WO2023246733A1

WO2023246733A1 - Methods and systems for nucleic acid detection

Info

Publication number: WO2023246733A1
Application number: PCT/CN2023/101234
Authority: WO
Inventors: Zhenxin HU; Bing GAO; Qiushi LI
Original assignee: Genevide Biotech Co., Ltd.
Priority date: 2022-06-21
Filing date: 2023-06-20
Publication date: 2023-12-28

Abstract

Disclosed herein include highly sensitive methods and systems for nucleic acid detection.

Description

METHODS AND SYSTEMS FOR NUCLEIC ACID DETECTION

This application claims priority to PCT Patent Application No. PCT/CN2022/100135, filed June 21, 2022, which is incorporated herein by reference in its entirety.

1. Reference to Sequence Listing Submitted Electronically

This application incorporates by reference a Sequence Listing with this application as an ASCII text file entitled “514A001WO02_SL. XML” created on June 8, 2023 and having a size of 22,856 bytes.

2. Field

The present invention relates to molecular biology. Provided herein include methods, compositions, and systems for nucleic acid detection.

3. Background

Rapid and sensitive methods for nucleic acid detection are greatly needed for the detection of pathogens and for point-of-care diagnostic. Although progress has been made, there is still an unmet need for methods and systems for nucleic acid detection with high sensitivity and specificity. The methods and compositions provided herein meet these needs and provide relative advantages.

4. Summary

Provided herein are methods of detecting a target nucleic acid in a sample, comprising (1) amplifying the target nucleic acid by subjecting a reaction mixture comprising the sample to an amplification condition, wherein the reaction mixture comprises: (a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; and (b) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; and (2) detecting the amplicons with an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; wherein (a) an RNase H cleaves the ribonucleotide (s) in the amplicons, generating nicking sites; (b) the DNA polymerase recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ; (c) the oligonucleotide probe hybridizes with the single-strand target; (d) the RNase H cleaves the ribonucleotide (s) on the hybridized probe, separating F from Q and producing a detectable signal; and (e) the cleaved probe fragments separate from the single-strand target, allowing the repetition of steps (c) and (d) .

In some embodiments, the probe is included in the reaction mixture during target amplification. In some embodiments, the RNase H is reversibly inactivated and included in the reaction mixture during target amplification, and becomes reactivated before the detection.

In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample, comprising (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; (d) a reversibly inactivated RNase H; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition for sufficient time to amplify the target nucleic acid; (3) subjecting the reaction mixture to a transition condition for sufficient time to reactivate RNase H; and (4) subjecting the reaction mixture to a detection condition and collecting fluorescent signals. In some embodiments, the transition condition is the same as the amplification condition.

In some embodiments of the methods provided herein, the RNase H is reversibly inactivated by chemical modification. In some embodiments, the reversibly inactivated RNase H comprises a lysine residue modified by a maleic anhydride or a derivative thereof having the structure below, wherein X and Y each independently is a negatively charged or neutral group.

In some embodiments, the reversibly inactivated RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride.

In some embodiments, the RNase H is reversibly inactivated by ligand association. In some embodiments, the reversibly inactivated RNase H is associated with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent.

In some embodiments, the RNase H is reversibly inactivated by physical separation. In some embodiments, the RNase H is physically trapped in a switchable chemical shell, a microsphere or an isolated chamber of a reaction vessel.

In some embodiments, the RNase H is reactivated by temperature change or pH change.

In some embodiments, the RNase H is Pyrococcus furiosus RNase HI, Pyrococcus horikoshi RNase HI, Pyrococcus abyssi RNase HI, Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coli RNase HI, E. coli RNase HII, Saccharomyces cerevisiae RNase HI, mouse RNase HI or human RNase H.

In some embodiments of the methods provided herein, the primer comprises 1-15, 2-12, 4-12, or 4-8 ribonucleotides. In some embodiments, the primer comprises about 4, about 6, or about 8 ribonucleotides.

In some embodiments of the methods provided herein, the DNA polymerase is Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; or SD polymerase; or a functional variant thereof.

In some embodiments, the reaction mixture further comprises a reverse transcriptase. In some embodiments, the reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.

In some embodiments, the target nucleic acid is amplified by isothermal amplification. In some embodiments, the isothermal amplification is enzyme-mediated amplification (EMA) , loop-mediated isothermal amplification (LAMP) , cross-priming amplification (CPA) , recombinase polymerase amplification (PRA) , helicase-dependent isothermal DNA amplification (HDA) , rolling circle amplification (RCA) , strand displacement amplification (SDA) , nicking enzyme amplification reaction (NEAR) , polymerase spiral reaction (PSR) , hybridization chain reaction (HCR) , primer exchange reaction (PER) , signal amplification by exchange reaction (SABER) , transcription-based amplification system (TAS) , self-sustained sequence replication reaction (3 SR) , or single primer isothermal amplification (SPIA) .

In some embodiments, methods provided herein include amplifying the nucleic acid target by EMA. In some embodiments, methods provided herein include amplifying the nucleic acid target by LAMP.

In some embodiments, methods provided herein include amplifying the nucleic acid target by PCR.

In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample comprising: (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) , (c) SAP Pol I and M-MLV reverse transcriptase; (d) an RNase H modified by 2, 3-dimethylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition at about 42 ℃ for about 5 to 30 minutes; and (3) collecting fluorescence signals.

In some embodiments, the reaction mixture further comprises a DNA helicase, a single-stranded binding protein, or both.

In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample, comprising (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a Bst polymerase having reverse transcriptase activity; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; and (2) subjecting the reaction mixture to an amplification condition at 65 ℃ for about 30 min; and (3) incubating the reaction mixture at about 95 ℃ for at least three minutes; and (4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.

In some embodiments, the reaction mixture comprises an outer primer pair and an inner primer pair, wherein each of the inner primer pair contains a looping fragment that is complementary to a region of the amplicon that allows for the formation of a loop at the end of the amplicon, and wherein one of the inner primer pair contains about 1-15 ribonucleotide (s) .

In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample, comprising (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) Tth Pol and SD Polymerase; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition for 25-45 thermal cycles alternating between 95 ℃ and 75 ℃; (3) incubating the reaction mixture at about 95 ℃ for at least three minutes; and (4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.

In some embodiments of the methods disclosed herein, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA.

In some embodiments of the methods disclosed herein, the probe has about 10-30 nucleotides. In some embodiments, the probe comprises at least 4 ribonucleotides between the F and Q. In some embodiments, the probe is an RNA probe.

In some embodiments, methods provided herein comprise detecting two or more target nucleic acids, wherein for each target nucleic acid, a probe that can hybridize with the target nucleic acid and has a distinct fluorophore (F) is used.

In some embodiments of the methods provided herein, during the detection step, the fluorescence spectrum is collected every 30 seconds.

In some embodiments of the methods provided herein, the reaction mixture is kept in a vessel that remains closed from the beginning of the amplification to the end of the detection.

In some embodiments, provided herein are nucleic acid detection systems comprising: (a) (i) a DNA polymerase with strand displacement activity and reverse transcriptase activity or (ii) a DNA polymerase with strand displacement activity and a reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H that is reversibly inactivated; and (d) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments of the nucleic acid detection systems disclosed herein, the RNase H is reversibly inactivated by chemical modification, ligand association, or physical separation.

In some embodiments, the RNase H is reversibly inactivated by chemical modification. In some embodiments, the RNase H comprises a lysine residue modified by a maleic anhydride or a derivative thereof having the structure below, wherein X and Y each independently is a negatively charged or neutral group.

In some embodiments, the RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride.

In some embodiments, the RNase H is associated with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent.

In some embodiments, the RNase H is physically trapped in a switchable chemical shell, a microsphere or an isolated chamber of a reaction vessel.

In some embodiments, the RNase H can be reactivated by temperature change or pH change.

In some embodiments of the nucleic acid detection systems disclosed herein, the primer contains 1-20, 2-15, 4-12, or 4-8 ribonucleotides. In some embodiments, the primer contains about 4, about 8, about 12, about 16 or about 20 ribonucleotides.

In some embodiments of the nucleic acid detection systems disclosed herein, the DNA polymerase is Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; SD polymerase; or a functional variant thereof.

In some embodiments, the reaction mixture comprises a reverse transcriptase. In some embodiments, the reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.

In some embodiments, provided herein are nucleic acid detection systems comprising: (a) SAP Pol I and M-MLV reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H modified by 2, 3-dimethylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments, provided herein are nucleic acid detection systems comprising: (a) a Bst polymerase having reverse transcriptase activity; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H modified by 3-methylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments, provided herein are nucleic acid detection systems, comprising (a) Tth Pol and SD Polymerase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H modified by 3-methylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments of the nucleic acid detection systems disclosed herein, the probe has about 10-30 nucleotides. In some embodiments, the probe comprises at least 4 ribonucleotides between the F and Q. In some embodiments, in the probe is an RNA probe.

In some embodiments, the nucleic acid detection systems provided herein comprise two or more different probes, wherein each can hybridize to a different nucleic acid and has a distinct fluorophore (F) .

5. Brief Description of Drawings

FIG. 1A provides a diagram illustrating an exemplary nucleic acid detection method disclosed herein. Step 1: Amplification: Primers (one of which includes RNA base (s) ) invade, search for homologous sequences, bind to matched target sequence and are extended by polymerase. The process repeats and the target nucleic acid is amplified exponentially. In some embodiments, the amplification reaches the plateau in, e.g., 10 minutes. RNase H remains largely inactive during Step 1, and it can gets reactivated gradually during Step 1, or after Step 1 is completed. Step 2: the reactivated RNase H cleaves the ribonucleotides in the amplicon, generating a nick site. Polymerase with strand-displacement activity extends the primer from the nick, displacing the original strand, which becomes ssDNA. RNA probe binds to the ssDNA and gets cleaved by RNase H, the cleaved RNA probe fragments generate detectable signal, and become replaced by additional intact RNA probe, which again gets cleaved by RNase H, and the cycle continues.

FIG. 1B provides a diagram illustrating another exemplary nucleic acid detection method disclosed herein. Step 1: Loop-Mediated Isothermal Amplification (LAMP) is used for amplifying the target nucleic acid (one of the inner primers, such as the forward inner primer can contain RNA base (s) ) . RNase H remains largely inactive during Step 1, and it can gets reactivated gradually during Step 1, or after Step 1 is completed. Step 2: the reactivated RNase H cleaves the ribonucleotides in the amplicon, generating a nick site. Polymerase with strand-displacement activity extends the primer from the nick, displacing the original strand, which becomes ssDNA. RNA probe binds to the ssDNA and gets cleaved by RNase H, the cleaved RNA probe fragments generate detectable signal, and become replaced by additional intact RNA probe, which again gets cleaved by RNase H, and the cycle continues.

FIG. 2 provides a diagram illustrating the reversible modification on lysine residue by maleic anhydride. As shown, the lysine residue reacts with maleic acid anhydride or its derivatives and forms covalent bond under basic condition, which breaks under low pH or with heat.

FIG. 3 provides results of RNase H activity assay. From top to bottom, the curves represent the following samples, respectively: (1) unmodified RNase H, (2) modified RNase H that had been incubated at 45℃ for 10 min, (3) modified RNase H, and (4) no enzyme.

FIG. 4 provides results of linear DNA detection (isothermal amplification) . From top to bottom, the curves represent the following samples, respectively: (1) positive control; (2) sample 1 (positive) ; (3) sample 2 (positive) ; (4) sample 3 (negative) ; and (5) negative control.

FIG. 5 provides results of cyclic DNA detection (isothermal amplification) . From top to bottom, the curves represent the following samples, respectively: (1) 5000 copies of pUC-VP72 plasmid; (2) 500 copies of pUC-VP72 plasmid; (3) 50 copies of pUC-VP72 plasmid; (4) 5 copies of pUC-VP72 plasmid; and (5) negative control.

FIG. 6 provides results of viral RNA detection (isothermal amplification) . From top to bottom, the curves represent the following samples, respectively: (1) positive control; (2) sample 1 (positive) ; and (3) negative control.

FIG. 7 provides results of linear DNA detection (PCR amplification) . From top to bottom, the curves represent the following samples, respectively: (1) positive control; (2) sample 1 (positive) ; and (3) negative control.

FIG. 8 provides results of multiplexing detection (PCR amplification) . From top to bottom, the curves represent following signals in the designated samples, respectively: (1) VP72 (FAM) in Sample 2 (10 copies/μL) ; (2) VP72 (FAM) in Sample 1 (100 copies/μL) ; (3) PCV2 (HEX) in sample 1 (100 copies/μL) ; (4) PCV2 (HEX) of sample 2 (10 copies/μL) ; (5) VP72 (FAM) of negative control (0 copies/μL) ; and (6) PCV2 (HEX) of negative control (0 copies/μL) .

6. Detailed Description

The present disclosure provides highly sensitive methods and systems for nucleic acid detection using a reversibly inactivated RNase H. The high sensitivity is achieved with dual amplification: the amplification of the target nucleic acid, and the amplification of the detectable signal. Target nucleic acid amplification can be done using any methods known in the art, including PCR and isothermal amplification, and the signal amplification is achieved by target recycling. Target recycling refers to the process wherein a single-strand target is detected for multiple times by multiple probes, thereby amplifying the detectable signals over time. To do so, oligonucleotide probes containing ribonucleotides are used, which can be cleaved by RNase H when forming DNA/RNA hybrid with a single-strand target nucleic acid, allowing their dissociation from the single-strand target and the recycling of the single-strand target for detection by additional probes. Additionally, methods and systems provided herein enable single tube testing. Usually, the amplification and detection of nucleic acid are conducted separately because the two steps require interfering enzymatic activities, namely, DNA polymerase activity for amplification and nuclease activity for signal detection. Separation of the two steps complicates the design of the detection system, and if the reaction chamber needs to be opened between two steps, increases the risk of aerosol contamination. Methods and systems disclosed in present disclosure use reversibly inactivated RNase H to allow completion of both the amplification step and the detection step in one reaction vessel that can remain closed during the entire process, allowing easy design as well as rapid and specific detection of target nucleic acids.

Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and that the terminology used herein is not intended to be limiting, but is for the purpose of describing particular embodiments.

6.1 Definitions

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The term “a” or “an” entity refers to one or more of that entity; for example, “an oligonucleotide, ” is understood to represent one or more oligonucleotides.

The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B, ” “A or B, ” “A” (alone) , and B” (alone) . Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .

The term “nucleic acid” and “oligonucleotide, ” as used herein and understood in the art, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) , polyribonucleotides (containing D-ribose) , and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid, ” “oligonucleotide” and “polynucleotide, ” and these terms are used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) , as well as double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) . As used herein, a nucleic acid or an oligonucleotide can have nucleotide analogs in which the base, sugar or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. Certain nucleotides not commonly found in natural nucleic acids or chemically synthesized can be included in the nucleic acids described herein; these include but not limited to base and sugar modified nucleosides, nucleotides, and nucleic acids, such as inosine, isocytosine and isoguanine. When nucleic acids are referred to as “double-stranded, ” it is understood by those of skill in the art that a pair of oligonucleotides exists in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100%complementary form of double-stranded oligonucleotides, the term “double-stranded” as used herein is also meant to include those form which include such structural features as bulges and loops (see Stryer, BIOCHEMISTRY, Third Ed. (1988) , incorporated herein by reference in its entirety) . Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al, 1979, Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al, 1981, Tetrahedron Lett. 22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, BIOCONJUGATE CHEMISTRY 1 (3) : 165-187, incorporated herein by reference.

As used herein and understood in the art, the term “hybridize, ” “anneal, ” or their grammatical equivalents are used interchangeably in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. In other words, there terms refer to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The resulting double-stranded polynucleotide is a “hybrid” or “duplex. ” Hybridization can occur between fully complementary nucleic acid strands or between substantially complementary nucleic acid strands that contain minor regions of mismatch.

In some embodiments, the step of hybridizing comprises heating and/or cooling. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as “stringent hybridization conditions” or “sequence-specific hybridization conditions. ” Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitable adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al, 1989, MOLECULAR CLONING-A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26 (3/4) : 227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference) .

As used herein and understood in the art, the term “complementary, ” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide comprising the second nucleotide sequence. Such conditions can, for example, be stringent conditions. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Also as used herein and understood in the art, the term “complementary” when used in the context of nucleotide sequences refers to standard Watson/Crick base pairing rules. For example, the sequence “5’-A-G-T-C-3’” is complementary to the sequence “3’-T-C-A-G-5. ’” “Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G: U Wobble or Hoogsteen base pairing. In other words, complementarity need not be perfect; stable duplexes can contain mismatched base pairs, degenerative, or unmatched nucleotides. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, incidence of mismatched base pairs, ionic strength, other hybridization buffer components and conditions.

Complementarity can be partial in which only some of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. Complementarity can be complete or total where all the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. Complementarity is absent where none of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. In some embodiments, two nucleic acid strands are at least 50%, at least 55%, at least 60%, at least 65%, 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%, at least 99%, or 100%complementary. The degree of complementarity between nucleic acid strands can have significant effects on the efficiency and strength of hybridization between nucleic acid strands.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The term “variant” as used herein in relation to a protein or an enzyme refers to a different protein or polypeptide having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference polypeptide. A functional variant of a protein or enzyme maintains the basic structural and functional properties of the reference protein or enzyme.

Exemplary genes and proteins are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .

Definitions of common terms in molecular biology can be found in e.g., The MERCK MANUAL OF DIAGNOSIS AND THERAPY, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421) ; Robert S. Porter et al. (eds. ) , THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908) ; and Robert A. Meyers (ed. ) , MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) ; IMMUNOLOGY by Werner Luttmann, published by Elsevier, 2006; JANEWAY'S IMMUNOBIOLOGY, Kenneth Murphy, Allan Mowat, Casey Weaver (eds. ) , W.W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053) ; LEWIN’S GENES XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055) ; Michael Richard Green and Joseph Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414) ; Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X) ; LABORATORY METHODS IN ENZYMOLOGY: DNA, Jon Lorsch (ed. ) Elsevier, 2013 (ISBN 0124199542) ; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (CPMB) , Frederick M. Ausubel (ed. ) , John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385) , CURRENT PROTOCOLS IN PROTEIN SCIENCE (CPPS) , John E. Coligan (ed. ) , John Wiley and Sons, Inc., 2005; and CURRENT PROTOCOLS IN IMMUNOLOGY (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds. ) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737) , the contents of which are all incorporated by reference herein in their entireties.

6.2 Methods of nucleic acid detection

Provided herein are highly sensitive methods of nucleic acid detection. In some embodiments, methods provided herein comprise: (1) amplifying the target nucleic acid to generate double-stranded amplicons; and (2) detecting the target nucleic acid with signals amplified by target recycling. High sensitivity of the methods is achieved with the two steps of amplification. High specificity is achieved because both amplification steps require sequence specificity. That is, primers with specific sequences are used to amplify the target nucleic acid, and probes with specific sequences are used to detect the target nucleic acid. Additionally, reduced contamination and increased efficiency can be achieved with the use of a reversibly inactivated RNase H, which allows the nucleic acid amplification and signal detection to be performed in one reaction vessel that can remain closed during the entire process.

Accordingly, in some embodiments, provided herein are methods of detecting a target nucleic acid in a sample, comprising (1) amplifying the target nucleic acid by incubating a reaction mixture comprising the sample under amplification condition, wherein the reaction mixture comprises: (a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; and (b) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; and (2) detecting the target nucleic acid with a complementary oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; wherein (a) an RNase H cleaves the ribonucleotides on the double-stranded amplicons generated in Step (1) ; (b) the DNA polymerase recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ; (c) the oligonucleotide probe hybridizes with the single-strand target; (d) the RNase H cleaves the ribonucleotide on the hybridized probe, separating F from Q and producing a detectable signal; and (e) the cleaved probe separates from the single-strand target, allowing the repetition of steps (c) , (d) and (e) . In some embodiments, a reversibly inactivated RNase H is included in the reaction mixture of Step (1) and becomes reactivated at the end of Step (1) .

6.2.1 Target nucleic acid

The target nucleic acid to be detected by methods disclosed herein can be any desired nucleic acid. For example, the target nucleic acid can be naturally occurring or synthetic. In some embodiments, the target nucleic acid is a naturally occurring nucleic acid. A naturally occurring nucleic acid can be a nucleic acid isolated and/or purified from a natural source. In some embodiments, the target nucleic acid is a synthetic nucleic acid.

The methods disclosed herein can be used to detect both DNA and RNA. In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA. For illustrative purposes, in some embodiments, the target nucleic acid that can be detected by methods disclosed herein can be a genomic DNA, a plasmid DNA, a dsDNA such as an amplification product by PCR or other amplification method, a ssDNA such as a cDNA prepared by the reverse transcription reaction from total RNA or messenger RNA, and the like. Additionally, those in which a dsDNA is denatured or un-stabilized in such a manner that it becomes a single-stranded DNA completely or partially can also be detected by methods disclosed herein. If the target nucleic acid is an RNA molecule or has ribonucleotide (s) , it can serve as the template to be reverse transcribed to DNA using enzymes and methods known in art.

In some embodiments, the target nucleic acid is DNA, e.g., a target DNA. Exemplary target DNAs include, but are not limited to, genomic DNA, viral DNA, cDNA, ssDNA, dsDNA, circular DNA, etc. In some embodiments, the target nucleic acid is ssDNA. In some embodiments, the target nucleic acid is dsDNA. In some embodiments, the target nucleic acid is an RNA, e.g., a target RNA. Generally, the RNA can be any known type of RNA. In some embodiments of the various aspects described herein, the target RNA is messenger RNA, ribosomal RNA, signal recognition particle RNA, transfer RNA, transfer-messenger RNA, small nuclear RNA, small nucleolar RNA, SmYRNA, small Cajal body-specific RNA, guide RNA, ribonuclease P, ribonuclease MRP, Y RNA, telomerase RNA component, spliced leader RNA, antisense RNA, cis-natural antisense transcript, CRISPR RNA, long noncoding RNA, microRNA, Piwi-interacting RNA, small interfering RNA, short hairpin RNA, trans-acting siRNA, repeat associated siRNA, 7SK RNA, enhancer RNA, parasitic RNAs, retrotransposon, viral genome, viroid, satellite RNA, or vault RNA. In some embodiments, the target nucleic acid is dsRNA. In some embodiments, the target nucleic acid is ssRNA.

In some embodiments, the target RNA can be a viral RNA. As used herein and understood in the art, “RNA virus” refers to a virus comprising an RNA genome. In some embodiments, the RNA virus is a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, or a reverse transcribing virus (e.g., retrovirus) . In some embodiments, the RNA virus is a Group III (i.e., dsRNA) virus. In some embodiments, the Group III RNA virus belongs to a viral family selected from the group consisting of: Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endomaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae (e.g., rotavirus) , Totiviridae, Quadriviridae. In some embodiments, the Group III RNA virus belongs to the Genus Botybirnavirus. In some embodiments, the Group III RNA virus is an unassigned species selected from the group consisting of: Botrytis porri RNA virus 1, Circulifer tenellus virus 1, Colletotrichum camelliae filamentous virus 1, Cucurbit yellows associated virus, Sclerotinia sclerotiorum debilitation-associated virus, and Spissistilus festinus virus 1.

In some embodiments, the RNA virus is a Group IV (i.e., positive-sense ssRNA) virus. In some embodiments, the Group IV RNA virus belongs to a viral order selected from the group consisting of: Nidovirales, Picornavirales, and Tymovirales. In some embodiments, the Group IV RNA virus belongs to a viral family selected from the group consisting of: Arteriviridae, Coronaviridae (e.g., coronavirus, SARS-CoV) , Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Mamaviridae, Picornaviridae (e.g., poliovirus, Rhinovirus (acommon cold virus) , hepatitis A virus) , Secoviridae (e.g., sub Comovirinae) , Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Bamaviridae, Benyviridae, Bromoviridae, Caliciviridae (e.g., Norwalk virus) , Carmotetraviridae, Closteroviridae, Flaviviridae (e.g., yellow fever virus, West Nile virus, hepatitis C virus, Dengue fever virus, Zika virus) , Fusariviridae, Hepeviridae, Hypoviridae, Leviviridae, Luteoviridae (e.g., barley yellow dwarf virus) , Polycipiviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Sarthroviridae, Statovirus, Togaviridae (e.g., Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus) , Tombusviridae, and Virgaviridae. In some embodiments of the various aspects described herein, the Group IV RNA virus belongs to a viral genus selected from the group consisting of: Bacillariornavirus, Dicipivirus, Labyrnavirus, Sequiviridae, Blunervirus, Cilevirus, Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sinaivirus, and Sobemovirus. In some embodiments of the various aspects described herein, the Group IV RNA virus is an unassigned species selected from the group consisting of: Acyrthosiphon pisum virus, Bastrovirus, Blackford virus, Blueberry necrotic ring blotch virus, Cadicistrovirus, Chara australis virus, Extra small virus, Goji berry chlorosis virus, Hepelivirus, Jingmen tick virus, Le Blanc virus, Nedicistrovirus, Nesidiocoris tenuis virus l, Niflavirus, Nylanderiafulva virus 1, Orsay virus, Osedaxjaponicus RNA virus 1, Picalivirus, Plasmopara halstedii virus, Rosellinia necatrix fusarivirus 1, Santeuil virus, Secalivirus, Solenopsis invicta virus 3, Wuhan large pig roundworm virus. In some embodiments of the various aspects described herein, the Group IV RNA virus is a satellite virus selected from the group consisting of: Family Sarthroviridae, Genus Albetovirus, Genus Aumaivirus, Genus Papanivirus, Genus Virtovirus, and Chronic bee paralysis virus.

In some embodiments, the RNA virus is a Group V (i.e., negative-sense ssRNA) virus. In some embodiments, the Group V RNA virus belongs to a viral phylum or subphylum selected from the group consisting of: Negarnaviricota, Haploviricotina, and Polyploviricotina. In some embodiments, the Group V RNA virus belongs to a viral class selected from the group consisting of: Chunqiuviricetes, Ellioviricetes, Insthoviricetes, Milneviricetes, Monjiviricetes, and Yunchangviricetes. In some embodiments, the Group V RNA virus belongs to a viral order selected from the group consisting of: Articulavirales, Bunyavirales, Goujianvirales, Jingchuvirales, Mononegavirales, Muvirales, and Serpentovirales. In some embodiments, the Group V RNA virus belongs to a viral family selected from the group consisting of: Amnoonviridae (e.g., Taastrup virus) , Arenaviridae (e.g., Lassa virus) , Aspiviridae, Bomaviridae (e.g., Boma disease virus) , Chuviridae, Cruliviridae, Feraviridae, Filoviridae (e.g., Ebola virus, Marburg virus) , Fimoviridae, Hantaviridae, Jonviridae, Mymonaviridae, Nairoviridae, Nyamiviridae, Orthomyxoviridae (e.g., influenza viruses) , Paramyxoviridae (e.g., measles virus, mumps virus, Nipah virus, Hendra virus, and NDV) , Peribunyaviridae, Phasmaviridae, Phenuiviridae, Pneumoviridae (e.g., RSV and metapneumovirus) , Qinviridae, Rhabdoviridae (e.g., Rabies virus) , Sunviridae, Tospoviridae, and Yueviridae. In some embodiments, the Group V RNA virus belongs to a viral genus selected from the group consisting of: Anphevirus, Arlivirus, Chengtivirus, Crustavirus, Tilapineviridae, Wastrivirus, and Deltavirus (e.g., hepatitis D virus) .

In some embodiments, the RNA virus is a Group VI RNA virus, which comprise a virally encoded reverse transcriptase. In some embodiments, the Group VI RNA virus belongs to the viral order Ortervirales. In some embodiments, the Group VI RNA virus belongs to a viral family or subfamily selected from the group consisting of: Belpaoviridae, Caulimoviridae, Metaviridae, Pseudoviridae, Retroviridae (e.g., retroviruses, e.g., HIV) , Orthoretrovirinae, and Spumaretrovirinae. In some embodiments, the Group VI RNA virus belongs to a viral genus selected from the group consisting of: Alpharetrovirus (e.g., avian leukosis virus; Rous sarcoma virus) , Betaretrovirus (e.g., mouse mammary tumour virus) , Bovispumavirus (e.g., bovine foamy virus) , Deltaretrovirus (e.g., bovine leukemia virus; human T-lymphotropic virus) , Epsilonretrovirus (e.g., Walleye dermal sarcoma virus) , Equispumavirus (e.g., equine foamy virus) , Felispumavirus (e.g., feline foamy virus) , Gammaretrovirus (e.g., murine leukemia virus; feline leukemia virus) , Lentivirus (e.g., human immunodeficiency virus 1; simian immunodeficiency virus; feline immunodeficiency virus) , Prosimiispumavirus (e.g., Brown greater galago prosimian foamy virus) , and Simiispumavirus (e.g., eastern chimpanzee simian foamy virus) .

In some embodiments, the RNA virus is selected from influenza virus, human immunodeficiency virus (HIV) , and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . In some embodiments, the RNA virus is influenza virus. In some embodiments of the various aspects described herein, the RNA virus is immunodeficiency virus (HIV) . In some embodiments, the RNA virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) .

In some embodiments, the viral RNA is an RNA produced by a virus with a DNA genome, i.e., a DNA virus. As a non-limiting example, the DNA virus is a Group I (dsDNA) virus, a Group II (ssDNA) virus, or a Group VII (dsDNA-RT) virus.

In some embodiments, the target nucleic acid is a viral nucleic acid. In some embodiments, the target nucleic acid is a viral RNA. In some embodiments, the target nucleic acid is a viral DNA.

Methods provided herein can detect a target nucleic acid in a sample. The sample can be obtained from any supply source having a possibility of containing the target nucleic acid. In some embodiments, a sample can be taken or isolated from a biological organism, e.g., a subject in need of testing. A sample can also be obtained from an environmental source, food, agricultural product, or fermented product. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample: blood; serum; plasma; urine; semen; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva, and/or tissue sample, etc. A sample can also be a mixture of the above-mentioned samples. A sample can be untreated or pretreated (or pre-processed) biological samples. In some embodiments, a sample can comprise cells from a subject.

In some embodiments, the sample can include a viral transport media (VTM) . Non-limiting examples of viral transport media include COPAN Universal Transport Medium; Eagle Minimum Essential Medium (E-MEM) ; Transport medium 199; and PBS-Glycerol transport medium. See e.g., Johnson, Transport of Viral Specimens, CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 120-31; Collecting, preserving and shipping specimens for the diagnosis of avian influenza A (H5N1) virus infection, GUIDE FOR FIELD OPERATIONS, October 2006.

In some embodiments, prior to amplification, target nucleic acids are isolated or purified from the sample. Nucleic acids can be isolated from a particular biological sample using any procedures known in the art and appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials (Roiff, A et al. PCR: CLINICAL DIAGNOSTICS AND RESEARCH, Springer (1994) ) . Non- limiting examples of methods for isolating/purifying nucleic acids from test samples include: (1) organic extraction, such as phenol-Guanidine Isothiocyanate (GITC) -based solutions (e.g., TRIZOL and TRI reagent) ; (2) silica-membrane based spin column technology (e.g., RNeasy and its variants) ; (3) paramagnetic particle technology (e.g., DYNABEADS mRNA DIRECT MICRO) ; (4) density gradient centrifugation using cesium chloride or cesium trifluoroacetate; (5) lithium chloride and urea isolation; (6) oligo (dt) -cellulose column chromatography; (7) non-column poly (A) +purification/isolation; (8) organic extraction; (9) CHELEX 100 extraction; and (10) solid phase extraction.

In some embodiments, the sample can be an untreated sample. An untreated sample refers to a sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the sample can be a frozen test sample. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the sample is a clarified sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, homogenization, sonication, filtration, thawing, purification, and any combinations thereof. In some embodiments, the sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed, for example, to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. In some embodiments, the sample can be a product completely or partially treated with a restriction enzyme or other nucleic acid-cleaving or degrading enzyme or the like. The skilled artisan can determine methods and processes appropriate for pre-processing of biological samples required for detection of a nucleic acid as described herein.

6.2.2 Amplification of target nucleic acids

Methods of nucleic acid detection provided herein include a step of nucleic acid of amplification. As used herein and understood in the art, amplification of a nucleic acid refers to the production of additional copies of the nucleic acid, i.e., amplicons or amplification products. An amplification reaction refers to an enzymatic reaction that results in increased copies of a target nucleic acid sequence. Amplification reactions can also include reverse transcription. In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample, comprising amplifying the target nucleic acid by incubating a reaction mixture comprising the sample under amplification condition. An amplification condition is a condition that allows the amplification of the target nucleic acid. As understood in the art, amplification conditions can vary, which depend on factors such as the specific enzymes used and the target nucleic acid to be amplified. Subjecting a reaction mixture to an amplification condition means that the reaction mixture is supplied with the necessary components (including, e.g., ATP, dNTP, primers, buffer, etc. ) and incubated at a temperature that allows the polymerase to carry out the amplification.

Methods for amplifying nucleic acids are well known in the art and amenable to the methods and systems described herein. Such methods include, but are not limited to, polymerase chain reaction (PCR) and variants of PCR such as Rapid amplification of cDNA ends (RACE) , ligase chain reaction (LCR) , multiplex RT-PCR, immuno-PCR, SSIPA, Real Time RT-qPCR and nanofluidic digital PCR.

In some embodiments, the nucleic acid detection methods disclosed herein can use PCR-based thermal cycling techniques for nucleic acid amplification. PCR is well known as a typical technique of nucleic acid amplification. This method synthesizes a target sequence in vitro by the action of DNA polymerase activity using two oligonucleotide primers which respectively hybridize with separate DNA chains at both termini of the target dsDNA region. As is also well known, PCR can be combined with reverse transcriptase for the purpose of amplifying a target sequence in RNA which is referred to as RT-PCR.

In these PCR methods, a specific double-stranded nucleic acid fragment specified by 5’-ends of the two primers is exponentially accumulated as the amplicons by repeating a reaction consisting of three steps of (1) dissociation (denaturation) of the double-stranded nucleic acids into single-stranded nucleic acids, (2) hybridization (annealing) of the primers to the single-stranded nucleic acids and (3) synthesis (elongation) of template-dependent complementary chain from the primers. Thus, repetition of these three steps by adjusting the reaction solution at temperatures which are respectively suited for these three steps (thermal cycle) is required. Since the denaturation step requires a high temperature (e.g., 95℃) , a heat-resistant DNA polymerase is used, and the PCR reactions are commonly automated by a temperature cycling device.

The reaction mixture for PCR typically contains oligonucleotide primers, a DNA polymerase (typically a thermostable DNA polymerase) , dNTPs, and a divalent metal cation in a suitable buffer.

PCR requires heating the amplification composition at each cycle to about 95℃ to denature the double strand target sequence. In some embodiments, the heating can also release the inactivating factor from the RNase H, partially or fully restoring the activity of the enzyme.

In some embodiments of methods disclosed herein, the amplification step comprises isothermal amplification. As used herein and understood in the art, “isothermal amplification” refers to amplification that occurs at a constant temperature. For example, the amplification process is performed at a single temperature or where the major aspect of the amplification process is performed at a single temperature. Generally, isothermal amplification relies on the ability of a polymerase to copy the template strand being amplified to form a bound duplex. In general, isothermal amplification comprises (i) sequence-specific hybridization of primers to sequences within a target nucleic acid, and (ii) subsequent amplification involving multiple rounds of primer annealing, elongation, and strand displacement (as a non-limiting example, using a combination of recombinase, single-stranded binding proteins, and DNA polymerase) .

The primers used in both PCR and isothermal amplification are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e., each primer is specifically designed to be complementary to a strand of the target nucleic acid to be amplified.

Non-limiting examples of isothermal amplification include: enzyme-mediated amplification (EMA) , Loop Mediated Isothermal Amplification (LAMP) , Recombinase Polymerase Amplification (RPA) , cross-priming amplification (CPA) , Helicase-dependent isothermal DNA amplification (HDA) , Rolling Circle Amplification (RCA) , Nucleic acid sequence-based amplification (NASBA) , strand displacement amplification (SDA) , nicking enzyme amplification reaction (NEAR) , polymerase Spiral Reaction (PSR) , hybridization chain reaction (HCR) , primer exchange reaction (PER) , signal amplification by exchange reaction (SABER) , transcription-based amplification system (TAS) , self-sustained sequence replication reaction (3 SR) , and single primer isothermal amplification (SPIA) . See e.g., Yan et al., 2014, Molecular BioSystems 10 (5) , the content of which is incorporated herein by reference in its entirety. For illustrative purposes, some of these methods are described in further detail below.

In some embodiments, the isothermal amplification reaction (s) is Enzyme-Mediated Amplification (EMA) . See e.g., CN104059905B, the content of which is incorporated herein by reference in its entirety. The EMA approach coordinates the activities of prokaryotic recombinase and eukaryotic RFC and PCNA, which enables rapid amplification of template nucleic acid at room temperature. In some embodiments, human Polymerase η can be used.

In some embodiments, the isothermal amplification reaction (s) is loop Mediated Isothermal Amplification (LAMP) , i.e., the step of amplifying the target nucleic acids comprises Loop Mediated Isothermal Amplification. LAMP is a single tube technique for the amplification of nucleic acids; LAMP uses 4-6 primers, which form loop structures to facilitate subsequent rounds of amplification. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a strand-displacing DNA polymerase and a set of primers, wherein the set of primers comprises 4 or 6 loop-forming primers. In the method, a loop structure is formed by introducing a region, in which the sequence becomes self-complementary, into a terminal region of a target nucleic acid. The 3’ end which becomes the starting point of the elongation reaction is provided by the self-complementary hybridization at the time of the formation of the loop structure or by annealing of a primer to a single-stranded loop region formed by the formation of the loop structure. Said 3’ end is elongated by the action of a strand displacement type DNA polymerase and its downstream DNA chain is displaced.

In some embodiments, the isothermal amplification reaction (s) is Recombinase Polymerase Amplification (RPA) , i.e., the step of amplifying the target nucleic acids comprises Recombinase Polymerase Amplification. RPA is a low temperature DNA and RNA amplification technique. The RPA process employs three core enzymes -a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing polymerase. Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. SSB bind to displaced strands of DNA and prevent the primers from being displaced. Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA. By using two opposing primers, much like PCR, if the target sequence is indeed present, an exponential DNA amplification reaction is initiated. No other sample manipulation such as thermal or chemical melting is required to initiate amplification. At optimal temperatures (e.g., 37-42 ℃) , the RPA reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 10 minutes, for rapid detection of the target nucleic acid. In some embodiments, the single-stranded DNA-binding protein is a gp32 SSB protein. In some embodiments, the recombinase is a uvsX recombinase. See e.g., US Patent 7,666,598, the content of which is incorporated herein by reference in its entirety. In some embodiments, RPA can also be referred to as Recombinase Aided Amplification (RAA) . Accordingly, in some embodiments, the amplification step comprises contacting the sample with a recombinase and single-stranded DNA binding protein. In some embodiments, the amplification step comprises contacting the sample with a strand-displacing DNA polymerase, a set of primers, a recombinase, and single-stranded DNA binding protein.

In some embodiments, the isothermal amplification reaction (s) is Helicase-dependent isothermal DNA amplification (HDA) . HDA uses the double-stranded DNA unwinding activity of a helicase to separate strands for in vitro DNA amplification at constant temperature. In some embodiments, the helicase is a thermostable helicase, which can improve the specificity and performance of HDA; as such, the isothermal amplification reaction (s) can be thermophilic helicase-dependent amplification (tHDA) . As a non-limiting example, the helicase is the thermostable UvrD helicase (Tte-UvrD) , which is stable and active from 45℃ to 65 ℃. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase, a set of primers, and a helicase, wherein the helicase is optionally a thermostable helicase.

In some embodiments, the isothermal amplification reaction (s) is Rolling Circle Amplification (RCA) . RCA starts from a circular DNA template and a short DNA or RNA primer to form a long single-stranded molecule. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample (e.g., a circular DNA) with a DNA polymerase and a set of primers, wherein the second set of primers comprises a single primer.

In some embodiments, the isothermal amplification reaction (s) is Nucleic acid sequence-based amplification (NASBA) , which is also known as transcription mediated amplification (TMA) . NASBA is an isothermal technique predominantly used for the amplification of RNA through the cyclic formation of complimentary DNA and destruction of original RNA sequence (e.g., using RNase H) . The NASBA reaction mixture contains three enzymes: reverse transcriptase (RT) , RNase H, and T7 RNA polymerase -and two primers. T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5' to 3' direction. Primer 1 (P1) contains a 3' terminal sequence that is complementary to a sequence on the target nucleic acid and a 5' terminal (+) sense sequence of a promoter that is recognized by the T7 RNA polymerase. Primer 2 (P2) contains a sequence complementary to the P1 -primed DNA strand. The NASBA enzymes and primers operate in concert to amplify a specific nucleic acid sequence exponentially. NASBA results in the amplification of the target RNA to cDNA to RNA to cDNA, etc., with alternating reverse transcription (e.g., RNA to DNA) and transcription steps (e.g., DNA to RNA) , and the RNA being degraded after each transcription. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample (e.g., a cDNA) with an RNA polymerase, a reverse transcriptase, RNase H, and a set of primers, wherein the set of primers comprise a 5’ sequence that is recognized by the RNA polymerase.

In some embodiments, the isothermal amplification reaction (s) is Strand Displacement Amplification (SDA) . SDA is an isothermal, in vitro nucleic acid amplification technique based upon the ability of the restriction endonuclease HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo-klenow) DNA polymerase to extend the 3’-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase (e.g., exo-klenow) , a set of primers, and a restriction endonuclease (e.g., HincII) .

In some embodiments, the isothermal amplification reaction (s) is nicking enzyme amplification reaction (NEAR) , which is a similar approach to SDA. In NEAR, DNA is amplified at a constant temperature (e.g., 55 ℃ to 59 ℃) using a polymerase and nicking enzyme. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase (e.g., exo-klenow) , a set of primers, and a nicking enzyme (e.g., N.BstNBI) .

In some embodiments, the isothermal amplification reaction (s) is Polymerase Spiral Reaction (PSR) . The PSR method employs a DNA polymerase (e.g., Bst) and a pair of primers. The forward and reverse primer sequences are reverse to each other at their 5’ end, whereas their 3’ end sequences are complementary to their respective target nucleic acid sequences. The PSR method is performed at a constant temperature 61-65 ℃, yielding a complicated spiral structure. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase (e.g., exo-klenow) and a set of primers that are reverse to each other at their 5’ end.

In some embodiments, the isothermal amplification reaction (s) is polymerase cross-linking spiral reaction (PCLSR) . PCLSR uses three primers (e.g., two outer-spiral primers and a cross-linking primer) to produce three independent prerequisite spiral products, which can be cross-linked into a final spiral amplification product. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase and a set of primers (e.g., two outer-spiral primers and a cross-linking primer) .

In some embodiments, the isothermal amplification step is performed at a temperature between from about 12℃ to about 45℃. As a non-limiting example, the isothermal amplification step is performed at least 12℃, at least 13℃, at least 14℃, at least 15℃, at least 16℃, at least 17℃, at least 18℃, at least 19℃, at least 20℃, at least 21℃, at least 22℃, at least 23℃, at least 24℃, at least 25℃, at least 26℃, at least 27℃, at least 28℃, at least 29℃, at least 30℃, at least 31℃, at least 32℃, at least 33℃, at least 34℃, at least 35℃, at least 36℃, at least 37℃, at least 38℃, at least 39℃, at least 40℃, at least 41℃, at least 42℃, at least 43℃, at least 44℃, or at least 45℃.

In some embodiments, the isothermal amplification step is performed at a temperature of at most 12℃, at most 13℃, at most 14℃, at most 15℃, at most 16℃, at most 17℃, at most 18℃, at most 19℃, at most 20℃, at most 21℃, at most 22℃, at most 23℃, at most 24℃, at most 25℃, at most 26℃, at most 27℃, at most 28℃, at most 29℃, at most 30℃, at most 31℃, at most 32℃, at most 33℃, at most 34℃, at most 35℃, at most 36℃, at most 37℃, at most 38℃, at most 39℃, at most 40℃, at most 41℃, at most 42℃, at most 43℃, at most 44℃, or at most 45℃.

In some embodiments, the isothermal amplification step is performed at a temperature of about 12℃, about 13℃, about 14℃, about 15℃, about 16℃, about 17℃, about 18℃, about 19℃, about 20℃, about 21℃, about 22 ℃, about 23 ℃, about 24℃, about 25℃, about 26℃, about 27℃, about 28℃, about 29℃, about 30℃, about 31℃, about 32℃, about 33℃, about 34℃, about 35℃, about 36℃, about 37℃, about 38℃, about 39℃, about 40℃, about 41℃, about 42℃, about 43℃, about 44℃, or about 45℃.

In some embodiments, the isothermal amplification step is performed at room temperature (e.g., 20-22℃) . In some embodiments, the isothermal amplification step is performed at body temperature (e.g., 37℃) . In some embodiments, the isothermal amplification step is performed at about 42℃, e.g., on a heat block set to approximately 42℃.

The amplification step such as isothermal amplification step can be performed for any period of time to produce sufficient double strand amplicons. For example, the amplification step can be for a period of from about 5 minutes to about 4 hours. In some embodiments, the amplification step can be for a period of from about 5 minutes to about 1 hour. In some embodiments, the amplification step can be for a period of from about 5 minutes to about 20minutes.

In some embodiments, the amplification step can be performed for about 5 minutes. As a non-limiting example, the isothermal amplification step is performed for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 70 minutes, about 75 minutes, about 80 minutes, or about 90 minutes.

In some embodiments, the amplification step is performed for at most 5 minutes, at most 6 minutes, at most 7 minutes, at most 8 minutes, at most 9 minutes, at most 10 minutes, at most 11 minutes, at most 12 minutes, at most 13 minutes, at most 14 minutes, at most 15 minutes, at most 16 minutes, at most 17 minutes, at most 18 minutes, at most 19 minutes, at most 20 minutes, at most 21 minutes, at most 22 minutes, at most 23 minutes, at most 24 minutes, at most 25 minutes, at most 26 minutes, at most 27 minutes, at most 28 minutes, at most 29 minutes, at most 30 minutes, at most 31 minutes, at most 32 minutes, at most 33 minutes, at most 34 minutes, at most 35 minutes, at most 36 minutes, at most 37 minutes, at most 38 minutes, at most 39 minutes, at most 40 minutes, at most 41 minutes, at most 42 minutes, at most 43 minutes, at most 44 minutes, at most 45 minutes, at most 46 minutes, at most 47 minutes, at most 48 minutes, at most 49 minutes, at most 50 minutes, at most 51 minutes, at most 52 minutes, at most 53 minutes, at most 54 minutes, at most 55 minutes, at most 56 minutes, at most 57 minutes, at most 58 minutes, at most 59 minutes, at most 60 minutes, at most 70 minutes, at most 75 minutes, at most 80 minutes, or at most 90 minutes.

6.2.2.1 Polymerase

DNA polymerase activity is required for nucleic acid amplification. As used herein and understood in the art, a polymerase is an enzyme that catalyzes the polymerization of nucleotides. Generally, the enzyme initiates synthesis at the 3’-end of the primer annealed to a nucleic acid template sequence. DNA polymerase catalyzes the polymerization of deoxyribonucleotides. In some embodiments, methods provided herein use DNA polymerase with strand displacement activity. As known in the art, the term strand displacement describes the ability of the enzyme to displace downstream nucleotides encountered during synthesis by elongating the 3’-end. When synthesis of a new complementary strand is carried out in accordance with the template strand, the old complementary strand which is present in the synthesis proceeding direction and is already formed a double strand with the template strand is released. DNA polymerase with strand displacement activity used herein does not have 5’ to 3’ exonuclease activity.

As a person of ordinary skill in the art would understand, methods provided herein are not limited to using specific DNA polymerases, and any DNA polymerase with strand displacement activity and lacking 5’ to 3’ exonuclease activity can be suitable. For illustrative purpose, the following DNA polymerase can be used in methods disclosed herein: Klenow fragment of DNA polymerase I derived from Escherichia coli; Phi29 DNA polymerase derived from bacteriophage phi29; 5’ to 3’ exonuclease-deficient DNA polymerase derived from bacteriophage T7 (e.g., Sequenase or the like) ; 5’ to 3’ exonuclease-deficient Bst DNA polymerase derived from Bacillus stearothermophilus; 5’ to 3’ exonuclease-deficient Bsu DNA polymerase derived from Bacillus subtilis DNA Polymerase; 5’ to 3’ exonuclease-deficient Bca DNA polymerase derived from Bacillus caldotenax (e.g., BcaBEST DNA polymerase or the like) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Pyrococcus sp. GB-D (e.g., Deep VentR DNA polymerase, Deep VentR (exo-) DNA polymerase or the like) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Pyrococcus furiosus (e.g., Pfu DNA polymerase, Pfu Turbo DNA polymerase or the like) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Staphylococcus aureus (e.g., Sau DNA Polymerase I, Large Fragment) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Thermus aquaticus (e.g., Stoffel Fragment, Z-Taq DNA polymerase, TopoTaq DNS polymerase or the like) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Thermus thermophilus (e.g., ΔTth DNA polymerase or the like) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Thermococcus sp. 9° N-7 (e.g., 9° Nm DNA polymerase, Therminator DNA polymerase or the like) ; 5’ to 3’ exonuclease-deficient DNA polymerase derived from Thermococcus litoralis (e.g., Tli DNA polymerase, VentR DNA polymerase, VentR (exo-) DNA polymerase or the like) , and 5’ to 3’ exonuclease-deficient DNA polymerase derived from Thermococcus kodakaraensis strain KOD1 (e.g., KOD DNA polymerase, KOD Dash DNA polymerase, KOD-Plus-DNA polymerase or the like) ; or SD polymerase (avariation of Taq polymerase) ; or functional variants thereof.

Combinations of the DNA polymerases disclosed herein can be used for the amplification as appropriate. In some embodiments of the methods disclosed herein, two or more DNA polymerases are used for nucleic acid amplification, wherein at least one of the DNA polymerases have strand-displacement activity.

Enzyme provided herein are commercially available from, e.g., New England GoldPCRandThe polymerase activity and the strand displacement activity of any of the above enzymes can be determined by means well known in the art.

Any of the disclosed DNA polymerase with strand displacement activity or functional variants thereof can be used in methods disclosed herein. For example, in some embodiments, methods provided herein use an ΔTth DNA polymerase or a functional variant thereof. In some embodiments, methods provided herein use Sau DNA Polymerase I, Large Fragment ( “SAP Pol” ) , or a functional variant thereof. In some embodiments, methods provided herein use Klenow fragment, or a functional variant thereof. In some embodiments, methods provided herein use Phi29 DNA polymerase, or a functional variant thereof.

In accordance with the use of DNA/RNA-mixed primers in methods disclosed herein. The double-stranded amplicons formed in the amplification step would include ribonucleotides. In some embodiments, the DNA polymerase with strand-displacement activity also has reverse transcriptase activity. In some embodiments, the reaction mixture further comprises a reverse transcriptase. The reverse transcriptase used in methods disclosed herein does not have exonuclease activity.

In some embodiments, the DNA polymerase used in methods disclosed herein further has reverse transcriptase activity. In some embodiments, the reaction mixture of the amplification step further includes a reverse transcriptase. Any reverse transcriptase known in the art that does not have exonuclease activity can be used in methods disclosed herein. In some embodiments, the reverse transcriptase can be M-MLV reverse transcriptase or AMV reverse transcriptase.

Regarding the DNA polymerase and reverse transcriptase to be used in the methods and systems disclosed herein, any one of from mesophilic to heat-resistant ones can be suitably used.

The strand displacement type DNA polymerase to be used in the present invention can be either a substance obtained by purifying from a natural resource or a recombinant protein produced by means of a genetic engineering. Additionally, said enzyme can be those to which modifications such as substitution, deletion, addition, insertion and the like were applied by a genetic engineering or other methods. Methods of genetic engineering and recombinant production are well known in the art.

6.2.2.2 Primers

The reaction mixtures used in the amplification steps include primers. The term “primer, ” as used herein and understood in the art, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (e.g., a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.

A primer for amplifying a specific target nucleic acid need not reflect the exact sequence of the target nucleic acid, but must be sufficiently complementary to hybridize with the nucleic acid. The design of suitable primers for the amplification of a given target sequence is well known in the art. In general, the primer is designed in such a manner that it can be annealed to the target nucleic acid by such a positional relation that the region to be amplified is set on the downstream side of said primer. The primer is designed in such a manner that it is substantially complementary for the nucleotide sequence of the region to which it is going to be annealed. It is conventionally known to those skilled in the art that primers should be designed to avoid or minimize the hybridization between primers. Primers to be used in methods disclosed herein can be substantially complementary with the nucleotide sequence of the target nucleic acid, and can anneal to the target nucleic acid under the conditions to be used. Melting temperature, GC content, nucleotide sequence, length and the like should be considered. Software and services for primer designing are commercially available and can be used.

In designing the primer to be used in methods disclosed herein, the nucleotide sequence of the target nucleic acid is generally used as reference. Nonetheless, it is not necessary that the exact nucleotide sequence of the target nucleic acid is completely know. It would be sufficient if the information for designing a primer substantially complementary with the annealing region is available. For example, the primer that would be proper for use in methods disclosed herein can be designed although the nucleotide sequence of the annealing region may have mutations such as unknown substitution, deletion, addition, insertion and the like.

The region of the primer which is sufficiently complementary to the target nucleic acid to hybridize is referred to herein as the hybridizing region. In addition to the hybridizing region, the primers to be used in the methods disclosed herein can have an additional sequence which does not anneal to the target nucleic acid, in the upstream side and/or downstream side of said sequence, which do not alter the basic of the primers of acting as a point of initiation of DNA synthesis. Examples of such additional sequence include a restriction enzyme recognition sequence, a DNA binding protein recognition sequence, a sequence which is recognized by other protein or nucleic acid or by a chemical reagent, a sequence which can form a hairpin structure or stem loop structure by self-annealing, or an optional nucleotide sequence, a nonsense nucleotide sequence and the like. Additional features which allow for the detection or immobilization of the primer can also be included, which do not alter the basic function of the primer of acting as a point of initiation of DNA synthesis.

The appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 50 nucleotides, preferably from 15-35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Primers used herein can include from 10 to 50 nucleotides, from 15 to 35 nucleotides, or from 18 to 26 nucleotides.

Primer pairs are a couple of primers that direct DNA elongation toward each other at opposite ends of the sequence being amplified. The primer pair can include a forward primer and a reverse primer that code for the specific upstream and downstream sites of the sequence being amplified. When used in PCR, pairs of primers should have similar melting temperatures (Tm) since annealing occurs for both strands simultaneously. In some embodiments, primer pairs can amplify a target of about 50 base pairs (bp) to about 50,000 bp, unless indicated otherwise.

A primer can be a ssDNA. A primer can also contains both deoxyribonucleotides and ribonucleotides, which is herein referred to as a DNA/RNA-mixed primer. In methods disclosed herein, primer pairs are used in the amplification step wherein one primer of the primer pair contains at least one ribonucleotide. In other words, one primer of the primer pairs used in methods disclosed herein is a DNA/RNA-mixed primer. Accordingly, double-stranded amplicons formed in the amplification step include ribonucleotides from the mixed primer, and the DNA/RNA hybrid formed between the ribonucleotides in the primer and the DNA template can be recognized and cleaved by RNase H, generating nicking sites on the double-stranded amplicon.

In some embodiments, the DNA/RNA-mixed primer contains about 1-20 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains 1-15, 2-12, 4-12, or 4-8 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains 2-12 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains 4-10 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains 4-8 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains 4-6 ribonucleotides.

In some embodiments, the DNA/RNA-mixed primer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains about 4 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains about 6 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains about 8 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains about 10 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains about 12 ribonucleotides. In some embodiments, the DNA/RNA-mixed primer contains about 16 ribonucleotides.

The DNA/RNA-mixed primers used in methods disclosed herein can contain ribonucleotides either at the 3’ end or between the 5’ and 3’ ends of the primer. The incorporation of a DNA/RNA-mixed primer in the amplification product results in double-stranded amplicons containing ribonucleotide (s) in one strand (FIG. 1A) . RNase H can recognize the DNA/RNA hybrid region and create nicking sites on the double-stranded amplicons; the DNA polymerase can then recognize the nicking sites and release a single strand of the double-stranded amplicon by strand-displacement. To form proper nicking sites recognizable by the RNA polymerase, the DNA/RNA-mixed primers used herein should contain deoxyribonucleotides at or near the 5’ end, or in other words, the ribonucleotide or fragment of ribonucleotides in the mixed primer should be accompanied by deoxyribonucleotides to its 5’s end. As such, after the removal of ribonucleotides by RNase H, the deoxyribonucleotides to the 5’ end of the cleaved ribonucleotides can remain hybridized to the template strand of the double-stranded amplicon, forming nicking sites recognizable by DNA polymerase with strand displacement activity. In some embodiments, the DNA/RNA-mixed primer used in the methods contains at least 6 deoxyribonucleotides to the 5’ end of the ribonucleotide (s) . In some embodiments, the DNA/RNA-mixed primer used in the methods contains at least 8, at least 10, at least 12, at least 15, at least 18, or at least 20 deoxyribonucleotides to the 5’ end of the ribonucleotide (s) . In some embodiments, the DNA/RNA-mixed primer used in the methods contains about 6, about 8, about 10, about 12, about 15, about 18, or about 20 deoxyribonucleotides to the 5’ end of the ribonucleotide (s) .

In methods disclosed herein, the second primer of the primer pair does not form a hybrid with the template nucleic acid that is recognizable by RNase H. In some embodiments, the second primer of the primer pair used is a DNA primer which does not contain any ribonucleotide. In some embodiments, the second primer of the primer pair also contains ribonucleotide (s) , but does not generate another site recognizable by RNase H during amplification albeit the presence of the ribonucleotide (s) . For example, in some embodiments wherein the second primer contains at least one ribonucleotide, none of the ribonucleotide (s) within the second primer can hybridize with the template nucleic acid. In some embodiments, all ribonucleotide (s) of the second primer are located outside the hybridizing region. In some embodiments, the ribonucleotide (s) of the second primer that are located within the hybridizing region are not complementary with the target nucleic acid.

The primers to be used in methods disclosed herein can be synthesized by any methods known in the art, for example the phosphoamidite method, phosphotrimester method, H-phosphonate method, thiophosphonate method or the like, using for example a commercially available automatic DNA synthesizer.

6.2.2.3 Reaction mixture

The term “reaction mixture, ” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. For example, a reaction mixture for nucleic acid amplification refers to a solution containing reagents necessary to carry out an amplification reaction. As understood by a person of ordinary skill in the art, the components of a reaction mixture vary depending on the types of the amplification methods, but typically include oligonucleotide primers, DNA polymerase, deoxyribonucleotide 3-phosphate (dNTP) , and a suitable buffer that commonly includes a divalent metal cation.

As known in the art, the reaction mixture for a nucleic acid amplification method requires a dNTP mixture, namely, a mixture of dATP, dCTP, dGTP and dTTP. In some embodiments, the dNTP to be used in methods disclosed herein can contain other dNTP or a derivative of the dNTP, as long as it can be used as the substrate of the DNA polymerase. Examples of the other dNTP or a derivative of the dNTP include dUTP, dITP, 7-deaza-dGTP, α-S-dNTP in which oxygen atom of the α-position phosphate group is replaced by sulfur atom, dNTP labeled with a radioisotope, a fluorescent material or the like.

The reaction mixture in the nucleic acid amplification step can comprise a buffer agent which provides the enzyme activities with suitable conditions (e.g., pH, metal ion concentration, salt concentration and the like) , a metal ion providing substance, salts and the like. Examples of buffer agents used in methods disclosed herein include conventionally known buffer agents generally used by those skilled in the art, such as Tris, Tricine, Bicine, HEPES, MOPS, TES, TAPS, PIPES, CAPS, a phosphate (e.g., sodium phosphate, potassium phosphate) and the like.

A metal ion providing substance is generally included in the reaction mixture for nucleic acid amplification. The metal ion providing substance can be a conventionally known substance generally used by those skilled in the art. For example, when the desired metal ion is Mg²⁺, examples of the provided substance include magnesium chloride, magnesium acetate, magnesium sulfate and the like. Additionally, the salts can also be conventionally known substances generally used by those skilled in the art. Examples thereof include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like. Also, as a matter of course, suitable selection and suitable concentration of these substances can be changed according to the kind and combination of the enzymes to be used. Additionally, as these substances can affect the melting temperature of nucleic acid, and the dNTP can also affect the concentration of a free metal ion by chelating the metal ion, those skilled in the art can select an optimum reaction mixture by taking into consideration these factors and the like.

For illustrative purposes, in some embodiments, concentration of buffer agent in the reaction mixture can be from 1 to 100 mM, or from 5 to 50 mM. In some embodiments, pH of the buffer agent is from 6.0 to 9.5, or from 7.0 to 8.8. Concentration of magnesium chloride, magnesium acetate, magnesium sulfate or another magnesium salt can be from 0.2 to 20 mM, or from 2 to 12 mM. Also, concentration of potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate or salt alike can be from 1 to 200 mM, or from 2 to 125 mM.

In some embodiments, concentration of each dNTP in the reaction mixture can be from 0.1 to 3.0 mM, or from 0.2 to 1.2 mM.

In some embodiments, the amount of each primer in the reaction mixture used in methods disclosed herein can be from 0.1 to 1 μM, from 0.1 to 0.5 μM, or about 0.2 μM.

In the reaction mixtures used in methods disclosed herein, the amounts of enzymes can vary according to the kind, property and combination of the enzymes to be used. Additionally, optimum amounts of the enzymes for achieving proper amplification can also be changed according to the using conditions, amount of the primer, amount of the template nucleic acid, other reaction composition and the like. In some embodiments, the DNA polymerase is provided (i.e., added to the reaction mixture) at a concentration sufficient to promote polymerization, e.g., 0.1 U/μL to 100 U/μL, 0.1 U/μL to 10 U/μL, 0.1 U/μL to 5 U/μL, or 0.1 U/μL to 1 U/μL. As used herein, one unit ( “U” ) of DNA polymerase is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at its optimal temperature (e.g., 37℃) .

In some embodiments, separate reverse transcriptase is also included in the reaction mixture at a concentration sufficient to promote ribonucleotide-based polymerase, e.g., about 0.1 U/μL, 0.5 U/μL, 1 U/μL, 5 U/μL, 10 U/μL, 20 U/μL, or 50 U/μL. Depending on the specific amplification methods adopted, additional enzymes can be included, such as DNA helicase, at a concentration of, e.g., about 0.1 U/μL, 0.5 U/μL, 1 U/μL, 5 U/μL, 10 U/μL, or 20 U/μL. A person of ordinary skill in the art would be able to determine and optimize the kind and concentration of enzyme used in the reaction mixture.

The reaction mixture used in methods disclosed herein can further include an additive agent to facilitate the amplification. Non-limiting examples of such additive agent include 10%or less of dimethyl sulfoxide (DMSO) , 3 M or less of betaine (N, N, N-trimethylglycine) , 5%or less of formamide, 100 mM or less of tetramethylammonium chloride (TMAC) , 1%or less of a surfactant (e.g., NP-40, Tween-20, Triton X-100 or the like) , 10%or less of glycerol, 10%or less of a saccharide (dextran or the like) , 10%or less of polyethylene glycol (PEG) , 10 mM or less of dithiothreitol (DTT) , 0.1%or less of bovine serum albumin (BSA) , SSB protein (single-stranded DNA-binding protein) and the like.

If PCR is used for amplification, melting temperature of the target nucleic acid can be adjusted by adding a melting temperature adjusting agent to the reaction mixture. Examples of said melting temperature adjusting agent include betaine, dimethylglycine, triethylamine N-oxide, DMSO and the like. In some embodiments, betaine is used. The concentration of betaine in the reaction mixture can be no more than about 5.2 M, which is its isostabilizing concentration. In some embodiments, the concentration of betaine in the reaction mixture from 0.3 to 1.5 M.

A single-stranded nucleic acid-stabilizing agent can also be included as an additive agent in the reaction mixture used in methods disclosed herein. Examples of the single-stranded nucleic acid-stabilizing agent include a single-stranded nucleic acid-binding protein ( “SSB” ) . Examples of the single-stranded nucleic acid-binding protein include Escherichia coli SSB protein (single-stranded DNA-binding protein) , Escherichia coli RecA protein, T4 phage gp32 or their corresponding proteins derived from other organisms or viruses, and the like. The concentration of these single-stranded nucleic acid-binding proteins in the reaction mixture can be determined by those skilled in the art. For example, in some embodiments, Escherichia coli SSB protein within the range of from 0.5 to 1.5 μg can be included in the reaction mixture. In some embodiments, Escherichia coli RecA protein within the range of from 0.01 to 1 μg/μl can be included in the reaction mixture. In some embodiments, T4 phage gp32 within the range of from 0.01 to 1 μg/μl can be included in the reaction mixture. Additionally, together with these single-stranded nucleic acid-binding proteins, their cofactors (e.g., ATP and its derivatives and the like) can be included as appropriate.

As one of ordinary skill in the art would understand, reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it is understood by one of skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components.

6.2.3 Detection

As described above, the nucleic acid detection methods provided herein include amplification of the nucleic acid and the amplification of the detectable signals. To generate and amplify the detectable signals, the double-stranded amplicons generated from the nucleic acid amplification step are subject to treatment by RNase H and the DNA polymerase with strand-displacement activity, producing single-stranded targets that can be detected by oligonucleotide probes. In particular, the RNase H generates nicking sites on the double-stranded amplicons by cleaving the ribonucleotides derived from the DNA/RNA-mixed primer; and the DNA polymerase then recognizes the nicking sites and releases single strands of the double-stranded amplicons with strand-displacement. The released single strands (or “the single-strand targets” ) can be detected by complementary ribonucleotide-containing oligonucleotide probes attached with both fluorophore (F) and quencher (Q) . In an intact probe, Q quenches the fluorescence signal from F. The probes that are not hybridized are not recognized by RNase H, and the Q prevents F from producing the florescence signal. When the probe is hybridized to a single-strand target, however, the DNA/RNA hybrid region can be recognized by RNase H, which cleaves the ribonucleotides on the probe, separating F from Q and producing a detectable signal. Additionally, as the cleaved probe fragments dissociate from the single-strand target, the single-strand target becomes available for detection by additional probes, and the repeated cleavage of probes by RNase H allows accumulation and linear amplification of the detectable signal. One single-strand target can be recycled and produce accumulated fluorescent signals that are 100 or more times higher than that of a single probe. (e.g., FIG. 1A)

In some embodiments, the oligonucleotide probe has about 10-50 nucleotides. The oligonucleotide probe can have about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, or about 50 nucleotides. In some embodiments, the oligonucleotide probe has about 20 nucleotides. In some embodiments, the oligonucleotide probe has about 25 nucleotides. In some embodiments, the oligonucleotide probe has about 30 nucleotides. In some embodiments, the oligonucleotide probe has about 40 nucleotides.

As described above, the oligonucleotide probes used herein comprise ribonucleotides, and when hybridized with the single-strand targets released from the double-stranded amplicons, the presence of the ribonucleotides result in DNA/RNA hybrid region that can be recognized by RNase H. After the ribonucleotides are cleaved by RNase H, the cleaved probe fragments need to dissociate from the single-strand target to recycle it for detection by additional probes. As such, the detection temperature is (1) permissible for the enzymatic activity of RNase H, (2) lower than Tm of the hybrids formed by intact probe and the single-strand target, and (3) higher than the Tm of the hybrids formed by the cleaved probe fragments the single-strand target. In some embodiments, the oligonucleotide probes comprise at least 4 ribonucleotides between the F and Q. In some embodiments, the probes comprise at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, or at least 30 ribonucleotides between the F and Q. In some embodiments, the probes contain 1 to 20 ribonucleotides between the F and Q. In some embodiments, the probes contain 2 to 15 ribonucleotides between the F and Q. In some embodiments, the probes contain 4 to 12 ribonucleotides between the F and Q. In some embodiments, the probes contain 4 to 8 ribonucleotides between the F and Q. In some embodiments, the probes contain about 4 ribonucleotides between the F and Q. In some embodiments, the probes contain about 8 ribonucleotides between the F and Q. In some embodiments, the probes contain about 12 ribonucleotides between the F and Q. In some embodiments, the probes contain about 16 ribonucleotides between the F and Q. In some embodiments, the probes contain about 20 ribonucleotides between the F and Q. A person of ordinary skill in the art can design the probes using conventional methods and available software according to the present disclosures.

In some embodiments, the probe is an RNA probe. RNA probes contain only ribonucleotides and can be completely hydrolyzed by RNase H.

The oligonucleotide probes used in the detection methods disclosed herein comprise a fluorophore (F) and a quencher (Q) . As used herein and understood in the art, a fluorophore, or a fluorescent label can be detected due to fluorescence when exposed to light of the proper wavelength. A wide variety of fluorophores can be used for detection in methods disclosed herein. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothi azole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound. Exemplary fluorophores include, but are not limited to, 1, 5 IAEDANS; 1, 8-ANS; 4-Methylumbelliferone; 5-carboxy-2, 7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM) ; 5-Carboxynapthofluorescein (pH 10) ; 5-Carboxytetramethylrhodamine (5-TAMRA) ; 5-FAM (5-Carboxyfluorescein) ; 5-Hydroxy Tryptamine (HAT) ; 5-ROX (carboxy-X-rhodamine) ; 5-TAMRA (5-Carboxytetramethylrhodamine) ; 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD) ; 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine) ; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein) ; Alexa Fluor 350^TM; Alexa Fluor 430^TM; Alexa Fluor 488^TM; Alexa Fluor 532^TM; Alexa Fluor 546^TM; Alexa Fluor 568^TM; Alexa Fluor 594^TM; Alexa Fluor 633^TM; Alexa Fluor 647^TM; Alexa Fluor 660^TM; Alexa Fluor 680^TM; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC) ; AMC, AMCA-S; AMCA (Aminomethylcoumarin) ; AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG^TM CBQCA; ATTO-TAG^TM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole) ; BCECF (high pH) ; BCECF (low pH) ; Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H) ; BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO^TM -1; BOBO^TM -3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO^TM -1; BO-PRO^TM -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson^TM; Calcium Green; Calcium Green-1 Ca²⁺ Dye; Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX) ; Cascade Blue^TM; Cascade Yellow; Catecholamine; CFDA; CFP -Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine ; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2^TM; Cy3.1 8; Cy3.5^TM; Cy3^TM; Cy5.1 8; Cy5.5^TM; Cy5^TM; Cy7^TM; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR) ; d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate) ; DDAO; DHR (Dihydorhodamine 123) ; Di-4-ANEPPS; Di-8-ANEPPS (non-ratio) ; DiA (4-Di-16-ASP) ; DIDS; Dihydorhodamine 123 (DHR) ; DiO (DiOC18 (3) ) ; DiR; DiR (DiIC18 (7) ) ; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1) ; Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline) ; FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine) ; Fluor-Ruby; FluorX; FM 1-43^TM; FM 4-46; Fura Red^TM (high pH) ; Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T) ; GFP red shifted (rsGFP) ; GFP wild type, non-UV excitation (wtGFP) ; GFP wild type, UV excitation (wtGFP) ; GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold) ; Hydroxytryptamine; Indodicarbocyanine (DiD) ; Indotricarbocyanine (DiR) ; Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Phloxin B) ; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH) ; Monochlorobimane; MPS (Methyl Green Pyronine Stilbene) ; NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green^TM; Oregon Green 488-X; Oregon Green^TM 488; Oregon Green^TM 500; Oregon Green^TM 514; Pacific Blue; Pararosaniline (Feulgen) ; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613) ; Phloxin B (Magdala Red) ; Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE] ; Phycoerythrin R [PE] ; PKH26 ; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI) ; PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200 ; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycoerythrin (PE) ; red shifted GFP (rsGFP, S65T) ; S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP^TM; sgBFP^TM (super glow BFP) ; sgGFP^TM; sgGFP^TM (super glow GFP) ; SITS; SITS (Primuline) ; SITS (Stilbene Isothiosulphonic Acid) ; SPQ (6-methoxy-N- (3-sulfopropyl) -quinolinium) ; Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine ; Texas Red^TM; Texas Red-X^TM conjugate; Thiadicarbocyanine (DiSC3) ; Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White) ; TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5) ; TRITC (Tetramethylrhodamine Isothiocyanate) ; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these fluorescent compounds are available and can be used. Additional fluorophore examples include, but are not limited to fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluorescamine, Cy3^TM, Cy5 ^TM , allophycocyanin, Texas Red, peridinin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5 ^TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green^TM , rhodamine and derivatives (e.g., Texas red and tetramethylrhodamine isothiocyanate (TRITC) ) , biotin, phycoerythrin, AMCA, CyDyes^TM, 6-carboxyfluorescein (commonly known by the abbreviations FAM and F) , 6-carboxy-2', 4', 7', 4, 7-hexachlorofluorescein (HEX) , 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfiuorescein (JOE or J) , N, N, N', N'-tetramethyl-6carboxyrhodamine (TAMRA or T) , 6-carboxy-X-rhodamine (ROX or R) , 5-carboxyrhodamine-6G (R6G5 or G5) , 6-carboxyrhodamine-6G (R6G6 or G6) , and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g., umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g., cyanine dyes such as Cy3, Cy5, etc.; BODIPY dyes and quinoline dyes.

As used herein and understood in the art, a quencher refers to a molecule or part of a compound that can reduce the emission from a fluorophore when attached to or in proximity to the fluorophore. Quenching can occur by any of several mechanisms including fluorescence resonance energy transfer, photo-induced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange coupling, and exciton coupling such as the formation of dark complexes. Fluorescence is “quenched” when the fluorescence emitted by the fluorophore is reduced as compared with the fluorescence in the absence of the quencher by, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. Commercially available quenchers include but are not limited to DABCYL, Black Hole^TM Quenchers (BHQ-1, BHQ-2, and BHQ-3) , Iowa FQ and Iowa RQ.

Methods disclosed herein are not limited to specific combinations of Fs and Qs used for detection. A person of ordinary skill in the art would be able to make and use probes using appropriate combinations of F and Q according to the present disclosures.

6.2.3.1 Reversibly inactivated RNase H

The RNase H and oligonucleotide probes need to be added to the reaction mixture for the detection step. As known in the art, RNase H is an endoribonuclease that cleaves the phosphodiester bond in an RNA strand when it is part of an RNA: DNA duplex. The enzyme does not cleave DNA or unhybridized ssRNA. In the methods disclosed herein, the RNase H carries dual roles: first, it generates a nicking site on the double-stranded amplicons containing ribonucleotide (s) recognizable by the DNA polymerase with strand-displacement activity; second, it cleaves the probe with ribonucleotide (s) , allows the recycling of the released single strands for multiple detection.

In some embodiments, the RNase H is added to the reaction mixture after the nucleic acid amplification is completed. In some embodiments, the oligonucleotide probes are added to the reaction mixture after the nucleic acid amplification is completed. In some embodiments, both the RNase H and the oligonucleotide probes are added to the reaction mixture after the nucleic acid amplification is completed. In some embodiments, a reversibly inactivated RNase H is included in the reaction mixture for the nucleic acid amplification (Step (1) ) , which is then reactivated at the end of Step (1) and participates in the release of single strands from double-stranded amplicons and the production and amplification of detectable signals (Step (2) ) .

As such, in some embodiments, methods provided herein use a reversibly inactivated RNase H. In some embodiments, RNase H used in the methods disclosed herein is reversibly inactivated, which becomes reactivated either during the nucleic acid amplification step (Step (1) ) , or after Step (1) is completed. In some embodiments, the reversibly inactivated RNase H can be added in the reaction mixture from the beginning of Step (1) , remain inactive for a period, which provides a time window for the target nucleic acid to amplify without the interfering nuclease activity, and become reactivated over time or at specific conditions, which then initiates the detection step. As used herein, a “transition condition” refers to a condition that allows reactivation of the reversibly inhibited RNase H. In some embodiments, the catalytic activity of a modified RNase H can be regulated by changing the pH of a solution containing the enzyme, and that subjecting the reaction mixture to a transition condition means adjusting the pH of the reaction mixture to allow the activation of RNase H. In some embodiments, the catalytic activity of a modified RNase H can be regulated changing the reaction temperature, and that subjecting the reaction mixture to a transition condition means adjusting the temperature of the reaction mixture to allow the activation of RNase H. A person of ordinary skill in the art would understand that the transition condition depends on the modification of RNase H and can be adopted and optimized with routine procedures.

RNase H can be reversibly inactivated using different approaches. An inactivated RNase H can lose its endonuclease activity by at least about 70%as compared to unmodified RNase H (considered as 100%) determined under the optimal condition for the enzyme or under otherwise identical experimental conditions. In some embodiments, the inactivated RNase H can lose at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 99%of its endonuclease activity. RNase H can be reversibly inactivated by chemical modification, ligand association, or physical separation via a mechanism that can be removed, released, or decoupled at certain condition, which allows the reversal of its nuclease activity.

The enzymatic activity of RNase H can be determined using methods that are well known in the art. For example, unit activity can be defined in terms of a specific increase in the relative fluorescence intensity of a reaction containing equimolar amounts of the probe and a complementary template DNA under defined assay conditions, wherein the cleavage of the probe by RNase H generates a fluorescent signal.

In some embodiments, the RNase H is reversibly inactivated by chemical modification that be reversed with, for example, heating or pH change. In some embodiments, the RNase H is reversibly inactivated by modifying of a positively charged amino acid of the enzyme such as lysine. For example, RNase H can be reversibly modified by crosslinking its lysine residue (s) with acylating agents such as imides, anhydrides, and chloroanhydrides. In some embodiments, RNase H can also be modified by subjecting the enzyme to acylation of lysine residues using an acylating agent, for example, a dicarboxylic acid.

In some embodiments, RNase can be reversibly inactivated using maleic anhydride and its derivatives: under alkaline condition, maleic anhydride and its derivatives would react with the lysine residues, reducing its positive charge and affinity with the negatively charged nucleic acids. The modification can be reversed at lower pH or with heating, which restores the positive charge of the lysine residues and allows the protein to regain its affinity with nucleic acids and enzymatic activity.

In some embodiments, the RNase H used in methods described herein comprises a lysine residue modified by a maleic anhydride or a derivative thereof. In some embodiments, the maleic

anhydride or a derivative thereof has the structure below, wherein X and Y each independently is a negatively charged or neutral group.

Exemplary neutral groups include, but are not limited to, H, an acyl group (e.g., -CH₃, -C₂H₅) , or a hydroxyl group. Exemplary negatively charged groups include, but are not limited to, Cl, F, or carboxyl group (e.g., -COOH, -CH₃COOH) . In some embodiments, X and Y are each independently selected from -H, -CH3, and -Cl. In some embodiments, the RNase H is modified by 2-methylmaleic anhydride. In some embodiments, the RNase H is modified by 2, 3-dimethylmaleic anhydride. In some embodiments, the RNase H is modified by 2, 3-dichloromaleic anhydride.

Methods of modifying RNase H with a maleic anhydride or a derivative thereof, or another acylating agents are known in the art. The reactions are carried out in a controlled manner. The pH of the reaction mixture is preferably maintained between about 7.5 to about 9.0 such that almost all of the lysine epsilon amino groups present on the enzyme are freed from their protonated form and become capable of reaction with the modifying agent. For illustrative purposes, RNase H can be incubated in a solution with 2, 3-dichloromaleic anhydride at 1: 3 or 1: 5 w/w under continuous stirring at room temperature for 18-24 h, with pH maintained constant by adding a 1 N NaOH solution.

RNase H modified by a maleic anhydride or a derivative thereof can be reactivated by lowering the pH to about 7.0 or less or by increasing the temperature to about 65℃ to about 95℃. For example, when Tris buffer is used as a buffering agent, the composition may be heated to about 95℃, resulting in the lowering of pH from about 8.7 (at 25℃) to about 6.5 (at 95℃) .

A person of ordinary skill in the art would be able to determine the specific condition for reversing a given modification with no more routine experimentation. For illustration purposes, RNase H modified with 2, 3-dimethylmaleic anhydride can be reactivated by incubation at 42℃ for, e.g., 10 minutes. In some embodiments, RNase H modified with 2-methylmaleic anhydride can be reactivated by incubation at 95℃ for, e.g., 5 minutes.

Additional methods are available for chemical enzyme modification for the off-/on control of the enzyme activity. (See e.g., Yu et al., Macromol. Rapid Commun. 2022, 2200195. ) In some embodiments, nonspecific covalent surface modification can be used, which undertakes modification at natural amino acid residues. In some embodiments, site-specific covalent modification can be used, which occurs in low-abundance natural amino acids (e.g., Cys, Trp, Ser, Tyr, and N-terminus, etc. ) or unnatural amino acids via bioorthogonal reactions.

Noncovalent modification can also be used to achieve the off/on control of the enzymatic activity of RNase H (Yu 2022, supra) . Noncovalent interaction enables dynamic responsiveness while minimizing the possibility of enzyme inactivation and stress on enzyme structure. In some embodiments, small molecule interactions, such as biotin-streptavidin and ligand pockets can be used. In some embodiments, nanomaterial such as electrostatic, hydrophilic, and hydrophobic interactions can be used.

In some embodiments, the RNase H can be reversibly inactivated via affinity interactions, such as antibodies and antigens, lectins and free saccharide chains or glycosylated macromolecules, nucleic acids and nucleic acid binding proteins, hormones, and their receptors, avidin, and biotin, polyhistidine, and metal ions, etc.

In some embodiments, the RNase H can be reversibly inactivated by ligand association. The ligand can be an antibody, an antigen-binding fragment, an aptamer, a receptor, a cofactor, or a chelating agent. In some embodiments, the RNase is reversibly inactivated by association with an antibody. In some embodiments, the RNase is reversibly inactivated by association with an antigen-binding fragment. In some embodiments, the RNase is reversibly inactivated by association with an aptamer. In some embodiments, the RNase is reversibly inactivated by association with a receptor. In some embodiments, the RNase is reversibly inactivated by association with a cofactor. In some embodiments, the RNase is reversibly inactivated by association with a chelating agent. The ligand can inhibit the enzymatic activity of RNase H by binding to either its active site or a site remote from the RNase’s active site. In some embodiment, the ligand can induce a conformational change.

In some embodiments, RNase H is reversibly inactivated by binding with an antibody or antigen binding fragment. In some embodiment, the binding can be reversed with pH change or heating. In some embodiments, RNase H is inactivated by binding with an aptamer. In some embodiments, the conformation of the aptamer changes with pH or temperature, which results in its dissociation from the enzyme and the reactivation of the enzyme.

An exemplary aptamer can have a stem-loop structure, such as the nucleic acid with the following sequence: 5’-ACGTGCCACGCATTCAArG*rC*rG*rU*rG*rG*rC*rA*rCAG-3’ (SEQ ID NO: 1) . The nucleotides preceded with letter “r” are ribonucleotides; and asterisks indicate thio-modification. As shown, the underlined sequences are complementary to each other and can form DNA/RNA hybrid that traps RNase H. With heating, the hybrid will denature and release the RNase from inhibition.

In some embodiments, the RNase H can be trapped in a microsphere or an isolated chamber of a reaction vessel, such that it is physically separated from the reaction mixture during nucleic acid amplification. When nucleic acid amplification is completed, RNase H can be released from its compartment and included in the reaction mixture by, for example, heating, shaking, or centrifugation.

In some embodiments, a switchable chemical shell in response to external stimuli (e.g., light, magnetic force, pH, or temperature) around or on the surface of RNase H can be used to block the active site or induce conformational changes, thus prohibiting substrate access. As an example of using the formation of a chemical shell via nonspecific surface modification for the off/on manipulation of enzyme activity, an ultrasmall platinum nanoparticle can be embedded into enzymes, which can be decorated with thermoresponsive copolymers exhibiting upper critical solution temperature (UCST) behavior. The Pt nanoparticle–embedded enzyme is modified via nonspecific covalent surface modification with N-acryloxysuccinimide (NAS) and the ∈-amino group of lysine on the surface of the enzymes to produce E/Pt-NAS. Thus, a poly (AAmco-AN) -engineered E/Pt (PE-E/Pt) with a chemical shell is obtained by copolymerization of AAm and AN on the surface of E/Pt-NAS. Herein, the chemical shell forms microscale aggregates in solution below the UCST, preventing the substrate approach, and the enzyme activity is switched off. Upon near-infrared irradiation, the chemical shell is disassembled above the UCST through a photothermal effect of Pt nanoparticles inside the shell, and the activity of the enzyme can be switched on.

Additionally, pH can also be employed for manipulating enzyme activity. For example, a pH-responsive dendrimer shell on the surface of active enzymes can be formed. Specifically, enzymes are pre-installed with boronic acid on the ∈-amino groups of lysines on the surface via NHS in boronic acids in a nonspecific covalent surface modification manner. Catalytic activity is efficiently reversible off/on controlled by the dendrimer shell assemblage or shell degradation responding to changes in pH between 5.0 and 7.4, which is accomplished by the bioorthogonal ligation method between boronic acid and salicyl hydroxamate. Thus, such nanomaterial shells provide off/on control of enzyme activity by the pH control.

In some embodiments, chemical modification of the enzymes with a switchable small molecule to block the substrate from entering the active site or resulting in structural and conformational changes in response to external stimuli can be used to reversibly regulate enzyme activity. In some embodiments, optical control can be used, employing small molecules such as azobenzene analogs, spiropyran, and salicylideneaniline, to allowing reversible photomodulation of the activity of enzymes. Azobenzene derivatives, due to their advantages of large geometrical changes resulting from cis-trans isomerization under exposure to ultraviolet (UV) light or blue light and their photostability, have undergone extensive utilization in photoresponsive systems and devices. For example, molecular switches and photoswitchable polymers can be blended with peptides or enzymes to regulate their activity.

Other designs for site-specific chemical modification have been developed for off/on control of enzymatic activity. For example, site-directed mutagenesis can be used to introduce an additional residue at the specific sites, which can be modified to produce, e.g., a polymer module conjugated on the enzyme surface to control its activity. In some embodiments, photocaged residues can be introduced to the enzyme, which can be activated by light. A person of ordinary skill in the art would understand that the methods and systems disclosed herein are not limited to any specific manner of RNase H modification, and any methods disclosed herein or otherwise known in the art can be adopted to achieve reversible control of the enzymatic activity of RNase H.

The RNase that can be used in the methods disclosed herein can be any RNase H. The RNase H can be a human RNase, a mouse RNase, or a bacterial RNase. In some embodiments where PCR is used for nucleic amplification and the RNase H is included in the amplification reaction mixture, a thermal stable RNase H can be used. For example, in some embodiments, the RNase H is derived from Pyrococcus. In some embodiments, the RNase H is Pyrococcus furiosus RNase HI. In some embodiments, the RNase H is Pyrococcus horikoshi RNase HI. In some embodiments, the RNase H is Pyrococcus abyssi RNase HI. In some embodiments, the RNase H is derived from Thermococcus. In some embodiments, the RNase H is Thermococcus litoralis RNase HI. In some embodiments, the RNase H is Thermus thermophilus RNase HI,

In some embodiment, the RNase H is derived from E. coli. The RNase H can be E. coli RNase HI. The RNase H can be E. coli RNase HII. In some embodiment, the RNase H is Saccharomyces cerevisiae RNase HI. In some embodiment, the RNase H is mouse RNase HI. In some embodiment, the RNase H is human RNase H. The RNase H can be a wildtype enzyme. The RNase can also be a functional variant of the wildtype enzyme.

In some embodiments, the methods disclosed herein use isothermal amplification of the target nucleic acids. In some embodiments, the RNase H is added after the nucleic acid amplification is completed. The RNase H that can be used in these methods can be either thermal stable or not. In some embodiments, methods disclosed herein use isothermal amplification of target nucleic acid, and any RNase H (thermal stable or not) can be used and included in the reaction mixture during the nucleic acid amplification. In some embodiments of methods disclosed herein, PCR is used for nucleic acid amplification, and an RNase H that is not thermal stable is used, but added to the reaction mixture after the DNA amplification step is completed.

6.2.4 Multiplexing

Methods provided herein can be used in a multiplexing format to simultaneously detect a plurality of target nucleic acids. In some embodiments, the methods provided herein can simultaneously detect at least two, at least three, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 target nucleic acids. In some embodiments, methods provided herein can simultaneously detect at least two target nucleic acids. In some embodiments, methods provided herein can simultaneously detect at least three target nucleic acids. In some embodiments, methods provided herein can simultaneously detect at least 5 target nucleic acids. In some embodiments, methods provided herein can simultaneously detect at least 8 target nucleic acids.

To achieve multiplexing detection, a plurality of primer pairs can be used. For example, in a multiplexing method for detecting two target nucleic acids, two primer pairs are used, wherein the first primer pair can amplify the first target nucleic acid and the second primer pair can amplify the second target nucleic acid. In Step (1) , both target nucleic acids are amplified to form double-stranded amplicons, of which single-strand targets are released by the combined action of RNase H and strand-displacement DNA polymerase and detected by oligonucleotide probes in Step (2) . To simultaneously identify two different single-strand targets, two different probes are used, wherein the first probe can hybridize to the first single-strand target, and the second probe can hybridize to the second single-strand target, and both probes carry different fluorophores that can produce distinct fluorescent signals.

As such, it is understood that simultaneous detection of multiple target nucleic acids can be achieved using multiple primer pairs and multiple probes with distinct fluorophores with the methods disclosed herein.

The multiplexing methods disclosed herein can be used to detect two or more nucleic acid molecules. In some embodiments, the multiplexing methods disclosed herein can detect different regions on the same target nucleic acid. Additionally, the multiplexing methods disclosed herein can be used to detect same types of nucleic acids or different types of nucleic acids. For illustrative purposes, in some embodiments, the multiplexing methods disclosed herein can be used to detect a plurality of viral nucleic acids, wherein the first target nucleic acid is a first viral nucleic acid, and the second target nucleicacid is a second viral nucleic acid. In some embodiments, the first and second viral nucleic acids are from the same virus. In some embodiments, the first and second viral nucleic acid are from two different viruses. In some embodiments, the first viral nucleic acid is a viral DNA, and the second viral nucleic acid is a viral RNA. In some embodiments, both the first and the second viral nucleic acids are viral RNAs.

The target nucleic acids can be amplified separately or together in the multiplexing detection methods disclosed herein. In some embodiments, the target nucleic acids can be amplified separately, and the double-stranded amplicons can be pooled together prior to the signal detection. Alternatively, or in addition, the target nucleic acids can be pooled together prior to amplification and amplified together. In some embodiments, the target nucleic acids are present in one sample and amplified together. For example, in some embodiments, provided herein are multiplexing detection methods that simultaneously detect two or more target nucleic acids from one sample, wherein the first target nucleic acid serves as positive control, and the second target nucleic acid is the tested nucleic acid, the presence of which, for example, can indicate the presence of a pathogen in the sample.

6.3 Systems for nucleic acid detection

Provided herein are also nucleic acid detection systems. The nucleic acid detection system can comprise any of the components and reagents discussed herein for carrying out the nucleic acid detection methods disclosed herein.

In some embodiments, the nucleic acid detection systems comprise: (a) a DNA polymerase with strand displacement activity and reverse transcriptase activity; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a RNase H that is reversibly inactivated; and (d) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments, the nucleic acid detection systems comprise: (a) a DNA polymerase with strand displacement activity and a reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a RNase H that is reversibly inactivated; and (d) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments, the nucleic acid detection systems can contain a RNase and reagents for reversibly inactivating the RNase. As such, in some embodiments, the nucleic acid detection systems comprise: (a) a DNA polymerase with strand displacement activity and reverse transcriptase activity; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a RNase H; (d) reagents for reversibly inactivating RNase; and (e) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

In some embodiments, the nucleic acid detection systems comprise: (a) a DNA polymerase with strand displacement activity and a reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a RNase H; (d) reagents for reversibly inactivating RNase; and (e) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

The DNA polymerase with strand displacement activity, reverse transcriptase, primer pairs, RNase H, reagents for reversibly inactivating the RNase H, and oligonucleotide probes described in present disclosure can be included in the nucleic acid detection systems. All permutations and combinations are expressly contemplated.

In some embodiments, the RNase H included in the nucleic acid detection systems disclosed herein is reversibly inactivated by chemical modification, ligand association, or physical separation. In some embodiments, the RNase H is reversibly inactivated by chemical modification. In some embodiments, the RNase H is modified by a maleic anhydride or a derivative thereof. In some embodiments, the RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride. In some embodiments, the RNase H is reversibly inactivated by association with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent. In some embodiments, the RNase H is physically trapped in a microsphere or an isolated chamber of a reaction vessel. In some embodiments, the inactivating modification or association can be reversed by, for example, temperature change or pH change. In some embodiments, the physical separation can be removed by, for example, heating, shaking, or centrifugation.

In some embodiments, RNase H that can be used in the nucleic acid detection systems described herein is Pyrococcus furiosus RNase HI, Pyrococcus horikoshi RNase HI, Pyrococcus abyssi RNase HI, Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coli RNase HI, Escherichia coli RNase HII, Saccharomyces cerevisiae RNase HI, mouse RNase HI, or human RNase H, or any functional variant thereof. In some embodiments, the RNase H included in the nucleic acid detection systems described herein is E. coli RNase HI. In some embodiments, the RNase H is human RNase H. In some embodiments, the RNase H is a thermal stable enzyme. In some embodiments, the RNase H is Thermus thermophilus RNase HI.

DNA polymerase that can be used in the nucleic acid detection systems described herein can be any DNA polymerase with strand-displacement activity that lacks exonuclease activity. In some embodiments, the DNA polymerase further has reverse transcriptase activity. In some embodiments, the DNA polymerase can be Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; SD polymerase; or a functional variant thereof.

In some embodiments, the nucleic acid detection systems described herein further include a reverse transcriptase, including but not limited to, M-MLV reverse transcriptase and AMV reverse transcriptase.

In some embodiments, the nucleic acid detection systems described herein further include other enzymes helpful for the nucleic acid amplification and/or signal detection. In some embodiments, the nucleic acid detection systems described herein further include a DNA helicase. the nucleic acid detection systems described herein further include SSB. The systems described herein are not limited to specific methods for nucleic acid amplification. As such, depending on the methods of nucleic acid amplification used in the detection methods, different reagents required for the amplification can be included in the system. For example, if EMA is used to amplify the nucleic acid at room temperature, eukaryotic RFC and PCNA, and prokaryotic SSBs can be further included in the systems described herein.

A person of ordinary skill in the art would be able to determine the necessary and appropriate components for the systems according to teachings in the present disclosure. For illustrative purposes, in some embodiments, provided herein are nucleic acid detection systems that comprise (a) SAP Pol I and M-MLV reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) RNase H modified by 2, 3-dimethylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q. The system can further include a DNA helicase, a single-stranded binding protein, or both.

In some embodiments, provided herein are nucleic acid detection systems that comprise (a) Tth Pol and SD Polymerase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) RNase H modified by 3-methylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

Primer pairs and oligonucleotide probes described above for the nucleic acid detection methods can be included in the nucleic acid detection systems disclosed herein. In some embodiments, provided herein are systems for multiplexing detection of nucleic acid targets, which would comprise a plurality of primer pairs, each for amplifying a different nucleic acid target, and a plurality of oligonucleotide probes, each for detecting a different nucleic acid and each capable of producing a distinct fluorescent signal.

In some embodiments, the nucleic acid detection systems provided herein can further comprise other reagents useful for nucleic amplification step and/or the detection step.

In some embodiments, the nucleic acid detection systems provided herein further comprise at least one of the following: reaction buffer, diluent, water, magnesium salt (such as magnesium acetate, magnesium chloride, or magnesium sulfate) and/or manganese salt, dNTPs, reducing agent (such as DTT) , and a surfactant (such as SDS) .

The components described herein can be provided singularly or in any combination as a kit. Such a kit includes the components described herein and packaging materials thereof. In addition, a kit optionally comprises informational material. In some embodiments, the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the aggregates for the methods described herein. For example, the informational material can describe methods for using the kits provided herein to perform an assay for detection of a target entity, e.g., a virus.

The kits can include any of the preprocessing reagents as described herein. In some embodiments, the kit further comprises reagents for isolating nucleic acid from the sample. In some embodiments, the kit further comprises reagents for isolating DNA from the sample. In some embodiments, the kit further comprises reagents for isolating RNA from the sample. In some embodiments, the kit further comprises detergent, e.g., for lysing the sample. In some embodiments, the kit further comprises a sample collection device, such a swab. In some embodiments of any of the aspects, the kit further comprises a sample collection container. In some embodiments, the sample collection contain can include transport media.

In some embodiments, the kit can contain separate containers, dividers or compartments for each component and informational material. For example, each different component can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, a collection of the magnetic particles is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.

In some embodiments the kit includes a carrier for organizing and protecting the components in the kit during transport or storage. The carrier can be in any form including a bag, a box or a case, including handles, straps and wheels for convenient movement or storage.

6.4 Exemplary methods and systems

The examples provided below are for purposes of illustration only, which are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

6.4.1 Embodiment 1: EMA and RNase H-mediated dual amplification

In some embodiments, methods provided herein include isothermal amplification of the target nucleic acid, e.g., EMA and signal amplification using an RNase H (see e.g., FIG. 1A) . A DNA polymerase with strand-displacement activity is used. If the DNA polymerase lacks reverse transcription activity, a reverse transcriptase can be used to amplify the ribonucleotides in the sequences. The reversibly inactivated RNase H can be included in the amplification mixture, which become reactivated for detection when the amplification is completed.

In some embodiments, Sau DNA Polymerase I, Large Fragment ( “SAP Pol I” ) is used as the EMA enzyme. SAP Pol I has strand-displacement activity and can amplify the target nucleic acid exponentially under isothermal condition. As SAP Pol I lacks reverse transcription activity, a reverse transcriptase such as M-MLV RT can be included in the reaction. In some embodiments, the reaction mixture can further comprise E. coli single-stranded binding protein (SSB) . In some embodiments, the reaction mixture can further comprise DNA helicase.

In some embodiments, an RNase H can be reversibly inactivated by 2, 3-dimethylmaleic anhydride modification and added to the amplification reaction mixture. At 42℃, it becomes gradually de-modified and therefore reactivated within about 5-30 minutes. As the reaction is carried out at 42℃, any RNase H (such as one that is not thermal stable) can be used, such as E. coli RNase H.

Accordingly, in some embodiments, provided herein are methods of detecting a target nucleic acid in a sample comprising: (1) amplifying the target nucleic acid to generate double-stranded amplicons by adding the sample to a reaction mixture comprising (a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) , and (b) SAP Pol I and M-MLV reverse transcriptase; and incubating the reaction mixture at about 42℃ for about 5 to 30 min; and (2) detecting the amplicons with an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; wherein (a) an RNase H cleaves the ribonucleotide (s) in the amplicons, generating nicking sites; (b) SAP Pol I recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ; (c) the oligonucleotide probe hybridizes with the single-strand target; (d) the RNase H cleaves the ribonucleotide (s) on the hybridized probe, separating F from Q and producing a detectable signal; and (e) the cleaved probe fragments separate from the single-strand target, allowing the repetition of steps (c) and (d) . The RNase H can be reversibly inactivated and included in the reaction mixture during target amplification, and becomes reactivated before the detection. In some embodiments, the RNase H can be modified by 2, 3-dimethylmaleic anhydride, which can be gradually reversed during Step (1) .

In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample comprising: (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) , (c) SAP Pol I and M-MLV reverse transcriptase; (d) an RNase H modified by 2, 3-dimethylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition, incubating at about 42℃ for about 5 to 30 minutes; and (3) continuing the incubation at about 42℃ and collecting fluorescence signals.

For illustrative purposes, the methods can include the following steps: (1) prepare the reaction mixture containing: a buffer having Tris Ac (e.g., pH 7.8; 30 mM) , KAc (e.g., 50 mM) and MgAc2 (e.g., 10 mM) ; SAP Pol I (e.g., 100 ng/μL) , M-MLV reverse transcriptase (e.g., 10 U/μL) , E. coli SSB (e.g., 600ng/μL) , phosphocreatine kinase (PK; e.g., 0.05U/μL) , DNA helicase (e.g., 100 ng/μL) , 2, 3-dimethylmaleic anhydride modified E. coli RNase H (e.g., 50ng/μL) , forward DNA/RNA mixed primer (e.g., 200nM) , reverse DNA primer (e.g., 200nM) , ATP (e.g., 3 mM) , phosphoinositide (e.g., 10 mM) , dNTP (e.g., 200 nM) , RNA probe modified with FAM and BHQ1 separately at the two ends (e.g., 2.5 μM) ; (2) incubate the mixture at 42℃ for 10 minutes; and (3) collect fluorescence signals.

To prepare 2, 3-dimethylmaleic anhydride modified E. coli RNase H, RNase H can be overexpressed in E. coli, purified by affinity column and size exclusion column, dialyzed and concentrated in physiological saline (PBS) to 3～5 mg/mL. 2, 3-dimethylmaleic anhydride (in DMSO) is added to RNase H solution to a final concentration of 1～10 mM and the mixture is incubated at 4℃ for about 4～6 hours. The mixture is then purified by size exclusion column to obtain modified RNase H.

During the 10 minutes of incubation, the target nucleic acid is exponentially amplified by SAP Pol I, wherein the ribonucleotides are reverser transcribed by M-MLV RT. 3-dimethylmaleic anhydride modified E. coli RNase H becomes de-modified and reactivated during the incubation and starts to cleave the ribonucleotides in the double-stranded amplicons, generating single-stranded amplicons that can hybridize with the RNA probe. The reactivated RNase H further cleaves the RNA probes that are hybridized with the single-stranded amplicons, generating the fluorescence signal. The cleaved fragments fall off the single-stranded amplicons, freeing them for detection by additional probes and allowing linear amplification of the fluorescence signal over time.

6.4.2 Embodiment 2: LAMP and RNase H-mediated dual amplification

In some embodiments, methods provided herein include amplifying the target nucleic acid using LAMP and signal amplification using an RNase H (see e.g., FIG. 1B) . A DNA polymerase with strand displacement activity is used. If the DNA polymerase lacks reverse transcription activity, a reverse transcriptase is also included. The reversibly inactivated RNase H can be included in the amplification mixture, which become reactivated for detection when the amplification is completed.

In some embodiments, Bst polymerase is used in LAMP. Bst 3.0 DNA Polymerase (available from e.g., NEB) , which has reverse transcriptase activity can be used. The target nucleic acid can be DNA or RNA. A thermal stable RNase H can be reversibly inactivated by 2-methylmaleic anhydride modification and added to the amplification reaction mixture. When heated, the RNase H becomes gradually de-modified and reactivated in the reaction mixture (e.g., 5 min at 95℃) .

Accordingly, in some embodiments, provided herein are methods of detecting a target nucleic acid in a sample comprising: (1) amplifying the target nucleic acid by subjecting a reaction mixture comprising the sample to amplification condition, wherein the reaction mixture comprises (a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (b) a Bst polymerase having reverse transcriptase activity; wherein the reaction mixture is incubated at 65℃ for about 30 min, allowing formation of double-stranded amplicons; and (2) detecting the target nucleic acid with a complementary oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q, wherein (a) an RNase H cleaves the ribonucleotide (s) in the amplicons, generating nicking sites; (b) the Bst polymerase recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ; (c) the oligonucleotide probe hybridizes with the single-strand target; (d) the RNase H cleaves the ribonucleotide (s) on the hybridized probe, separating F from Q and producing a detectable signal; and (e) the cleaved probe fragments separate from the single-strand target, allowing the repetition of steps (c) and (d) .

The RNase H can be reversibly inactivated and included in the reaction mixture during target amplification, and becomes reactivated before the detection. In some embodiments, the RNase H can be a thermal stable RNase H modified by 2-methylmaleic anhydride. In some embodiments, the reaction mixture can be incubated at 95℃ for at least three minutes after Step (1) is completed to reactivate RNase H and then at 60 ℃ for detection.

In some embodiments, provided herein are methods of detecting a target nucleic acid in a sample comprising: (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a Bst polymerase having reverse transcriptase activity; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; and (2) subjecting the reaction mixture to an amplification condition and incubating at 65 ℃ for about 30 min; and (3) incubating the reaction mixture at about 95 ℃ for at least three minutes; and (4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.

In some embodiments, the reaction mixture comprises an outer primer pair (e.g., F3 and B3 in FIG. 1B) and an inner primer pair (e.g., forward inner primer and back inner primer in FIG. 1B) , wherein each of the inner primer pair contains a looping fragment (e.g., F1C and B1C in FIG. 1B) that can hybridize with a region of the amplicon (e.g., F1 and B1 in FIG. 1B) that allows for the formation of a loop at the end of the amplicon, and wherein one of the inner primer pair contains about 1-15 ribonucleotide (s) .

For illustrative purposes, the methods can be used to detect canine distemper virus (CDV) and include the following steps: (1) prepare the reaction mixture containing: a buffer having Tris (e.g., 30 mM, pH 7.9) , potassium acetate (e.g., 50 mM) , betaine (e.g., 3 mM) , magnesium sulphate (e.g., 2.5 mM) , dNTPs (e.g., 0.1 mM) , Bst3.0 polymerase (e.g., 8.0 U) , 2-methylmaleic anhydride modified thermostable RNase H (e.g., 75 μg/mL) ; FIP (forward inner primer) and BIP (back inner primer) (e.g., 2.4 μM) ; F3 and B3 primers (outer primers; e.g., 300 nM) ; and RNA probe (e.g., 10 μM) ; (2) incubate the mixture at about 65 ℃ for about 30 minutes (reverse transcription and amplification of the viral RNA) ; (3) incubate the mixture at about 95℃ for about 5 minutes (reactivation of modified RNase H) ; and (4) incubate the mixture at about 60℃ (strand-displacement, generation and recycling of single-strand target) and collect fluorescence signal every 30 seconds.

6.4.3 Embodiment 3: qPCR and RNase H-mediated dual amplification

In some embodiments, methods provided herein include nucleic acid amplification by qPCR and signal amplification using a thermal stable RNase H. If the DNA polymerase lacks reverse transcription activity, a reverse transcriptase can also be included. The reversibly inactivated RNase H can be included in the amplification mixture, which become reactivated for detection when the amplification is completed.

In some embodiments, SD polymerase (avariant of Taq that has strand-displacement activity) can be used for qPCR. 5’ to 3’ exonuclease-deficient DNA polymerase derived from Thermus thermophilus ( “Tth Pol” ) , which has reverse transcriptase activity can be used to amplify the ribonucleotides in the sequences.

A thermal stable RNase H can be reversibly inactivated by 2-methylmaleic anhydride modification and added to the amplification reaction mixture. The modification can be reversed with heating (e.g., 5 min at 95 ℃) , which reactivates the RNase H for signal detection.

Accordingly, in some embodiments, provided herein are methods of detecting a target nucleic acid in a sample comprising: (1) amplifying the target nucleic acid to generate double-stranded amplicons by adding the sample to a reaction mixture comprising (a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; and (b) Tth Pol and SD Polymerase; and subjecting the reaction mixture to 25-45 thermal cycles alternating between 95 ℃ and 75 ℃; (2) detecting the amplicons with an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; wherein (a) an RNase H cleaves the ribonucleotide (s) in the amplicons, generating nicking sites; (b) SD Polymerase recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ; (c) the oligonucleotide probe hybridizes with the single-strand target; (d) the RNase H cleaves the ribonucleotide (s) on the hybridized probe, separating F from Q and producing a detectable signal; and (e) the cleaved probe fragments separate from the single-strand target, allowing the repetition of steps (c) and (d) .

The RNase H can be reversibly inactivated and included in the reaction mixture during target amplification, and becomes reactivated before the detection. In some embodiments, the RNase H can be a thermal stable RNase H modified by 2-methylmaleic anhydride. In some embodiments, the reaction mixture can be incubated at 95℃ for about 3 minutes after Step (1) is completed to reactivate RNase H and then at 60 ℃ for detection.

For illustrative purposes, the methods can include the following steps: (1) prepare the reaction mixture containing: Tris-HAc (e.g., pH8.5; 30 mM) , KAc (e.g., 50 mM) , MgAc2 (e.g., 5 mM) , MnAc2 (e.g., 0.5 mM) , Tth Pol (e.g., 0.25 U/μL) , SD Pol (e.g., 0.1 U/μL) , 2-methylmaleic anhydride modified RNase H (e.g., 50 ng/μL) , forward mixed primer (e.g., 200 nM) , reverse DNA primer (e.g., 200 nM) , dNTP (e.g., 200 nM) , RNA probe modified with FAM and BHQ1 separately at the two ends (e.g., 2.5 μM) ; (2) subject the reaction mixture to 40 thermal cycles alternating between 95℃ (10 seconds) and 75℃ (10 seconds) ; (3) incubate the reaction mixture at 95℃ for 3 minutes; and (4) incubate the reaction mixture to 60℃ and collect fluorescence.

Both SD Polymerase and Tth Pol are thermal stable polymerase. Tth Pol also has reverse transcription activity. During thermal cycles, the target sequence is exponentially amplified with the primer pair (one DNA primer and one mixed primer) . After the amplification is completed, the mixture is heated to 95℃ for 3 minutes to de-modify and reactivate the thermal stable RNase H. Then when the reaction is cooled to 60℃, the RNase H starts to cleave the ribonucleotides in the double-stranded amplicons, allowing the SD Polymerase to recognize and generate single-strand targets that can be hybridized with the RNA probe. The reactivated RNase H further cleaves the RNA probes as they are hybridized with the single-strand targets, generating the fluorescence signal. The cleaved fragments fall off the single-strand targets, freeing them for detection by additional probes and allowing linear amplification of the fluorescence signal over time.

Any thermal stable RNase H can be used in this method, such as T. thermophilus RNase H. To prepare 2-methylmaleic anhydride, T. thermophilus RNase H can be recombinantly expressed in E. coli, purified by affinity column and size exclusion column, dialyzed and concentrated to 3～5mg/mL in physiological saline (PBS) . 2-methylmaleic anhydride (in DMSO) is added to the RNase H solution to a final concentration of 10～50 mM and the mixture is incubated at 4℃overnight. The mixture is then purified by size exclusion column to obtain modified RNase H.

The methods and systems of nucleic acid detection have the following exemplary advantages: 1. High sensitivity. The two-step amplification enables highly sensitive detection. In addition to the amplification of the target nucleic acid, RNase H-mediated cleavage of probes allows recycling of the target nucleic acid for detection, and the accumulation and liner amplification of fluorescent signals for detection. 2. High specificity. Both amplification steps are sequence specific. Specific primers are designed to specifically amplify the target nucleic acids, and only the double-stranded amplicons containing the ribonucleotides from the primer are recognized and cleaved by RNase H. Additionally, RNase H only cleaves the probe that is hybridized to the target nucleic acid, but not the unhybridized probes, resulting in very low background noise. 3. Single-tube testing. Use of reversibly inactivated RNase H avoids the interference of enzymatic activities required for nucleic amplification (polymerase activity) and that for the signal detection (nuclease activity) , allowing the entire testing to be completed in one test tube that needs not to be opened in the interim. 4. Easy multiplexing. Multiplexing detection can be achieved by using multiple primers for amplifying multiple target nucleic acids and differential detection by using probes labeled with distinct fluorophores. 5. High compatibility. A variety of nucleic acid amplification technologies, including PCR and isothermal amplification technologies can be adapted into the methods disclosed herein. To do so, primers need not to be redesigned. One can replace certain deoxyribonucleotides in one primer of the existing primers with corresponding ribonucleotides and use the modified primer pairs in methods disclosed herein. 6. Simplicity in probe design. The oligonucleotide probes can hybridize to any region in the amplicon, allowing simple and flexible design.

6.5 Other Embodiments

Embodiment 1. A method of detecting a target nucleic acid in a sample, comprising (1) amplifying the target nucleic acid by subjecting a reaction mixture comprising the sample to an amplification condition, wherein the reaction mixture comprises: (a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; and (b) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; and (2) detecting the amplicons with an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; wherein (a) an RNase H cleaves the ribonucleotide (s) in the amplicons, generating nicking sites; (b) the DNA polymerase recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ; (c) the oligonucleotide probe hybridizes with the single-strand target; (d) the RNase H cleaves the ribonucleotide (s) on the hybridized probe, separating F from Q and producing a detectable signal; and (e) the cleaved probe fragments separate from the single-strand target, allowing the repetition of steps (c) and (d) .

Embodiment 2. The method of embodiment 1, wherein the probe is included in the reaction mixture during target amplification.

Embodiment 3. The method of embodiment 1 or 2, wherein the RNase H is reversibly inactivated and included in the reaction mixture during target amplification, and becomes reactivated before the detection.

Embodiment 4. A method of detecting a target nucleic acid in a sample, comprising (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; (d) a reversibly inactivated RNase H; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition for sufficient time to amplify the target nucleic acid; (3) subjecting the reaction mixture to a transition condition for sufficient time to reactivate RNase H; and (4) subjecting the reaction mixture to a detection condition and collecting fluorescent signals.

Embodiment 5. The method of embodiment 4, wherein the transition condition is the same as the amplification condition.

Embodiment 6. The method of any one of embodiments 3 to 5, wherein the RNase H is reversibly inactivated by chemical modification.

Embodiment 7. The method of embodiment 6, wherein the reversibly inactivated RNase H comprises a lysine residue modified by a maleic anhydride or a derivative thereof having the structure below, wherein X and Y each independently is a negatively charged or neutral group.

Embodiment 8. The method of embodiment 7, wherein the reversibly inactivated RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride.

Embodiment 9. The method of any one of embodiments 3 to 5, wherein the RNase H is reversibly inactivated by ligand association.

Embodiment 10. The method of embodiment 9, wherein the reversibly inactivated RNase H is associated with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent.

Embodiment 11. The method of any one of embodiments 3 to 5, wherein the RNase H is reversibly inactivated by physical separation.

Embodiment 12. The method of embodiment 11, wherein the RNase H is physically trapped in a switchable chemical shell, a microsphere or an isolated chamber of a reaction vessel.

Embodiment 13. The method of any one of embodiments 3 to 12, wherein the RNase H is reactivated by temperature change or pH change.

Embodiment 14. The method of any one of embodiments 1 to 13, wherein the RNase H is Pyrococcus furiosus RNase HI, Pyrococcus horikoshi RNase HI, Pyrococcus abyssi RNase HI, Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coli RNase HI, Escherichia coli RNase HII, Saccharomyces cerevisiae RNase HI, mouse RNase HI or human RNase H.

Embodiment 15. The method of any one of embodiments 1 to 14, wherein the primer comprises 1-15, 2-12, 4-12, or 4-8 ribonucleotides.

Embodiment 16. The method of embodiment 15, wherein the primer comprises about 4, about 6, or about 8 ribonucleotides.

Embodiment 17. The method of any one of embodiments 1 to 16, wherein the DNA polymerase is Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; or SD polymerase; or a functional variant thereof.

Embodiment 18. The method of any one of embodiments 1 to 17, wherein the reaction mixture further comprises a reverse transcriptase.

Embodiment 19. The method of embodiment 18, wherein the reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.

Embodiment 20. The method of any one of embodiments 1 to 19, wherein the target nucleic acid is amplified by isothermal amplification.

Embodiment 21. The method of embodiment 20, wherein the isothermal amplification is enzyme-mediated amplification (EMA) , loop-mediated isothermal amplification (LAMP) , cross-priming amplification (CPA) , recombinase polymerase amplification (PRA) , helicase-dependent isothermal DNA amplification (HDA) , rolling circle amplification (RCA) , strand displacement amplification (SDA) , nicking enzyme amplification reaction (NEAR) , polymerase spiral reaction (PSR) , hybridization chain reaction (HCR) , primer exchange reaction (PER) , signal amplification by exchange reaction (SABER) , transcription-based amplification system (TAS) , self-sustained sequence replication reaction (3 SR) , or single primer isothermal amplification (SPIA) .

Embodiment 22. The method of embodiment 20, wherein the isothermal amplification is EMA.

Embodiment 23. The method of embodiment 20, wherein the isothermal amplification is LAMP.

Embodiment 24. The method of any one of embodiments 1 to 19, wherein the target nucleic acid is amplified by PCR.

Embodiment 25. A method of detecting a target nucleic acid in a sample comprising: (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) , (c) SAP Pol I and M-MLV reverse transcriptase; (d) an RNase H modified by 2, 3-dimethylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition at about 42℃ for about 5 to 30 minutes; and (3) collecting fluorescence signals.

Embodiment 26. The method of embodiment 25, wherein the reaction mixture further comprises a DNA helicase, a single-stranded binding protein, or both.

Embodiment 27. A method of detecting a target nucleic acid in a sample, comprising (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a Bst polymerase having reverse transcriptase activity; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition at 65℃ for about 30 min; (3) incubating the reaction mixture at about 95 ℃ for at least three minutes; and (4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.

Embodiment 28. The method of embodiment 27, wherein the reaction mixture comprises an outer primer pair and an inner primer pair, wherein each of the inner primer pair contains a looping fragment that is complementary to a region of the amplicon that allows for the formation of a loop at the end of the amplicon, and wherein one of the inner primer pair contains about 1-15 ribonucleotide (s) .

Embodiment 29. A method of detecting a target nucleic acid in a sample, comprising (1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) Tth Pol and SD Polymerase; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; (2) subjecting the reaction mixture to an amplification condition for 25-45 thermal cycles alternating between 95℃ and 75℃; (3) incubating the reaction mixture at about 95℃ for at least three minutes; and (4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.

Embodiment 30. The method of any one of embodiments 1 to 29, wherein the target nucleic acid is DNA.

Embodiment 31. The method of any one of embodiments 1 to 29, wherein the target nucleic acid is RNA.

Embodiment 32. The method of any one of embodiments 1 to 31, wherein the probe has about 10-30 nucleotides.

Embodiment 33. The method of any one of embodiments 1 to 32, wherein the probe comprises at least 4 ribonucleotides between the F and Q.

Embodiment 34. The method of any one of embodiments 1 to 32, wherein the probe is an RNA probe.

Embodiment 35. The method of any one of embodiments 1 to 34, comprising detecting two or more target nucleic acids, wherein for each target nucleic acid, a probe that can hybridize with the target nucleic acid and has a distinct fluorophore (F) is used.

Embodiment 36. The method of any one of embodiments 1 to 35, wherein during the detection step, the fluorescence spectrum is collected every 30 seconds.

Embodiment 37. The method of any one of embodiments 1 to 36, wherein the reaction mixture is kept in a vessel that remains closed from the beginning of the amplification to the end of the detection.

Embodiment 38. A nucleic acid detection system comprising: (a) (i) a DNA polymerase with strand displacement activity and reverse transcriptase activity or (ii) a DNA polymerase with strand displacement activity and a reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H that is reversibly inactivated; and (d) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

Embodiment 39. The nucleic acid detection system of embodiment 38, wherein the RNase H is reversibly inactivated by chemical modification, ligand association, or physical separation.

Embodiment 40. The nucleic acid detection system of embodiment 39, wherein the RNase H is reversibly inactivated by chemical modification.

Embodiment 41. The nucleic acid detection system of embodiment 40, wherein the RNase H comprises a lysine residue modified by a maleic anhydride or a derivative thereof having the structure below, wherein X and Y each independently is a negatively charged or neutral group.

Embodiment 42. The nucleic acid detection system of embodiment 40, wherein the RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride.

Embodiment 43. The nucleic acid detection system of embodiment 39, wherein the RNase H is associated with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent.

Embodiment 44. The nucleic acid detection system of embodiment 39, wherein the RNase H is physically trapped in a switchable chemical shell, a microsphere or an isolated chamber of a reaction vessel.

Embodiment 45. The nucleic acid detection system of any one of embodiments 38 to 44, wherein the RNase H can be reactivated by temperature change or pH change.

Embodiment 46. The nucleic acid detection system of any one of embodiments 38 to 45, wherein the RNase H is Pyrococcus furiosus RNase HI, Pyrococcus horikoshi RNase HI, Pyrococcus abyssi RNase HI, Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coli RNase HI, E. coli RNase HII, Saccharomyces cerevisiae RNase HI, mouse RNase HI or human RNase H.

Embodiment 47. The nucleic acid detection system of any one of embodiments 38 to 46, wherein the primer contains 1-20, 2-15, 4-12, or 4-8 ribonucleotides.

Embodiment 48. The nucleic acid detection system of embodiment 47, wherein the primer contains about 4, about 8, about 12, about 16 or about 20 ribonucleotides.

Embodiment 49. The nucleic acid detection system of any one of embodiments 38 to 48, wherein the DNA polymerase is Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; SD polymerase; or a functional variant thereof.

Embodiment 50. The nucleic acid detection system of any one of embodiments 38 to 49, wherein the reaction mixture comprises a reverse transcriptase.

Embodiment 51. The nucleic acid detection system of embodiment 50, wherein the reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.

Embodiment 52. A nucleic acid detection system comprising: (a) SAP Pol I and M-MLV reverse transcriptase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H modified by 2, 3-dimethylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

Embodiment 53. The nucleic acid detection system of embodiment 52, wherein the reaction mixture further comprises a DNA helicase, a single-stranded binding protein, or both.

Embodiment 54. A nucleic acid detection system comprising: (a) a Bst polymerase having reverse transcriptase activity; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H modified by 3-methylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

Embodiment 55. A nucleic acid detection system, comprising (a) Tth Pol and SD Polymerase; (b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) an RNase H modified by 3-methylmaleic anhydride; and (d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.

Embodiment 56. The nucleic acid detection system of any one of embodiments 38 to 55, wherein the probe has about 10-30 nucleotides.

Embodiment 57. The nucleic acid detection system of any one of embodiments 38 to 56, wherein the probe comprises at least 4 ribonucleotides between the F and Q.

Embodiment 58. The nucleic acid detection system of any one of embodiments 38 to 57, where in the probe is an RNA probe.

Embodiment 59. The nucleic acid detection system of any one of embodiments 38 to 58, comprising two or more different probes, wherein each can hybridize to a different nucleic acid and has a distinct fluorophore (F) .

6.6 Experimental

6.6.1 Example 1: Preparation of reversibly inactivated RNase H

An E. coli RNase H gene (rnh) was cloned into an E. coli strain for recombinant production. The recombinantly produced RNase H enzyme was dialyzed against phosphate buffer 8.0 and concentrated to 5 mg/mL. To reversibly inactivated RNase H, dimethyl maleic anhydride in DMSO was added to the RNase solution to a final concentration of 10 mM, and the mixture was kept at 4℃for 16 hours, until Tris buffer was added to the solution to stop the reaction. The modified enzyme was further purified by size exclusion and stored at -80℃.

Both the unmodified and the modified RNase H were characterized using the assay below: A reaction mixture was prepared, which included 30 mM Tris7.9, 50 mM potassium acetate (KAc) , 10 nM template single-stranded DNA, and 10 μM RNA probe. In four parallel reactions, (1) 5 U RNase H; (2) 30 μg/mL modified RNase H; (3) 30 μg/mL modified RNase H that had been reactivated by incubation at 45℃ for 10 minutes, and (4) no enzyme, were added to the reaction mixtures, respectively. The reactions were initiated by adding Mg (II) to the solution and incubated at 30℃. The fluorescence spectrum was collected every 30 seconds.

As shown in FIG. 3, modified RNase H lost its enzymatic activity, which was restored after the modified RNase H was incubated at 45℃ for 10 minutes.

6.6.2 Example 2: Detection of linear DNA fragment

African swine fever virus (ASFV) is a linear double-stranded DNA arbovirus with a genome size about 180 kilobase pairs (ranging between 170-194 kbp in variant strains) . The following procedures were followed to detect fragments of the ASFV genome in swine nasopharyngeal swab samples.

(1) Sample preparation: Three swine nasopharyngeal swab samples were processed using the nucleic acid extraction kit according to the manufacturer’s instructions. (2) Preparation of the reaction mixture: The following components were included: 30 mM Tris7.9, 50 mM potassium acetate (KAc) , 10 mM magnesium acetate (MgAc2) , 8%PEG8000, 0.1 mM dNTPs, 1 mM ATP, 10 mM phosphocreatine, 0.1mg/mL SAP I fragment (DNA polymerase) , 10 U M-MLV reverse transcriptase, 0.1 mg/mL UvsX, 1.0 mg/mL gp32 (single-stranded binding protein) , 50 μg/mL dimethyl maleic anhydride modified RNase H, 0.1 U phosphocreatine kinase, 200 nM primer F and R, respectively, and 10 μM RNA probe. (3) Detection: 10 μL of each sample was added to the reaction tube and the reaction was carried out at 45℃ in an ABI 7500 machine, with the fluorescence spectrum collected every 30 seconds after 10 minutes incubation.

As shown in FIG. 4, strong signals were detected in positive samples 1 and 2, but not negative sample 3.

6.6.3 Example 3: Detection of cyclic plasmid DNA

The following procedures were followed to detect cyclic plasmid DNA.

(1) Sample preparation: the VP72 gene of ASFV was synthesized and cloned into pUC57 plasmid. The plasmid was purified and diluted to different concentrations. (2) Preparation of the reaction mixture: The following components were included: 30 mM Tris7.9, 50 mM potassium acetate (KAc) , 10 mM magnesium acetate (MgAc2) , 8%PEG8000, 0.1 mM dNTPs, 1 mM ATP, 10 mM phosphocreatine, 0.1 mg/mL SAP I fragment (DNA polymerase) , 10 U M-MLV reverse transcriptase, 0.1 mg/mL UvsX, 1.0 mg/mL gp32 (single-stranded binding protein) , 50 μg/mL dimethyl maleic anhydride modified RNase H, 0.1 U phosphocreatine kinase, 200 nM primer F and R, respectively, and 10 μM RNA probe. (3) Detection: 10 μL of each sample was added to the reaction tube and the reaction carried out at 45℃ in an ABI 7500 machine for 10 minutes (amplification of the target gene and reactivation of the modified RNase H) . Afterwards, the fluorescence signals were collected every 30 seconds.

As shown in FIG. 5, the method was sufficiently sensitive to detect as low as 5 copies of the pUC-VP72 plasmid.

6.6.4 Example 4: Detection of viral RNA

Canine distemper virus (CDV) is a non-segmented, negative-stranded, enveloped RNA virus that belongs to the family Paramyxoviridae and the genus Morbillivirus. The following procedures were followed to detect CDV in nasopharyngeal swab samples.

(1) Sample preparation: The nasopharyngeal swab samples were processed using the nucleic acid extraction kit according to the manufacturer’s instruction. (2) Preparation of reaction mixtures: The following components were included: buffer (30 mM Tris7.9, 50 mM potassium acetate (KAc) , 3 mM betaine, 2.5 mM magnesium sulphate (MgSO4) , 0.1 mM dNTPs) ; enzymes (8.0 U Bst3.0 polymerase, 75 μg/mL citraconic anhydride (or 2-methylmaleic anhydride) modified thermostable RNase H) ; 2.4 μM FIP (forward inner primer) and BIP (back inner primer) ; 300 nM F3 and B3 primers (outer primers) ; and 10 μM RNA probe. (3) Detection: 5 μL of each sample was added into each tube, the reaction mixtures were first incubated at 65 ℃ for 30 minutes (reverse transcription and amplification of the viral RNA) , followed with 95 ℃ incubation for 5 minutes (reactivation of modified RNase H) and then 60 ℃ incubation (strand-displacement/generation of ss-RNA) , during which time fluorescence spectrum was collected every 30 seconds.

As shown in FIG. 6, strong signals were detected in the positive sample.

6.6.5 Example 5: Detection with PCR amplification

The following procedures were followed to detect fragments of the ASFV genome in swine nasopharyngeal swab samples.

(1) Sample preparation: Three swine nasopharyngeal swab samples were processed using the nucleic acid extraction kit according to the manufacturer’s instructions. (2) Preparation of the reaction mixture: The following components were included: buffer (30 mM Tris7.9, 50 mM potassium acetate (KAc) , 2.5 mM magnesium sulphate (MgSO4) , and 0.25 mM manganese sulfate (MnSO4) ) ; enzyme (5.0 U Tth polymerase, 1.0 U SD polymerase, and 75 μg/mL citraconic anhydride (or 2-methylmaleic anhydride) modified thermostable RNase H) ; 200 nM primer F and R and 10 μM probe. (3) Detection: 5 μL of each sample was added to the reaction tube, which was subjected to 40 two-temperature cycles (95℃ 15 seconds, 68℃ 15 seconds) . The reaction tubes were then incubated at 95 ℃ for 5 minutes to activate modified RNase H and then incubated at 60℃, during which time fluorescence spectrum was collected every 30 seconds.

As shown in FIG. 7, strong signals were detected in the positive sample.

6.6.6 Example 6: Multiplexing detection

This study demonstrated that the methods provided herein could achieve sensitive multiplex detection. The following procedures were followed to detect fragments of the ASFV VP72 gene in pUC57 (pUC-VP72) and PCV2 sequence in pUC57 (pUC-PCV2) .

(1) Sample preparation: mixtures of two plasmids (pUC-VP72 and pUC-PCV2) at different concentration in TE buffer were prepared. (2) Preparation of the reaction mixture: The following components were included: buffer (30 mM Tris7.9, 50mM potassium acetate (KAc) , 2.5 mM magnesium sulphate (MgSO4) , and 0.25 mM manganese sulphate (MnSO4) ) , enzymes (5.0 U Tth polymerase, 1.0 U SD polymerase, 75 μg/mL citraconic anhydride (or 2-methylmaleic anhydride) modified thermostable RNase H) , 100 nM primer F1, F2 and R1and R2, each, and 10 μM probe 1 and probe 2. (3) Detection: 5 μL of each plasmid-mixture sample was added to the reaction tube, which was subjected to 40 two-temperature cycles (95℃ 15 seconds, 68℃ 15 seconds) . The reaction mixtures were then incubated at 95 ℃ for 5 minutes to activate modified RNase H and then incubated at 60℃, during which time fluorescence spectrum was collected every 30 seconds.

As shown in FIG. 8, sensitive multiplexing detection was achieved in which strong positive signals were detected in samples containing only 10 copies of each plasmid.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, the group. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

7. Sequences

*The nucleotides preceded with letter “r” are ribonucleotides; the nucleotides followed by asterisks indicate thio-modification.

Claims

A method of detecting a target nucleic acid in a sample, comprising

(1) amplifying the target nucleic acid by subjecting a reaction mixture comprising the sample to an amplification condition, wherein the reaction mixture comprises:

(a) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; and

(b) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; and

(2) detecting the amplicons with an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q; wherein

(a) an RNase H cleaves the ribonucleotide (s) in the amplicons, generating nicking sites;

(b) the DNA polymerase recognizes the nicking sites and releases a strand of the double-stranded amplicons by strand-displacement (the “single-strand target” ) ;

(c) the oligonucleotide probe hybridizes with the single-strand target;

(d) the RNase H cleaves the ribonucleotide (s) on the hybridized probe, separating F from Q and producing a detectable signal; and

(e) the cleaved probe fragments separate from the single-strand target, allowing the repetition of steps (c) and (d) .
The method of claim 1, wherein the probe is included in the reaction mixture during target amplification.
The method of claim 1 or 2, wherein the RNase H is reversibly inactivated and included in the reaction mixture during target amplification, and becomes reactivated before the detection.
A method of detecting a target nucleic acid in a sample, comprising

(1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a DNA polymerase with strand-displacement activity; wherein (i) the DNA polymerase also has reverse transcriptase activity, or (ii) the reaction mixture further comprises a reverse transcriptase; (d) a reversibly inactivated RNase H; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q;

(2) subjecting the reaction mixture to an amplification condition for sufficient time to amplify the target nucleic acid;

(3) subjecting the reaction mixture to a transition condition for sufficient time to reactivate RNase H; and

(4) subjecting the reaction mixture to a detection condition and collecting fluorescent signals.
The method of claim 4, wherein the transition condition is the same as the amplification condition.
The method of any one of claims 3 to 5, wherein the RNase H is reversibly inactivated by chemical modification.
The method of claim 6, wherein the reversibly inactivated RNase H comprises a lysine residue modified by a maleic anhydride or a derivative thereof having the structure below, wherein X and Y each independently is a negatively charged or neutral group.
The method of claim 7, wherein the reversibly inactivated RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride.
The method of any one of claims 3 to 5, wherein the RNase H is reversibly inactivated by ligand association.
The method of claim 9, wherein the reversibly inactivated RNase H is associated with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent.
The method of any one of claims 3 to 5, wherein the RNase H is reversibly inactivated by physical separation.
The method of claim 11, wherein the RNase H is physically trapped in a switchable chemical shell, a microsphere or an isolated chamber of a reaction vessel.
The method of any one of claims 3 to 12, wherein the RNase H is reactivated by temperature change or pH change.
The method of any one of claims 1 to 13, wherein the RNase H is Pyrococcus furiosus RNase HI, Pyrococcus horikoshi RNase HI, Pyrococcus abyssi RNase HI, Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coli RNase HI, Escherichia coli RNase HII, Saccharomyces cerevisiae RNase HI, mouse RNase HI or human RNase H.
The method of any one of claims 1 to 14, wherein the primer comprises 1-15, 2-12, 4-12, or 4-8 ribonucleotides.
The method of claim 15, wherein the primer comprises about 4, about 6, or about 8 ribonucleotides.
The method of any one of claims 1 to 16, wherein the DNA polymerase is Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; or SD polymerase; or a functional variant thereof.
The method of any one of claims 1 to 17, wherein the reaction mixture further comprises a reverse transcriptase.
The method of claim 18, wherein the reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.
The method of any one of claims 1 to 19, wherein the target nucleic acid is amplified by isothermal amplification.
The method of claim 20, wherein the isothermal amplification is enzyme-mediated amplification (EMA) , loop-mediated isothermal amplification (LAMP) , cross-priming amplification (CPA) , recombinase polymerase amplification (PRA) , helicase-dependent isothermal DNA amplification (HDA) , rolling circle amplification (RCA) , strand displacement amplification (SDA) , nicking enzyme amplification reaction (NEAR) , polymerase spiral reaction (PSR) , hybridization chain reaction (HCR) , primer exchange reaction (PER) , signal amplification by exchange reaction (SABER) , transcription-based amplification system (TAS) , self-sustained sequence replication reaction (3 SR) , or single primer isothermal amplification (SPIA) .
The method of claim 20, wherein the isothermal amplification is EMA.
The method of claim 20, wherein the isothermal amplification is LAMP.
The method of any one of claims 1 to 19, wherein the target nucleic acid is amplified by PCR.
A method of detecting a target nucleic acid in a sample comprising:

(1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) , (c) SAP Pol I and M-MLV reverse transcriptase; (d) an RNase H modified by 2, 3-dimethylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q;

(2) subjecting the reaction mixture to an amplification condition at about 42℃ for about 5 to 30 minutes; and

(3) collecting fluorescence signals.
The method of claim 25, wherein the reaction mixture further comprises a DNA helicase, a single-stranded binding protein, or both.
A method of detecting a target nucleic acid in a sample, comprising

(1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) a Bst polymerase having reverse transcriptase activity; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q;

(2) subjecting the reaction mixture to an amplification condition at 65℃ for about 30 min;

(3) incubating the reaction mixture at about 95 ℃ for at least three minutes; and

(4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.
The method of claim 27, wherein the reaction mixture comprises an outer primer pair and an inner primer pair, wherein each of the inner primer pair contains a looping fragment that is complementary to a region of the amplicon that allows for the formation of a loop at the end of the amplicon, and wherein one of the inner primer pair contains about 1-15 ribonucleotide (s) .
A method of detecting a target nucleic acid in a sample, comprising

(1) preparing a reaction mixture comprising (a) the sample, (b) a primer pair for amplifying the target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ; (c) Tth Pol and SD Polymerase; (d) a thermal stable RNase H modified by 2-methylmaleic anhydride; and (e) an oligonucleotide probe complementary to the target nucleic acid comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q;

(2) subjecting the reaction mixture to an amplification condition for 25-45 thermal cycles alternating between 95℃ and 75℃;

(3) incubating the reaction mixture at about 95℃ for at least three minutes; and

(4) incubating the reaction mixture at about 60 ℃ and collecting fluorescence signals.
The method of any one of claims 1 to 29, wherein the target nucleic acid is DNA.
The method of any one of claims 1 to 29, wherein the target nucleic acid is RNA.
The method of any one of claims 1 to 31, wherein the probe has about 10-30 nucleotides.
The method of any one of claims 1 to 32, wherein the probe comprises at least 4 ribonucleotides between the F and Q.
The method of any one of claims 1 to 32, wherein the probe is an RNA probe.
The method of any one of claims 1 to 34, comprising detecting two or more target nucleic acids, wherein for each target nucleic acid, a probe that can hybridize with the target nucleic acid and has a distinct fluorophore (F) is used.
The method of any one of claims 1 to 35, wherein during the detection step, the fluorescence spectrum is collected every 30 seconds.
The method of any one of claims 1 to 36, wherein the reaction mixture is kept in a vessel that remains closed from the beginning of the amplification to the end of the detection.
A nucleic acid detection system comprising:

(a) (i) a DNA polymerase with strand displacement activity and reverse transcriptase activity or (ii) a DNA polymerase with strand displacement activity and a reverse transcriptase;

(b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ;

(c) an RNase H that is reversibly inactivated; and

(d) an oligonucleotide probe comprising a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.
The nucleic acid detection system of claim 38, wherein the RNase H is reversibly inactivated by chemical modification, ligand association, or physical separation.
The nucleic acid detection system of claim 39, wherein the RNase H is reversibly inactivated by chemical modification.
The nucleic acid detection system of claim 40, wherein the RNase H comprises a lysine residue modified by a maleic anhydride or a derivative thereof having the structure below, wherein X and Y each independently is a negatively charged or neutral group.
The nucleic acid detection system of claim 40, wherein the RNase H is modified by 2-methylmaleic anhydride; 2, 3-dimethylmaleic anhydride; or 2, 3-dichloromaleic anhydride.
The nucleic acid detection system of claim 39, wherein the RNase H is associated with a ligand that is an antibody, an antigen-binding fragment, an aptamer, or a chelating agent.
The nucleic acid detection system of claim 39, wherein the RNase H is physically trapped in a switchable chemical shell, a microsphere or an isolated chamber of a reaction vessel.
The nucleic acid detection system of any one of claims 38 to 44, wherein the RNase H can be reactivated by temperature change or pH change.
The nucleic acid detection system of any one of claims 38 to 45, wherein the RNase H is Pyrococcus furiosus RNase HI, Pyrococcus horikoshi RNase HI, Pyrococcus abyssi RNase HI, Thermococcus litoralis RNase HI, Thermus thermophilus RNase HI, E. coli RNase HI, E. coli RNase HII, Saccharomyces cerevisiae RNase HI, mouse RNase HI or human RNase H.
The nucleic acid detection system of any one of claims 38 to 46, wherein the primer contains 1-20, 2-15, 4-12, or 4-8 ribonucleotides.
The nucleic acid detection system of claim 47, wherein the primer contains about 4, about 8, about 12, about 16 or about 20 ribonucleotides.
The nucleic acid detection system of any one of claims 38 to 48, wherein the DNA polymerase is Thermus thermophilus DNA Polymerase (Tth Pol) ; DNA Polymerase I Large Fragment, Exonuclease Minus (Klenow Fragment) ; Bacillus subtilis DNA Polymerase (Bsu Pol) ; Bacillus stearothermophilus DNA Polymerase (Bst Pol) ; Stoffel fragment of Thermus aquaticus DNA polymerase I (Stoffel Fragment) ; SD polymerase; or a functional variant thereof.
The nucleic acid detection system of any one of claims 38 to 49, wherein the reaction mixture comprises a reverse transcriptase.
The nucleic acid detection system of claim 50, wherein the reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.
A nucleic acid detection system comprising:

(a) SAP Pol I and M-MLV reverse transcriptase;

(b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ;

(c) an RNase H modified by 2, 3-dimethylmaleic anhydride; and

(d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.
The nucleic acid detection system of claim 52, wherein the reaction mixture further comprises a DNA helicase, a single-stranded binding protein, or both.
A nucleic acid detection system comprising:

(a) a Bst polymerase having reverse transcriptase activity;

(b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ;

(c) an RNase H modified by 3-methylmaleic anhydride; and

(d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.
A nucleic acid detection system, comprising

(a) Tth Pol and SD Polymerase;

(b) a primer pair for amplifying a target nucleic acid, wherein one primer contains about 1-15 ribonucleotide (s) ;

(c) an RNase H modified by 3-methylmaleic anhydride; and

(d) an oligonucleotide probe that can hybridize with the target nucleic acid and comprises a fluorophore (F) , a quencher (Q) , and at least one ribonucleotide between the F and Q.
The nucleic acid detection system of any one of claims 38 to 55, wherein the probe has about 10-30 nucleotides.
The nucleic acid detection system of any one of claims 38 to 56, wherein the probe comprises at least 4 ribonucleotides between the F and Q.
The nucleic acid detection system of any one of claims 38 to 57, where in the probe is an RNA probe.
The nucleic acid detection system of any one of claims 38 to 58, comprising two or more different probes, wherein each can hybridize to a different nucleic acid and has a distinct fluorophore (F) .