, relative to that of the reaction using SEQ ID NO: 2. In this example, the experiments were performed with SEQ ID NOs: 63-65, which were the label/quencher moiety (ies) coupled version, directed to SEQ ID NOs: 1-3, respectively.

Thus, decreasing the number of complementary nucleotides between the mediator probe and reporter probe can increase the detection sensitivity of the identification of the target nucleic acid using the method described herein.

Table 1: Exemplary sequences used in Example 2

Example 3: Determining the effects of having a quencher group between the first and second template-binding sequences of the mediator probe

Provided herein are methods for determining the effects of having or not having a quencher moiety between the first and second template-binding sequences of the mediator probe on determining a presence or absence of a target nucleic acid. The general method for detecting a presence or absence of the target nucleic acid is described in Examples 1-2 using the melting curve analysis described herein.

Four mediator probes (SEQ ID NOs: 26-29) were tested, each having 36 nucleotides in length, the same first and second template-binding sequences, a same label moiety and quencher moiety coupled to the 5’ and 3’ end of the mediator probe, respectively. Three mediator probes (as referred to as the first/second/third mediator probe, directed to SEQ ID NOs: 26, 27, and 28, respectively) had an additional quencher moiety BHQ1 (that could form a label/quencher pair) coupled to the 18^th, 19^th, 20^th nucleotide from 5’ of the mediator probe (3’ to the 3’ most nucleotide of the first template-binding sequence) , and one mediator probe (as referred to as the fourth mediator probe, directed to SEQ ID NO: 29) was not coupled with the additional quencher moiety. As shown in FIG. 12, coupling the additional quenching group between the first/second template-binding sequences in the first/second/third mediator probes reduced the fluorescence signal value and the melting peak value. Additionally, the Ct (threshold cycle) value also increased from 32 to 34, as compared to the fourth mediator probe.

Thus, adding an additional quenching group between the first/second template-binding sequences in the mediator probe could reduce the total fluorescence signal and increase the sensitivity of the methods, compared to those without the modification described herein.

Example 4: Exemplary sequences

Provided herein in Table 2 below are exemplary sequences for practicing the methods described herein.

Table 2: Exemplary sequences for practicing the methods described herein

/iBHQ1dT/depicts a BHQ1 moiety is modified on base T.

Example 5: Comparison of the methods described herein and the methods using a common reporter probe

Provided herein comparison showing beneficial advantages of using the methods as described herein. The general method for detecting a presence or absence of the target nucleic acid is described in Examples 1-2 using the melting curve analysis described herein.

For detecting 3 target nucleic acids as the methods as described herein, a 50 ul PCR reaction system was used to perform real-time fluorescence PCR having the following reagents: PCR Buffer (50 mM Tris-HCl (pH=8.0) , 3 mM MgCl2, 2%glycerol, 1%DMSO, 0.5%BSA) ; 40U Taq DNA polymerase (Beijing Zhong Keomei Biotechnology Co., Ltd. ) ; 200 U reverse transcriptase (Beijing Zhongkeomei Biotechnology Co., Ltd. ) ; 200uM dNTPs; 6 upstream and downstream primers for 3 different target nucleic acids (3 pairs, 400 nM for each primer; SEQ ID NOs: 30-31, 36-37, and 48-49) ; 3 different mediator probes (one for each target gene, 200 nM each; SEQ ID NOs: 32, 38, and 50) ; 3 different reporter probes (one for each target gene, 100 nM each; SEQ ID NOs: 40, 41, and 51) ; and 15 ul of sample containing the three target nucleic acids. The following real-time PCR reaction condition was used for this experiment: reverse transcription at 50℃ was carried out for 1 min. The reaction mixture was then incubated for pre-denaturation at 98℃ for 5 seconds (s) , followed with 45 cycles of (95℃ 2s, 55℃ 0s, 72℃ 2s) . Fluorescence was collected at 55℃. After the PCR amplification reaction was completed, melting curve analysis was performed: The temperature of the PCR reaction mixture was increased from 68℃ to 100℃, and fluorescence was collected at every 0.5℃ interval. The PCR instrument used was Flash10 fully automatic nucleic acid analysis system (Beijing Cayudi Biotechnology Co., Ltd. ) . The sequences used were all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The results of this experiment was shown in Table 3 &FIG. 13A (labeled as detection method #1 in Table 3) .

For comparison, another 50 ul PCT reaction system having the following reagents was used to detect the same 3 target nucleic acids: PCR Buffer (50 mM Tris-HCl (pH=8.0) , 3 mM MgCl2, 2%glycerol, 1%DMSO, 0.5%BSA) ; 40U Taq DNA polymerase (Beijing Zhong Keomei Biotechnology Co., Ltd. ) ; 200 U reverse transcriptase (Beijing Zhongkeomei Biotechnology Co., Ltd. ) ; 200uM dNTPs; 6 upstream and downstream primers for 3 different target nucleic acids (3 pairs, 100 nM for each primer; SEQ ID NOs: 53-54, 56-57, and 59-60) ; 1 universal primer (complementary to 5’ of upstream/downstream primers1.2 uM; SEQ ID NO: 52); 1 fluorescent probe (600 nM; SEQ ID NO: 62) ; 3 different mediator probes (one for each target gene, 200 nM each; SEQ ID NOs: 55, 58, and 61) ; and 15 ul of sample containing the three target nucleic acids. The following real-time PCR reaction condition was used for this experiment: reverse transcription at 50℃ was carried out for 1 min. The reaction mixture was then incubated for pre-denaturation at 98℃ for 5 seconds (s) , followed with 45 cycles of (95℃ 2s, 55℃ 0s, 72℃ 2s) . Fluorescence was collected at 55℃. After the PCR amplification reaction was completed, melting curve analysis was performed: The temperature of the PCR reaction mixture was increased from 68℃ to 100℃, and fluorescence was collected at every 0.5℃ interval. The PCR instrument used was Flash10 fully automatic nucleic acid analysis system (Beijing Cayudi Biotechnology Co., Ltd. ) . The sequences used were all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The method used for the comparison is also described in United States Patent No. 11,788, 118 and Wang et al., Heliyon. 2022 Nov 26; 8 (11) : e11856., each of which is herein incorporated by reference in its entirety. The results of this experiment was shown in Table 3 &FIG. 13B (labeled as detection method #2 in Table 3) .

Table 3: Summary of CT value, Tm, and fluorescence intensities detected in the experiments described in Example 5

As shown in FIGs 13A-B and Table 3, while both methods could detect the 3 different target nucleic acids, using the method described herein (method #1) , the fluorescence intensities of the melting peak for the 3 different target nucleic acids are detected at a much higher level. Thus, method 1 can provide a higher sensitivity due to the sharpening of the melting peak, making it easier to detect a melting peak, relative to the comparison method #2. Accordingly, such configuration of the label/quencher moieties of the mediator/reporter probes in method #1 would provide a higher signal-to-noise ratio, relative to those of method #2. For example, in method #2, the separation/uncoupling of the quencher and label moieties triggered by the binding of the cleaved fragment of the mediator probe may not be complete, since both the quencher and label moieties are coupled to the same reporter probe. Such configuration thus decreases the fluorescence intensities of the melting peak and sensitivity of the method, relative to method #1.

Example 6: Comparison of the methods described herein and the methods having different label/quencher moieties configuration

For detecting 3 target nucleic acids as the methods as described herein, a 50 ul PCR reaction system was used to perform real-time fluorescence PCR having the following reagents: PCR Buffer (50 mM Tris-HCl (pH=8.0) , 3 mM MgCl2, 2%glycerol, 1%DMSO, 0.5%BSA) ; 40U Taq DNA polymerase (Beijing Zhong Keomei Biotechnology Co., Ltd. ) ; 200 U reverse transcriptase (Beijing Zhongkeomei Biotechnology Co., Ltd. ) ; 200uM dNTPs; 6 upstream and downstream primers for 3 different target nucleic acids (3 pairs, 400 nM for each primer; SEQ ID NOs: 30-31, 33-34, and 36-37) ; 3 different mediator probes (one for each target gene, 200 nM each; SEQ ID NOs: 32, 35, and 38) ; 3 different reporter probes (one for each target gene, 100 nM each; SEQ ID NOs: 39-41) ; and 15 ul of sample containing the three target nucleic acids. The following real-time PCR reaction condition was used for this experiment: reverse transcription at 50℃ was carried out for 1 min. The reaction mixture was then incubated for pre-denaturation at 98℃ for 5 seconds (s) , followed with 45 cycles of (95℃ 2s, 55℃ 0s, 72℃ 2s) . Fluorescence was collected at 55℃. After the PCR amplification reaction was completed, melting curve analysis was performed: The temperature of the PCR reaction mixture was increased from 68℃ to 100℃, and fluorescence was collected at every 0.5℃ interval. The PCR instrument used was Flash10 fully automatic nucleic acid analysis system (Beijing Cayudi Biotechnology Co., Ltd. ) . The sequences used were all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The results of this experiment was shown in Table 4 &FIG. 14A (labeled as detection method #1 in Table 4) .

For comparison, another 50 ul PCT reaction system having the following reagents was used to detect the same 3 target nucleic acids: PCR Buffer (50 mM Tris-HCl (pH=8.0) , 3 mM MgCl2, 2%glycerol, 1%DMSO, 0.5%BSA) ; 40U Taq DNA polymerase (Beijing Zhong Keomei Biotechnology Co., Ltd. ) ; 200 U reverse transcriptase (Beijing Zhongkeomei Biotechnology Co., Ltd. ) ; 200uM dNTPs; 6 upstream and downstream primers for 3 different target nucleic acids (3 pairs, 100 nM for each primer; SEQ ID NOs: 30-31, 33-34, and 36-37) ; 3 different mediator probes (one for each target gene, 200 nM each; SEQ ID NOs: 42-44) ; 3 different reporter probes (one for each target gene, 100 nM each; SEQ ID NOs: 45-47) ; and 15 ul of sample containing the three target nucleic acids. The following real-time PCR reaction condition was used for this experiment: reverse transcription at 50℃ was carried out for 1 min. The reaction mixture was then incubated for pre-denaturation at 98℃ for 5 seconds (s) , followed with 45 cycles of (95℃ 2s, 55℃ 0s, 72℃ 2s) . Fluorescence was collected at 55℃. After the PCR amplification reaction was completed, melting curve analysis was performed: The temperature of the PCR reaction mixture was increased from 68℃ to 100℃, and fluorescence was collected at every 0.5℃ interval. The PCR instrument used was Flash10 fully automatic nucleic acid analysis system (Beijing Cayudi Biotechnology Co., Ltd. ) . The sequences used were all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The method used for the comparison is also described in United States Patent No. 88,092,39, which is herein incorporated by reference in its entirety. The results of this experiment was shown in Table 4 &FIG. 14B (labeled as detection method #2 in Table 4) .

Table 4: Summary of CT value, Tm, and fluorescence intensities detected in the experiments described in Example 6

As shown in FIGs 14A-B and Table 4, while both methods could detect the 3 different target nucleic acids, using the method described herein (method #1) , the fluorescence intensities of the melting peak for the 3 different target nucleic acids are detected at a much higher level (except for the replicate #2 of the target #3, which was likely an experimental error) . Thus, method 1 can provide a higher sensitivity due to the sharpening of the melting peak, making it easier to detect a melting peak, relative to the comparison method #2. Accordingly, such configuration of the label/quencher moieties of the mediator/reporter probes in method #1 would provide a higher signal-to-noise ratio, relative to those of method #2. For example, in method #2, the separation/uncoupling of the quencher and label moieties triggered by the binding of the cleaved fragment of the mediator probe may not be complete, since both the quencher and label moieties are coupled to the same reporter probe. Such configuration thus decreases the fluorescence intensities of the melting peak and sensitivity of the method, relative to method #1. Accordingly, in the configuration of

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A probe comprising, in a 5’ to 3’ direction, (1) a label moiety, a first template-binding nucleotide sequence, a second template-binding nucleotide sequence, and a quencher moiety or (2) the quencher moiety, the first template-binding nucleotide sequence, the second template-binding nucleotide sequence, and the label moiety; wherein the first and second template-binding nucleotide sequences binds two different template nucleotide sequences.
The probe of claim 1, wherein each of the first and second template-binding nucleotide sequences is complementary to only one of the two different template nucleotide sequences.
The probe of claim 2, wherein the first and second template-binding nucleotide sequences are complementary to the two different template nucleotide sequences of two different template nucleic acid molecules.
The probe of any one of claims 1-3, wherein the first template-binding nucleotide sequence is complementary to a non-naturally occurring nucleotide sequence.
The probe of any one of claims 1-3, wherein the second template-binding nucleotide is complementary to a pathology-associated nucleotide sequence.
The probe of claim 5, wherein the pathology-associated nucleotide sequence comprises a nucleotide sequence of a pathogen.
The probe of claim 6, wherein the pathogen comprises a virus, bacterium, protozoan, fungus, or a combination thereof.
The probe of claim 5, wherein the pathology-associated nucleotide sequence comprises a sequence of a cell.
The probe of claim 8, wherein the cell comprises a eucaryotic cell.
The probe of claim 8 or 9, wherein the cell comprises a human cell.
The probe of any one of claims 8-10, wherein the cell is associated with a disease condition.
The probe of claim 11, wherein the disease condition comprises a cancer, a genetic disorder, an infectious disease, or a combination thereof.
A probe comprising, in a 5’ to 3’ direction: (1) a label moiety, a nucleotide sequence comprising at least 25 nucleotides, and a quencher moiety; or (2) the quencher moiety, the nucleotide sequence comprising the at least 25 nucleotides, and the label moiety.
The probe of claim 13, wherein the nucleotide sequence comprises at most 100 nucleotides, at most 80 nucleotides, or at most 50 nucleotides.
The probe of claim 13 or 14, wherein the nucleotide sequence comprises at least 26 nucleotides, at least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides, at least 30 nucleotides, at least 31 nucleotides, or at least 32 nucleotides.
The probe of any one of claims 13-15, wherein the nucleotide sequence comprises at least 32 nucleotides.
The probe of any one of claims 1-16, wherein the label moiety comprises 2, 3, 4, 5, or more label moieties.
The probe of claim 17, wherein the label moiety comprises FAM, SYBR, JOE, VIC, NED, Cy3, TAMRA, ROX, Texas Red, Cy5, TET, HEX, Quasar 670, or Cy5.5.
The probe of any one of claims 1-18, wherein the quencher moiety comprises 2, 3, 4, 5, or more quencher moieties.
The probe of claim 19, wherein the quencher moiety comprises Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, or BBQ 650.
The probe of any one of claims 1-20, wherein the label moiety is configured to provide a label signal that is quenchable, absorbable, or alterable by the quencher moiety.
A composition comprising the probe of any one of claim 1-21 and at least one of: a target nucleic acid or an amplification product thereof, an upstream primer, a downstream primer, a second probe, or an enzyme.
A composition comprising: a first probe comprising (a) a nucleotide sequence and (b) a first label moiety and a first quencher moiety; and a second probe comprising a second label moiety or a second quencher moiety coupled thereto, wherein the first label moiety is coupled to a first terminal end of the first probe, and wherein the first quencher moiety is coupled to a second terminal end of the first probe different from the first terminal end.
The composition of claim 23, wherein the first quencher moiety and the second quencher moiety are a same quencher moiety.
Th composition of claim 23 or 24, wherein the first quencher moiety and the second quencher moiety are different quencher moieties.
The composition of any one of claims 23-25, wherein the label moiety comprises 2, 3, 4, 5, or more label moieties.
The composition of any one of claims 23-26, wherein the label moiety comprises FAM, SYBR, JOE, VIC, NED, Cy3, TAMRA, ROX, Texas Red, Cy5, TET, HEX, Quasar 670, or Cy5.5.
The composition of any one of claims 23-27, wherein the second probe comprises the second quencher moiety coupled thereto.
The composition of any one of claims 23-28, wherein the second quencher moiety comprises 2, 3, 4, 5, or more quencher moieties.
The composition of any one of claims 23-29, wherein the second quencher moiety comprises Dabcyl, Eclipse, MGB, BHQ1, BHQ2, BHQ3, or BBQ 650.
The composition of any one of claims 23-30, wherein the first label moiety is configured to provide a label signal that is quenchable, absorbable, or alterable by the first and second quencher moieties.
The composition of claim 31, wherein the second quencher moiety of the second probe is configured to quench, absorb, or alter a label signal generated by the first label moiety on the first probe.
The composition of any one of claims 23-32, wherein the composition further comprises at least one of: a target nucleic acid or an amplification product thereof, an upstream primer, a downstream primer, or an enzyme.
A method for detecting a target nucleic acid, comprising:

(a) providing a reaction mixture comprising

(i) the probe of any one of claims 1-22; and

(ii) the target nucleic acid or an amplification product thereof, an upstream primer of the target nucleic acid, a downstream primer of the target nucleic acid, a second probe, and an enzyme;

(b) detecting a label signal generated by the label moiety.
A method for detecting a target nucleic acid, comprising:

(a) providing a reaction mixture comprising

(i) the composition of any one of claims 23-33; and

(ii) the target nucleic acid or an amplification product thereof, an upstream primer of the target nucleic acid, a downstream primer of target nucleic acid, and an enzyme;

(b) detecting a label signal generated by the first label moiety.
A method comprising:

(a) hybridizing a first mediator probe to a target nucleic acid, wherein the first mediator probe comprises at least a nucleotide sequence complementary to a sequence of the target nucleic acid or an amplification product thereof, wherein the first mediator probe comprises, in a 5’ to 3’ direction, a first template-binding nucleotide sequence, a second template-binding nucleotide sequence and a quencher moiety;

(b) cleaving a cleaved fragment from the first mediator probe resulting in a first detectable signal or signal change, wherein the cleaved fragment comprises a second sequence is not complementary to a second sequence of the target nucleic acid;

(c) hybridizing the cleaved fragment to a nucleotide sequence of the first reporter probe to generate a duplex molecule and result in a second detectable signal or signal change;

(d) heating the duplex molecule to generate at least one third detectable signal or signal change;

(e) identifying a presence, an absence, or an amount of the target nucleic acid by matching the at least one third detectable signal or signal change and a temperature or temperature range at which the at least one third detectable signal or signal change is generated.
The method of claim 36, further comprising, hybridizing an upstream primer or/and a downstream primer to the target nucleic acid to generate the amplification product of the target nucleic acid.
The method of claim 36 or 37, further comprising, providing an enzyme.
The method of claim 38, wherein the enzyme comprises a comprises a nuclease activity or a nucleic acid polymerase activity.
The method of claim 39, wherein the enzyme comprises a Flap nuclease.
The method of claim 38 or 39, wherein the cleaved fragment is cleaved from the first mediator probe using the enzyme.
The method of any one of claims 36-41, wherein (c) comprises extending the cleaved fragment to generate the duplex molecule.
The method of claim 42, wherein (c) comprises extending the cleaved fragment to generate the duplex molecule using the cleaved fragment as a primer in a nucleic acid polymerization reaction.
The method of claim 42 or 43, wherein (c) comprises extending the cleaved fragment to generate the duplex molecule using a sequence of the first reporter probe as a template in a nucleic acid polymerization reaction.
The method of claim 44, wherein the nucleic acid polymerization reaction comprises a nucleic acid amplification reaction.
The method of claim 45, wherein the nucleic acid amplification reaction comprises a polymerase chain reaction (PCR) .
The method of any one of claims 36-46, further comprising, detecting the first detectable signal or signal change.
The method of any one of claims 36-47, further comprising, detecting the second detectable signal or signal change.
The method of any one of claims 36-48, further comprising, detecting the third detectable signal or signal change.
The method of any one of claims 36-49, further comprising, repeating (a) - (e) , wherein a second mediator probe is used in place of the first mediator probe, wherein the second mediator probe comprises at least a nucleotide sequence complementary to a second sequence of the target nucleic acid or an amplification product thereof different from the nucleotide sequence of the target nucleic acid or the amplification product thereof, and wherein a cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to a second nucleotide sequence of the first reporter probe that is different from the nucleotide sequence of the first reporter probe.
The method of any one of claims 36-50, further comprising, repeating (a) - (e) , wherein a second mediator probe is used in place of the first mediator probe and a second reporter probe is used in place of a first reporter probe, wherein the second mediator probe comprises at least a nucleotide sequence complementary to a second sequence of the target nucleic acid or an amplification product thereof different from the nucleotide sequence of the target nucleic acid or the amplification product thereof, wherein the cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to sequence of the second reporter probe, wherein the first and second reporter probes are different probe molecules.
The method of claim 51, wherein the cleaved fragment cleaved from the second mediator probe does not hybridize to the first reporter probe or the cleaved fragment cleaved from the first mediator probe does not hybridize to the second reporter probe.
The method any one of claims 36-52, further comprising, repeating (a) - (e) , wherein a second mediator probe is used in place of the first mediator probe, wherein the second mediator probe comprises at least a nucleotide sequence complementary to a sequence of a second target nucleic acid or an amplification product thereof different from the target nucleic acid or the amplification product thereof, and wherein a cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to a second nucleotide sequence of the first reporter probe that is different from the nucleotide sequence of the first reporter probe.
The method of any one of claims 50-53, wherein a cleaved fragment cleaved from the second mediator probe comprises a sequence complementary to a second nucleotide sequence of the first reporter probe that is different from the nucleotide sequence of the first reporter probe.
The method of claim 54, wherein the first signal change generated when using the first mediator probe in (a) is the same as a first signal or signal change generated when using the second mediator probe in (a) .
The method of claim 54, wherein the first signal change generated when using the first mediator probe in (a) is different from a first signal or signal change generated when using the second mediator probe in (a) .
The method of claim 55 or 56, wherein the second signal change generated when using the first mediator probe in (c) is the same as a second signal or signal change generated when using the second mediator probe in (c) .
The method of claim 55 or 56, wherein the second signal change generated when using the first mediator probe in (c) is different from a second signal or signal change generated when using the second mediator probe in (c) .
The method of any one of claims 55-58, wherein the third signal change generated when using the first mediator probe in (d) is the same as a third signal or signal change generated when using the second mediator probe in (d) .
The method of any one of claims 55-58, wherein the third signal change generated when using the first mediator probe in (d) is different from a third signal or signal change generated when using the second mediator probe in (d) .
The method of any one of claims 36-60, wherein the quencher moiety is coupled to a nucleotide that is at most about 10 nucleotides 3’ to a 3’ terminal nucleotide of the first template-binding nucleotide sequence.
The method of claim 61, wherein the quencher moiety is coupled to a nucleotide that is at most about 7 nucleotides 3’ to the 3’ terminal nucleotide of the first template-binding nucleotide sequence.
The method of claim 61, wherein the quencher moiety is coupled to a nucleotide that is at most about 5 nucleotides 3’ to the 3’ terminal nucleotide of the first template-binding nucleotide sequence.